key: cord- - syyl y authors: matsubayashi, makoto; takayama, hideko; kusumoto, masahiro; murata, misato; uchiyama, yuka; kaji, masaya; sasai, kazumi; yamaguchi, ryosaku; shibahara, tomoyuki title: first report of molecular identification of cystoisospora suis in piglets with lethal diarrhea in japan date: - - journal: acta parasitol doi: . /ap- - sha: doc_id: cord_uid: syyl y cystoisospora suis is a pathogen that causes diarrhea in pigs and can lead to serious disease. species identification, especially by histopathological examination, is often difficult because of morphologically similar parasites such as eimeria species. in this study, we used histopathological, bacteriological, virological, and parasitological methods to identify the cause of the disease in two piglets with severe diarrhea. villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of examined piglets, and coccidian parasites were found in the cytoplasm of the epithelial cells. in some merozoites in the meronts, the presence of two nuclei indicated type merozoites, characteristic of c. suis. according to cystoisospora-specific pcr targeting the rrna internal transcribed spacer (its ) gene, the sequences of the products were . % similar to those of c. suis. escherichia coli (o serogroup) exhibiting a virulence factor profile (lt, stb, and east as toxins and f as a colonization factor) was detected in one piglet. no other bacteria or significant enteric viruses were found. co-infection with c. suis and e. coli could imply aggravation of the disease, although further study is needed to assess the pathogenicity of this interaction. this study is the first to clarify by molecular analysis the sequences of c. suis detected in piglets in japan. many pathogens cause diarrhea in suckling or growing piglets. these include bacteria such as escherichia coli or salmonella enterica, parasites such as coccidian protozoa, and viruses such as porcine epidemic diarrhea (ped) virus. infection is often closely related to poor farm management, including inappropriate hygiene. cystoisospora suis and eimeria species are common protozoan parasites in pigs. c. suis in particular leads to serious disease in suckling piglets, causing diarrhea and dehydration mainly in animals - weeks of age (mundt et al. ) . eimeria species are thought to be less pathogenic (rommel ) , although some species, including eimeria suis, eimeria polita, and eimeria spinoisa, may cause clinical signs such as fever, diarrhea, and weight loss in young weaned pigs (jones et al. ; lindsay et al. ) . parasites of both genera infect intestinal mucosa, mostly of the small intestine, and generate asexually and sexually at the infected sites; oocysts are then shed in the feces. until now, morphological characterization of oocysts by direct smear or flotation techniques has been used to detect parasites and identify their species (four sporozoites in two sporocysts in c. suis, and two sporozoites in four sporocysts in eimeria species). however, it is impossible to identify the species of intracellular developmental zoites based only on histopathological examinations of intestinal tissue sections. recently, differentiation of c. suis and eimeria species has been accomplished with molecular diagnostic methods such as polymerase chain reaction (pcr) with species-specific *corresponding author: tshiba@affrc.go.jp first molecular identification of c. suis primers or subsequent sequencing (ruttkowski et al. ; samarasinghe et al. ) . pcr is reportedly more sensitive than microscopic examination (joachim et al. ) and is not complicated by morphological ambiguity in species identification. identification of coccidial isolates is important for determining the pathological agents causing disease and for evaluating pathogenicity. however, surveys of these parasites in japan have been limited, and their occurrence has not been well documented. in this study, we used histopathological, bacteriological, virological, and genetic methods to identify the causes of disease in piglets with severe diarrhea. this study is the first to clarify by molecular analysis the sequences of c. suis detected in piglets in japan. six piglets ( - days old) at a farm rearing sows in the kumamoto prefecture of japan, located in eastern kyushu island, exhibited diarrhea and died in november . two piglets (no. , days old; no. , days old) underwent histopathological, bacteriological, virological, and molecular analysis. at this farm, chronic diarrhea was not seen in piglets and intestinal lesions were not found in the examinations at slaughter houses. however, loose stools were shedded in some piglets for a week before their death. following necropsy, the stomach, small intestine (duodenum, jejunum, and ileum), colon, lung, spleen, heart, kidney, liver, and central nerves of each of the two animals were fixed in % buffered formalin at room temperature and then embedded in paraffin or examined by electron microscopy. parts of intestine were cryopreserved for additional analyses. tissue embedded blocks were cut into -μm thick and the sections were stained with hematoxylin and eosin (h & e) or periodic acid-schiff (pas). bacterial strains were isolated from the contents of the duodenum and ileum of the piglets using sheep blood agar (sba) and deoxycholate-hydrogen sulfide-lactose agar (dhl) as the culture media. sba and dhl are used to identify hemolytic bacteria and enterobacteriaceae (such as e. coli, s. enterica, or brachyspira hyodysenteriae), respectively; these bacteria are closely associated with porcine diarrhea. these intestinal contents were further examined by culture for clostridium perfringens on clostridium welchii agar with % egg yolk (ycw agar) (eiken chemical, tokyo, japan) for hours at ° c. the agar was incubated anaerobically. the bacterial strains isolated were identified by biochemical tests using api e (sysmex-biomerieux, tokyo, japan). o serogroups of the e. coli isolates were determined by agglutination using commercially available antisera (statens serum institut, copenhagen, denmark) according to the manufacturer's instructions. from these isolates, genes encoding virulence factors including toxins (lt, sta, stb, east , stx , and stx ) and colonization factors (f , f , f , f , f , and intimin) were examined by pcr as described by vu-khac et al. ( ) . all sections were stained with anti-e. coli o antibody (statens serum institut). sections of jejunum and ileum were also stained with antibodies to transmissible gastroenteritis (tge) virus and ped virus (both provided by dr. a. miyazaki, national institute of animal health, japan). immunolabeling and visualization were performed with the universal immunoenzyme polymer method using a histofine simple stain max-po ® kit (nichirei co., tokyo, japan). the sections were lightly counterstained with hematoxylin and assessed using light microscopy. for virological examination, contents of the small intestines of the two piglets were inspected by species-specific pcr using ped and tge viruses (kim et al. ). colon contents were tested for rotavirus using rapidtesta rota adeno (sekisui medical, tokyo, japan). following identification of cystoisospora parasites in piglet no. by histopathological examination, the formalin-fixed ileum tissue was examined by transmission electron microscopy (tem) as described previously (matsubayashi et al. ) . the sample was rinsed with . m phosphate buffer (ph . ), post-fixed with % oso , dehydrated in an ethanol series, and embedded in epon resin. ultra-thin sections were stained with saturated uranyl acetate (taab laboratories equipment, ltd., aldermaston, united kingdom) and examined with an electron microscope (h- ; hitachi, tokyo, japan). the presence of parasites in the ileum was confirmed by microscopy, and dna was purified from serial sections using a takara dxpat (takara bio, shiga, japan) for identification of cystoisospora species. to obtain more dnas of the parasites, dna was also purified from the cryopreserved ilea of two piglets using a qiaamp dna mini kit (qiagen gmbh, hilden, germany) according to the manufacturer's instructions. to identify cystoisospora species, pcr was performed to target the rrna internal transcribed spacer (its ) gene (approximately base pair [bp] primer set itsgf-itsr ) (samarasinghe et al. ) . each pcr amplification was conducted in a reaction volume of μl. a negative control containing reagents without a template was included in each pcr batch. pcr products were separated by electrophoresis on . % agarose gels, stained with ethidium bromide, and visualized on a uv transilluminator. pcr products were purified using a qi-aquick gel extraction kit (qiagen gmbh) and sequenced in makoto matsubayashi et al. both directions on an abi automated sequencer (applied biosystems inc., ca, usa). the obtained sequences were aligned with the representative nucleotide sequences of entamoeba species or genotypes using clustalw with initial fixed parameter values (dna data bank of japan [ddbj], http://clustalw.ddbj.njg.ac.jp/top-j.html). homology searches using the obtained partial gene sequences were performed using the fasta program (http://www.ddbj.nig.ac.jp/search/fastaj.html). a phylogenetic tree was constructed using the neighbor joining algorithm with evolutionary distances computed using the tamura-nei model and , bootstrap replicates. the resulting tree was generated using the mega software package (version ; tamura et al. ). veterinarians or civil servants who were employed by prefectural governments and belonged to the livestock hygiene service center conducted the examination of the animals or field hygienic surveys of pig farms. all examinations in this study were permitted by the farm owners and conducted as part of governmental affairs. ethical approval of animal experimentation was not necessary. human participants were not involved in this study. necropsy of piglet no. revealed no gross findings; but thinning of the small intestine was seen in piglet no. . histopathological findings in the jejunum included villous atrophy, formation of vacuoles, and adhesions of bacilli at the surface. epithelial cells with adherent bacilli were removed into the lumen. villous atrophy, diffuse necrosis, and flattening of mucosal epithelial cells were found in the ilea of both piglets (fig. a) . in these regions, coccidian parasites were found in the epithelial cells and intestinal gland cells of peyer's patch (table i) . these parasites were located in the cytoplasm of epithelial cells and stained positively with pas. most of them formed parasitophorous vacuoles or meronts containing one or more merozoites (figs. b and ) . the presence of two nuclei in a few merozoites indicated that these were type merozoites, as is characteristic of c. suis (lindsay et al. ) . bacteriological and virological examinations of intestinal contents are summarized in table i . strains of beta-hemolytic bacteria were isolated by sba agar culture from the contents of the duodenum and ileum of piglet no. . all of the strains formed red colonies on dhl agar and were identified as e. coli by biochemical tests using api e. c. perfringens, by contrast, was not detected by culture on ycw agar. all strains isolated with dhl agar belonged to the o serogroup and exhibited the same virulence factor profile (lt, stb, and east as toxins and f as a colonization factor). by immunohistochemical analysis of the sections, e. coli o antigens were also detected in the lumen of the stomach, duodenum, jejunum, ileum, colon, liver, and cerebrum of piglet no. . the detection of the e. coli from the internal organs was considered as the result of postdiarrheal septicemia as previously reported (fairbrother et al. ; berberov et al. ). the thinning of the small intestine at necropsy was thought to be due to villous atrophy, which was caused by the e. coli o . ped virus, tge virus, and rotavirus were not detected in either animal. e. coli o is one of the serogroups most frequently isolated from pigs, along with o , o , o , o , o , and o (fairbrother et al. ) . enterotoxins (lt and st) are associated with lethal diarrhea in pigs. therefore, the findings of this study, including characteristics of the isolated bacteria, strongly indicate that coccidian parasites cause diarrhea and imply that e. coli worsens the disease. by using cystoisospora-specific pcr, the predicted -bp product of the its gene was successfully amplified in dnas from the cryopreserved ileum and serial sections only of the no. piglet. the reason why the product could not be amplified from dna sample of no. remained unknown. the sequence of this product (accession no. lc ) was almost identical to that of c. suis (eu ), except for bp nucleotide substitutions ( . % identity). it was clearly different from those of cystoisospora anthochaerae from a bird (kf ) ( . % identity, with bp nucleotide substitutions) and cystoisospora belli (hm ) ( . % identity, with bp nucleotide substitutions). we constructed a phylogenetic tree of the its gene (fig. ) , although only a small number of cystoisospora species (seven species from five hosts) have been makoto matsubayashi et al. deposited in genbank. consequently, the isolate in the present study clustered with c. suis, and c. anthochaerae from anthochaera carunculata was closely related as a sister clade. the other species formed each host-specific clade. it can be taken as a valid result that the isolate detected are c. suis. we amplified the its region of cystoisospora species using a method whose efficacy for identification has been previously reported (samarasinghe et al. ) . although primers targeting the s rrna gene for detection or identification of cystoisospora species are available (ruttkowski et al. ) , some primers can amplify other organisms in addition to cystoisospora species (clark and diamond ) . in these cases, isolation of a single oocyst is needed for amplification of the target gene (matsubayashi et al. ). in the present study, we successfully identified cystoisospora isolates and demonstrated the potential for diagnostic amplification from tissue sections of lesions. the effectiveness of this method of targeting its is in accordance with that of previous reports (samarasinghe et al. ) . infection with c. suis is common in piglets worldwide. farms in japan are reported to have a high positive rate of c. suis (about %), with a prevalence in pigs of - % (saitoh and hattori ) ; however, only one report has been published. it is possible that this parasite is widespread in japan. although the age of the piglets at the infection has been previously shown to be crucial for the outcome like diarrhea and mortality (stuart et al. ; mundt et al. ) , disturbances in the intestinal flora caused by the infection are also considered to play a role for pathogenesis such as clostridial overgrowth (mundt et al. ; mengel et al. ) . until now, interactions with other pathogens such as bacteria and viruses have not been fully investigated. here we describe coinfection with e. coli, which possesses genes encoding toxin and colonization factors, and indicate the implication for aggravation of disease. further study is needed to assess the pathogenicity of this interaction. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic escherichia coli that produces multiple enterotoxins the laredo strain and other 'entamoeba histolytica-like' amoebae are entamoeba moshkovskii escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies extraintestinal escherichia coli infections in pigs detection of isospora suis (biester and murray first molecular identification of c journal of veterinary medicine. b, infectious diseases and veterinary public health coccidia associated with enteritis in grower pigs differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr ) diseases of swine porcine enteritis associated with eimeria spinosa henry, infection phylogenetic identification of cystoisospora spp. from dogs, cats, and raccoon dogs in japan ultrastructural characteristics and molecular identification of entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity necrotic enteritis due to simultaneous infection with isospora suis and clostridia in newborn piglets and its prevention by early treatment with toltrazuril occurrence of isospora suis in germany, switzerland and austria. journal of veterinary medicine b infectious diseases and veterinary public health accepted for publication population biology studies on isospora suis in piglets investigations into the synergic effect of postnatal experimental infection with isospora suis and natural infection with clostridium perfringens β for the development of necrotic enteritis in piglets körting) veterinärmedizinische parasitologie pcr-based differentiation of three porcine eimeria species and isospora suis survey of porcine neonatal coccidium isospora suis (biester and murray ) in japan. proceedings of phylogenetic analysis of cystoisospora species at the rrna its locus and development of a pcr-rflp assay coccidiosis in swine: dose and age response to isospora suis mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods serotypes, virulence genes, intimin types and pfge profiles of escherichia coli isolated from piglets with diarrhoea in slovakia key: cord- -ymsl snv authors: miura, fuminari; matsuyama, ryota; nishiura, hiroshi title: estimating the asymptomatic ratio of norovirus infection during foodborne outbreaks with laboratory testing in japan date: - - journal: j epidemiol doi: . /jea.je sha: doc_id: cord_uid: ymsl snv background: foodborne norovirus outbreak data in japan from – , involving virological surveillance of all symptomatic and asymptomatic individuals, were reanalyzed to estimate the asymptomatic ratio of norovirus infection along with the risk of infection and the probability of virus shedding. methods: employing a statistical model that is considered to capture the data-generating process of the outbreak and virus surveillance, maximum likelihood estimation of the asymptomatic ratio was implemented. results: assuming that all norovirus outbreaks (n = ) were the result of random sampling from an identical distribution and ignoring genogroup and genotype specificities, the asymptomatic ratio was estimated at . % ( % confidence interval [ci], . – . ). although not significant, separate estimation of the asymptomatic ratio of the gii. genotype appeared to be greater than other genotypes and was estimated at . % ( % ci, . – . ). conclusion: the present study offered the first explicit empirical estimates of the asymptomatic ratio of norovirus infection in natural infection settings. the estimate of about % was consistent with those derived from volunteer challenge studies. practical difficulty in controlling gii. outbreaks was supported by the data, considering that a large estimate of the asymptomatic ratio was obtained for the gii. genotype. noroviruses are an evolutionarily diverse group of singlestranded positive-sense rna viruses without the envelope, belonging to the caliciviridae family, and are responsible for a substantial part of acute viral gastroenteritis in humans. , norovirus outbreaks have happened both in developing and industrialized nations. once infected, symptoms are characterized by non-bloody diarrhea, vomiting, nausea, and abdominal cramps, and there has been no specific treatment to accelerate the cure of patients infected with the virus. vaccines have yet to be fully developed and brought into practice, and the presence of various genotypes prevents infected human from acquiring sufficient specific immunity. published studies have reported that around % of norovirus infection remains asymptomatic, while such asymptomatically infected individuals are known to excrete substantial volume of viruses. , vomit from infectious individuals contains over copies=gram of noroviruses, , and infectious vomitus is known to be sometimes aerosolized, , making it difficult to prevent secondary transmissions. the virus spreads through fecal-oral routes, such as via contaminated water, food, and people's contaminated hands. , published studies indicate that infections associated with environmental contaminants are mainly caused by contaminated water or foods (especially oysters), accounting for seasonal outbreaks that are frequently observed in winter. given these features, workers who are directly engaged in cooking and handling of foods, such as cooks, food servers, and food factory workers, are considered as one of the key subjects for prevention of secondary transmissions. [ ] [ ] [ ] one of the notable virological features of noroviruses is genotype variation, which is divided into five genogroups and many genotypes in each genogroup. as of , the most prevalent genotype in humans is gii. , , which accounts for as many as % of all reported norovirus infections with virus isolation. some pieces of evidence indicate that this genotype evolves rapidly, escaping from selection pressure. , in japan, the majority of outbreaks are caused by genogroup ii, and a large proportion is caused by gii. . gii. -associated epidemics in japan were first observed in , and the genotype has been continuously observed every year since then. to decipher the most effective preventive measures against this virus, it is vital to quantitatively clarify the natural history characteristics, including asymptomatic ratio, the risk of infection per exposure, and the probability of virus shedding. nevertheless, explicit estimates are very scarce, except for rigorous challenge studies that helped quantify the natural history , ; moreover, it is unclear if the natural history of experimentally infected individuals are similar to those based on natural infection. a small number of mathematical modelling studies estimated a part of the abovementioned values, - but explicit model-based estimates of the asymptomatic ratio have yet to be offered. the present study aims to estimate the asymptomatic ratio of norovirus infection, reanalyzing foodborne outbreak data with laboratory testing in japan, along with other parameters, including virus shedding frequency and the risk of infection. in addition to estimating the asymptomatic ratio for all noroviruses, the present study also compares the estimate across different genogroups and genotypes. we reanalyzed a published dataset that tested both symptomatic and asymptomatic individuals during norovirus outbreaks at food catering settings in japan from november , to december , (n = outbreaks). in that study, a total of , involved individuals, including food-handlers, other workers, and potentially exposed customers, were examined for the presence of symptoms (ie defined as either non-bloody diarrhea or vomiting) and also virus shedding from their feces, conducting a survey shortly after each outbreak (within week). asymptomatic individuals were defined as persons without diarrhea and vomiting, and the asymptomatic ratio was defined as the proportion of asymptomatically infected individuals among the total of infected individuals. through stool specimen tests using highly sensitive real-time reverse transcription polymerase chain reaction (rt-pcr), which has the highest detection limit among all existing genome testing methods the virus and its genogroup=genotype were identified. due to its highly sensitive nature, the real-time rt-pcr was shown to detect positive norovirus specimens if > copies were included in a well. since asymptomatic healthy individuals as well as symptomatic cases were tested, the results in each outbreak are classified into four categories, ie, (i) symptomatic and virus positive, (ii) symptomatic but virus negative, (iii) asymptomatic and virus positive, and (iv) asymptomatic and virus negative. in addition to the number of people in each category for each outbreak, genotyping results were obtained. figure summarizes the survey results, and the coded data are given in etable . it should be noted that the number of asymptomatic and virusnegative individuals includes both uninfected individuals and infected cases without virus shedding. the abovementioned study has been very rigorous, in that all individuals involved in each food-borne outbreak were fully surveyed, which has been extremely rare and not routinely attainable. in addition, such a study must employ the highly sensitive real-time rt-pcr, a condition that was also satisfied by the - study. moreover, all events were observed in the same year in japan within food handling facilities of comparable sizes. here, we describe a statistical model with which the asymptomatic ratio and other parameters were jointly estimated using the abovementioned foodborne outbreak data. first, the data generating process is schematically illustrated in figure . three pieces of information are considered: the infection process, the illness onset, and virus shedding results. we assume that all situations and population were similar across outbreaks and shared identical parameters. let p be the risk (or the probability) of norovirus infection given an exposure at food handling center, and similarly, let s be the asymptomatic ratio of norovirus infection, representing the probability that infected individuals escape from symptomatic illness. moreover, let q be the probability of virus detection from an infected individual. while these parameters are allocated, all these processes are not directly observed in the original survey. the epidemiological information we have an access includes (i) the total number of people involved (including healthy individuals) in each outbreak, (ii) the number of symptomatic cases, and (iii) the numbers of symptomatic virus shedding cases and asymptomatic virus shedding cases. from these pieces of information, we estimate p, q and s jointly. let i be an identity of an outbreak (ie, i ranges from to ). all three parameters were dealt with as deterministic parameters; we did not consider demographic stochasticity in the data generating process. since observed processes are the single and independent success=failure process, the resulting process is a bernoulli trial, and thus, we assume that each observed event was a result of binomial sampling (because every observed step was binary and mutually independent). the probability that n i cases experience symptomatic illness among a total of n i symptomatic and m i asymptomatic individuals is described by similarly, the probability that y i cases are asymptomatic but positive among a total of m i asymptomatic individuals (that include uninfected individuals) is p ðy i ; m i ; p; q; sÞ the probability that x i cases shed the virus among a total of n i symptomatic cases is it should be noted that q was assumed as independent of symptoms because the rt-pcr testing employed was highly sensitive and the cut-off value must have allowed q to be comparable between symptomatic and asymptomatic infected individuals. , sensitivity analysis was conducted to address potential difference of q between symptomatic and asymptomatic infection (see below). it should also be noted that the parameter p should have ideally reflected the demographic stochasticity of the data generating process, but p is a mixture of food-borne and human-to-human transmissions and we do not have a sufficient dataset to characterize the distribution. thus, the abovementioned process in principle adopted a deterministic modeling approach and measured the uncertainty of p based on sampling error alone. a maximum likelihood method was employed to estimate parameter values, p, q and s. these parameters were estimated assuming several different interpretation scenarios of empirical miura f, et al. data. first, a single set of p, q, and s was estimated, assuming that each outbreak was a random sampling result from the abovementioned mechanisms ( )-( ). second, parameters were assumed to be different between genogroup and , so there were six unknown parameters. third, parameters were assumed to be different by genotype. when estimating parameters by genotype, it should be noted that the available number of outbreaks differed by genotype (eg, seven outbreaks for gii. ), and we excluded outbreaks in which multiple genotypes were identified. to identify any improved fit despite increased number of parameters, akaike information criterion (aic) was computed for each scenario. using ( )- ( ), we derive the total likelihood l for each single interpretation scenario, computed as where l ð p; s; n; mÞ ¼ y i n i þ m i n i ð pð À sÞÞ ni ð À pð À sÞÞ mi ; l ð p; q; s; m; yÞ x i q xi ð À qÞ ðniÀxiÞ : maximum likelihood estimates of p, q, and s were obtained by minimizing the negative logarithm of ( ), and the % confidence intervals (ci) were computed using the profile likelihood. to analyze the sensitivity of parameter estimates to differential frequency of virus shedding between symptomatic and asymptomatic infections, we used the following α, ie, where q is the probability of virus shedding from an asymptomatic case and q is the probability from a symptomatic case. we set the baseline value of α as . , which indicates that asymptomatic and symptomatic cases have the same probability of virus shedding (due to a highly sensitive pcr testing method , ), which is supported by empirical observation. , however, as part of sensitivity analysis, the value of α was changed from . to . , anticipating that virus shedding frequency from asymptomatic cases might potentially be smaller than that from symptomatic cases. calculating the aic for each interpretation scenario, the aic of the genogroup-combined model (ie, a model with three parameters only) yielded the value of , . , while that of the genogroupseparated model (ie, a model with six parameters) was , . . the smaller value of aic for a genogroup-separated model indicated that the model with genogroup information was better fitted to outbreak data, so there might potentially be a difference between gi and gii. figure compared observed and predicted numbers of people by symptom and pcr testing result, visually demonstrating the satisfactory description of observed patterns. a χ goodness-of-fit test revealed no significant deviations between predicted and observed numbers for both genogroup-combined and genogroup-separated models (p > . for both models with degrees of freedom at and , respectively). varying the value of α from . to . , we examined the sensitivity of model parameters to differential virus shedding frequency between symptomatic and asymptomatic infections (figure ). when α gets smaller, both the risk of infection ( p) and the asymptomatic ratio (s) were estimated to be greater. the probability of virus shedding among symptomatic cases remained stable ( figure c ). of the total of outbreaks, there were two outbreaks involving large number of subjects (n = , for two outbreaks; . % of the total), which could lead us to feel that the overall results may be highly influenced by those two outbreaks. removing the two outbreaks and analyzing the remaining altogether, parameters p, s, and q were estimated at . % ( % ci, . - . %), . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. the risk of infection (p) was significantly greater than the estimate in which all outbreaks were analyzed together, but two other parameters, including asymptomatic ratio (s) and the risk of virus shedding (q), were not significantly deviated. the present study reanalyzed foodborne norovirus outbreaks in japan from - that involved laboratory testing of all individuals including healthy people. to statistically infer asymptomatic ratio in joint with the risk of infection and the probability of virus shedding, a statistical model was developed to describe the data generating process. assuming that all outbreaks occurred due to repeated random samplings, the risk of infection (p) and the asymptomatic ratio (s) were estimated to be . % and . %, respectively. the probability of virus shedding (q) was estimated at . %. possible differences by genogroup were implicated by penalized likelihoods. the risk of infection was small for gii. , while the asymptomatic ratio was estimated to be high for this common genotype. an important contribution of the present study to the literature of norovirus is that the asymptomatic ratio was estimated to be about %, which is consistent with existing literature based on volunteer challenge studies. , , not based on human volunteer challenge, our study has successfully estimated the asymptomatic ratio from naturally occurred outbreaks, as has also been achieved for other infectious diseases. in particular, the asymptomatic ratio of common genotype gii. was estimated to be high, which is in line with the conventional understanding that the control of gii. is likely more difficult than that of other genotypes. in addition to original findings of similar virus shedding frequency inducing asymptomatic infections among food handlers. not only spreading the virus via food handlers, but the greater asymptomatic ratio allows infected individuals to be freely mobile and have more opportunities to pass the virus to others. when we assumed that the actual frequency of virus shedding from asymptomatic individuals is smaller than symptomatic cases, the asymptomatic ratio was estimated to be greater than the baseline. in other words, the estimated value around % might be even greater than our result. there was no statistically significant difference between gi and gii, while the aic value was indicative that there might be a difference between gi and gii. this was caused by limited sample size, and slight non-significant differences (eg, greater risk of infection for gi than gii and smaller asymptomatic ratio for gi than gii) were identified, implying that gi might be more controllable than gii. to our knowledge, the present study is the first to epidemiologically endorse different patterns of outbreaks by genogroups in natural settings, though differences have been indicated in experimental studies. , , while a greater asymptomatic ratio for gii. than others was consistent with our existing understanding, the smaller infection risk with gii. than others calls for careful interpretation. due to the frequent circulation of gii. , a portion of exposed individuals might have been immune in advance of outbreaks (that were not widespread before but may have remained unrecognized); moreover, evolutionary changes in gii. (eg, antigenic evolution) might have later varied the virus characteristics, including the infectiousness of gii. . evolutionary epidemiological study of gii. to be adapted to human populations is a subject of our ongoing study. two limitations must be discussed. first, the survey data contained epidemiologically limited information. that is, to define symptomatic cases, diarrhea or vomiting was employed, but they were qualitative definitions that did not explicitly involve the frequency, such as the number of defecations=vomiting per day. such qualitative definition is favored for clearly defining what a symptomatic case is, and an important advantage of the present study is that an identical research group has conducted the entire survey and minimized the measurement of error that could potentially arise from researchers. nevertheless, when it comes to additionally measuring the severity of illness, which is out of the scope of the present study, it might have been unavoidable to include human perception error. such misclassification could have potentially affected the asymptomatic ratio (eg, due to broad definition of asymptomatic persons). moreover, the exact timing of stool sampling was unavailable; however, it is likely that all sampling took place in a matter of week from the onset of outbreaks, during which the virus shedding level of both symptomatic and asymptomatic cases should be substantial. second, the model involved a few simplifications. the simplified points include the ignorance of the dependent happening in the risk of infection. since the majority of outbreaks were considered as purely foodborne, a single parameter p was allocated, but if human-to-human transmission was involved, it might have been better to account for that information, which was not attainable in the present study using the same dataset. another simplification was the use of binomial sampling process for each likelihood. as a consequence, the uncertainty bound of p in the present study should be regarded as an underestimate. one of the drawbacks has been seen in significantly greater p that we obtained when two big outbreaks were removed from analysis; in other words, the estimate might not have been significantly different if the uncertainty also reflected demographic stochasticity. despite the abovementioned limitations, our study has successfully demonstrated that the asymptomatic ratio can be estimated from outbreak data in which all involved individuals undertook laboratory testing and also that the asymptomatic ratio was about % in natural outbreak settings. practical difficulty in controlling gii. outbreaks was supported by its large estimate of the asymptomatic ratio. collecting similar datasets from further outbreaks, we will be able to gain further insights into the natural history of norovirus infection, including genotype-specific differences. understanding the natural history better, similar explicit estimates based on natural infection in 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divergent haplotypes in the - norovirus gii= epidemic population by genomewide tracing of evolutionary history shedding of norovirus in symptomatic and asymptomatic infections heterogeneity in norovirus shedding duration affects community risk mathematical model for the control of nosocomial norovirus duration of immunity to norovirus gastroenteritis fitting outbreak models to data from many small norovirus outbreaks norovirus infections in symptomatic and asymptomatic food handlers in japan broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr identification of the asymptomatic ratio mers-cov scenario and modeling working group. estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia supporting online information accompanies the paper on the journal website.financial support: fm acknowledges the graduate program for social ict global creative leaders (gcl), the university of tokyo for financially supporting his study at hokkaido university. hn received funding support from the japan agency for medical research and development, jsps kakenhi grant numbers kt , k , and h , the japan science and technology agency (jst) crest program (jpmjcr ) and ristex program for science of science, technology and innovation policy. rm acknowledges the jsps kakenhi grant number h . the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.conflicts of interest: none declared. ethical standards: not applicable, as our study used published and anonymized data. key: cord- -bvalr bl authors: nomura, shuhei; sakamoto, haruka; sugai, maaya kita; nakamura, haruyo; maruyama-sakurai, keiko; lee, sangnim; ishizuka, aya; shibuya, kenji title: tracking japan’s development assistance for health, – date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: bvalr bl background: development assistance for health (dah) is one of the most important means for japan to promote diplomacy with developing countries and contribute to the international community. this study, for the first time, estimated the gross disbursement of japan’s dah from to and clarified its flows, including source, aid type, channel, target region, and target health focus area. methods: data on japan tracker, the first data platform of japan’s dah, were used. the dah definition was based on the organisation for economic co-operation and development’s (oecd) sector classification. regarding core funding to non-health-specific multilateral agencies, we estimated dah and its flows based on the oecd methodology for calculating imputed multilateral official development assistance (oda). results: japan’s dah was estimated at . ( ), . ( ), . ( ), . ( ), and . million usd ( ) in constant prices of . multilateral agencies received the largest dah share of . – . % in these periods, followed by bilateral grants ( . – . %) and bilateral loans ( . – . %). ministry of foreign affairs (mofa) was the largest contributors to the dah ( . – . %), followed by ministry of finance (mof) ( . – . %). japan’s dah was most heavily distributed in the african region with . – . % share. the channel through which the most dah went was global fund to fight aids, tuberculosis, and malaria ( . – . %). between and , approximately % was allocated to primary health care and the rest to health system strengthening. conclusions: with many major high-level health related meetings ahead, coming years will play a powerful opportunity to reevaluate dah and shape the future of dah for japan. we hope that the results of this study will enhance the social debate for and contribute to the implementation of japan’s dah with a more efficient and effective strategy. universal health coverage (uhc) is the cornerstone of sustainable and inclusive growth. the promotion of uhc to ensure that all people receive quality health services they need without financial hardship contributes to the development of human resources and security [ ] . at the joint session of finance and health ministers of the group of (g ) held in osaka, japan in june , the "g shared understanding on the importance of uhc financing in developing countries" was confirmed, and agreement was reached to accelerate our global efforts to promote uhc through fair, equitable and preferential use of domestic resources and further invest in primary health care (phc) services [ ] . furthermore, at the seventh tokyo international conference on african development (ticad vii) held in yokohama, japan in august , the yokohama declaration was adopted to promote a robust and sustainable society for human security in africa region, including the achievement of the sustainable development goals (sdgs) and african union (au) agenda as well as uhc [ ] . prioritizing domestic resources and promoting private investment in africa were also agreed as priority areas [ ] . for many donors, development assistance for health (dah) is one of the most important tools for promoting diplomacy and international cooperation with developing countries. for japan, in , the ministry of foreign affairs (mofa) issued the global health diplomacy strategy, officially positioning health as a pillar of government foreign diplomacy [ , ] . as one of the concrete measures, the strategy aims to ensure human security by promoting uhc in developing countries using dah [ ] . human security, as an universal definition, "protects the vital core of all human lives in ways that enhance human freedoms and human fulfillment" [ ] . through its contribution to health, the dah helps build the human capital necessary for economic development [ ] , resulting in greater self-reliance of recipient countries [ ] . also, in an increasingly interconnected world, epidemics, antimicrobial resistance (amr), and other health threats are easily spread, and efforts to prevent or suppress these threats in one country often benefit neighboring and distant countries [ ] . the dah therefore can provide a way for donors and recipient countries to benefit and share global prosperity [ ] . importantly, dah functions as a support for issues that cannot be adequately addressed with domestic and private funds to achieve sdgs, including the sdg target . on uhc. in addition, there is an increasing need to act as a catalyst to strengthen capacity to mobilize and properly manage and disburse domestic and private funds [ ] . dah must also adapt to the growing health effects of climate change, conflict, and refugees/migrants crisis, and global political trends that emphasize national interests [ ] . in early , japanese former foreign minister mr. taro kono (until september ) stated that there was no guarantee that official development assistance (oda) would increase in the future because of japan's fiscal deficit [ ] . as stated in the government official documents and by mr. kono himself, there is a need for more efficient and effective implementation of oda/dah and greater transparency and accountability because oda/ dah is funded by taxes [ , ] . in japan, decision-making and implementation of oda is led by mofa based on the development cooperation charter, reflecting the wishes of other ministries and agencies, such as the ministry of finance (mof) and japan international cooperation agency (jica: an implementation agency that coordinates bilateral oda). in , the oda charter was created by the cabinet office with asia as a priority region, indicating the basic principles of supporting the economic growth and social development of developing countries through oda [ ] . in , it was revised for the second time since the revision in , and the name was changed to "development cooperation charter" [ ] . the core of this revised, new charter is a deep commitment to a proactive contribution to peace standing on the concept of human security [ ] . it also added measures that have not been considered within the framework of oda, such as strengthening cooperation with private sectors; and clarified that oda for military operations must be limited to non-military purposes, such as disaster relief. it puts emphasis not only on the interests of developing countries, but also on securing 'national interests', indicating japan's willingness to actively engage in international cooperation that would contribute to japan's security and economic growth. in the same year, mofa also published the "basic design for peace and health (global health cooperation)" as a guideline for global health policy under the new charter. in this guideline, they put three areas as their priority: promotion of resilient global health governance able to respond to public health crises and natural disasters based on the concept of human security, including the realization of uhc; utilization of japanese expertise, experience, medical products and technologies; and tailored support in response to diversification of regional needs [ ] . in practice, however, due to its complex and fragmented administrative procedures and structures, the actual overall picture and flow of dah has been unclear to date, and strategic decision-making and implementation across ministries and agencies are not sufficient. for example, there is no shared priority among ministries on their own commitments and no inter-ministry collaboration in the budget acquisition process [ ] . jica and other relevant organizations and domestic stakeholders also have their own policies, limiting strategic policy coordination with the government [ ] . here, we present, for the first time, an overview of japan's dah, by examining the tracking of dah using data on japan tracker, the first data platform of japan's dah that the authors were in charge of [ ] . the results of this study will contribute to an effective and strategic dah decision-making and implementation across/within ministries and agencies. data on oda projects from to administered by mofa were used. this data includes, for each project and year, gross disbursements of oda, source (contributing ministry/agency), aid type (bilateral grant, including technical assistance; bilateral loan; earmarked funding to multilaterals [that is also called as 'bi-multi' and was reported as bilateral oda]; and core funding to multilateral agencies [i.e. assessed contributions and non-earmarked funding]), target country/region, and target health focus area. health focus area was based on purpose codes (also known as creditor reporting system [crs] codes) for sector classification defined by organisation for economic co-operation and development's (oecd) development assistance committee (dac) [ ] . purpose codes used for dah were (health) and (population policies/ programs and reproductive health) based on the previous studies, including the five-digit purpose codes [ ] [ ] [ ] . [ ] . regarding core funding to non-health-specific multilateral agencies (e.g., world bank), where it was not possible to directly identify dah out of the oda and its flows to target country/region and health focus area, they were estimated based on the oecd methodology for calculating imputed multilateral oda as follows [ ] . step : based on reports from multilateral agencies to the oecd [ ] , oda flows to the health sector of each agency (i.e., dah) were calculated as a percentage of total oda disbursements (α: health sector share of the agency's total oda). step : based on this report [ ] , each agency's dah flows to each target country/region and each health focus area were calculated (β: target country/region-specific share of the agency's dah, and γ: health focus area-specific share of the agency's dah). step : multiplying α, β, and γ obtained for each multilateral agency by the total oda from japan, we estimated flows of japan's dah through the agency. for example, the mof's multilateral dah through the world bank was estimated by multiplying the total oda from the mof to the world bank by α. in addition, mof's dah through the world bank to a particular target country/region and health focus area was estimated as total oda × α × β and total oda × α × γ, respectively. primary health care and health system strengthening in the spirit of both alma-ata and astana, a wellfunctioning phc system is regarded as the foundation for countries that successfully finance and provide quality health services to their entire population; this is essential to achieve uhc [ , ] . although the current crs system does not facilitate standardized measurement of dah for phc, shaw et al. ( ) attempted to define dah on 'phc delivery' versus on 'health system strengthening (hss)' in support of phc delivery, using crs purpose code data, and our study followed their definition and methodology and estimated how much japan's dah was invested in phc and hss [ ] . in short, as in the previous study, our working definition of phc focused only on inputs that are under the control of the health system itself, so intersectoral interventions (e.g., safe water, sanitation, and hygiene) were not considered. our scope of phc therefore included treatment of diseases and injuries, including the provision of essential medicines; reproductive health; prevention, detection and treatment of hiv/aids, tuberculosis and malaria; public health measures, preventive health care, promotion and education of healthy behavior, good nutrition, and immunization. in this study, we referred to dha for such scope as being most relevant to 'phc delivery'. meanwhile, in order for phc to function properly, system-wide investments are necessary: for example, effective priority setting system; sound management, administrative, financial, and technical capacities; adequate human resources and institution capacity; up-to-date health information systems for monitoring and evaluation of policies and programs; and appropriate regulatory, governance, finance, and accountability mechanisms. in this study, we referred to dah for such investment as being most relevant to 'hss' in support of phc delivery, as in the previous study [ ] . our working definition of hss is therefore much narrower than the extensive discussion of hss often found in the literature, where most public expenditures aimed at improving health care can be interpreted as hss. a discussion of their detailed definition and justification can be found in previous studies [ ] . a list of corresponding crs purpose codes for phc and hss can be found in the resulting table of this study. table . except for in , dah contributed to multilateral agencies had the largest share of about % among all aid types. the share of bilateral (grants) was about % and that of bilateral (loans) was about % in the periods. mofa accounted for majority of the dah contribution ( . %, ), with mof ( . %, ) and ministry of health, labour and welfare (mhlw) ( . %, ) following in that order. japan's dah was most heavily distributed in the african region, with a range of . - . % share between and , followed by south and central asia ( . - . %). figure shows japan's dah by aid type and target region. in bilateral (grants) and multilateral, japan's dah was allocated the most to africa. as for bilateral (loans), the dominant focus was on south and central asia, with the exception of . exact values for fig. table . in japan's dah, health policy and administrative management, medical services, infectious disease control, and sexually transmitted disease (std) control including hiv/aids were the priority areas, which occupied a share over about % every year. however, by aid type the trend was different (fig. ). infectious disease control had largest shares in bilateral (loans) except for - , while std control including hiv/aids had the largest share in multilateral. for bilateral (grants), on the other hand, japanese funds also focused around basic health infrastructure and health policy and administrative management. basic nutrition demonstrated a steep decline in its share in - among the bilateral grants. it was . % in , but fell to . % in . exact values for fig. can be found in supplementary table . to quantify levels and trends in dah to phc and hss, the five-digit purpose codes were grouped into two broad clusters (phc and hss). as reported in fig. with exact values presented in supplementary this study provided, for the first time, an estimated gross disbursement of japan's dah and its flows. japan's dah was found to be approximately million usd in . the main source of dah was mofa. according to the oecd statistics, japan's gross disbursements of oda in amounted to . billion usd [ ] , which means that the share of dah in oda was about . %. in accordance with the oda charter, japan has traditionally placed asia, which has a close relationship with japan, as a priority region [ ] . meanwhile, we revealed that approximately half of japan's bilateral and multilateral dah were allocated to the african region in the study periods (fig. ) . this finding may reflect japan's recent efforts to strengthen its diplomatic relations with african countries through various efforts including ticad as well as agenda and sdgs. for example, japan hosted ticad vi in kenya in and launched the "uhc in africa: framework for action" in partnership with the world bank, who, the global fund, and the african development bank [ ] . this is a roadmap for african countries to accelerate progress towards uhc and to monitor and assess their progress. in the same year, japan hosted the ise-shima group of seven (g ) summit, which was held in the aftermath of the ebola crisis in western africa, providing an important opportunity for japan to advance global health governance issues [ ] . in addition, in may , jica signed, for the first time, an oda loan agreement of up to . million usd (at current price in ) with the government of the federal republic of nigeria for the polio eradication project [ ] . this project aimed to contribute to the early eradication of polio in nigeria by ensuring smooth vaccination of children under five years of age throughout the country through the procurement of polio vaccines. oda loans to africa in in fig. refer to this project. this study examined the distribution of earmarked funding (bi-multi) and core funding to multilateral agencies for dah in - . bi-multi funding is a resource to multilateral agencies over which the donor retains some degree of control on decisions regarding disposal of the funds. such flows may be earmarked for a specific country, project, region, sector or theme. it is aid for bilateral functions channelled through on the other hand, core funding to multilateral agencies are used for a variety of purposes, some of which are channeled to global functions (e.g., provision of global public goods, management of cross-border externalities, and fostering of leadership and stewardship). schäferhoff et al. ( ) estimated the total share of core funding going to global functions by agencies, as follows: who %, unaids %, unfpa %, unicef %, world bank (international development association) %, global fund %, and gavi % [ ] . in japan, core funding accounted for the majority of the dah channeled through multilateral agencies, except for those through unicef and unfpa. in particular, japan's core funding to who, which primarily focuses on global functions, has ranked nd in the world after the united states [ ] . it may be said that japan's global functions in relation to the global trends is relatively high. this finding may be consistent with the direction of the basic design for peace and health, which emphasizes the strengthening of global functions based on the concept of human security. for example, at the ise-shima g summit, japan emphasized the promotion of aid for global functions both in the g ise-shima leaders' declaration and g ise-shima vision for global health [ , ] . while the effective dah allocation has long been discussed, it might be guided by a number of factors, including historical and traditional diplomatic relations, geographic proximity, strategic reciprocity, and trade-related considerations, particularly in bilateral aid; and not necessarily aligned with disease priorities for health aid in recipient countries and cost-effectiveness of interventions [ ] . globally, however, dah growth has been stagnant over the past years and limited financial resources are a universal constraint [ ] ; japan is not exception. it is, therefore, an urgent policy issue to implement dah strategies wisely, efficiently and effectively, while ensuring transparency. both in the global health diplomacy strategy and the basic design for peace and health, protecting human security has been a core concept of japanese foreign policy [ , ] . human security is at a convergence that combines the competing policy issues that could threaten vital core of all human lives, including infectious disease epidemics (as exemplified by the recent ebola outbreak [ ] or pandemic influenza) as well as refugee and migration crises and climate change. human security approach thus enriches the synergy between measures to address these issues. for example, among the nearly one million rohingyas, an islamic minority group, living in a refugee camp in bangladesh, there is a growing concern about a serious infectious disease epidemic, including measles, cholera, and typhoid [ ] . also, as global warming progresses, the distribution of vectors such as mosquitoes that transmit japanese encephalitis, dengue fever, malaria, and yellow fever, may expand [ , ] . human security approach will also contribute to the achievement of sdgs as well as au agenda by building a healthy, sustainable, and stable society. an important issue in the dah strategy for donors is therefore to consider how donors should fund their human security efforts from a limited oda budget, and in particular what is the optimal role of dah in this context. for example, japan is one of the founding partners of the global fund and a major donor who contributed . - . % of dah to the global fund in - . since its establishment in , an accumulated . billion usd has been contributed from japan [ ] . at the meeting of the sustainable development goals promotion headquarters on june , prime minister shinzo abe announced japan's new pledge of million usd to the global fund's sixth replenishment [ ] . infectious disease control is an important dah strategy of japan, which covered . - . % of japan's dah shares overall between and , and was mostly channeled through the global fund. in the context of human security approach to climate change and refugees and migrants crisis, further scale-up of dah investments in effective infectious disease control is expected. note that human security approach in this context means supporting people-centered, comprehensive, context-specific, and prevention-oriented responses that strengthen the protection and empowerment of all people, adopting partnerships across sectors, developing context-sensitive solutions, and supporting the realization of a world without fear, want, and dignity [ ] . caution is needed that while human security as a rationale for linking foreign policy and health introduces significant political power, sufficient attention must be paid to the possibility that national security interests may be skewed towards health and humanitarian issues [ ] . it should also be noted that treating global health issues as national security threats, rather than universal issues to be concerned with the humanity, may cause an excessive concern surrounding diseases surveillance and a divide between affected countries and non-affected countries. in the past, for example, securitization was misused as a rationale for implementing hiv-based travel, migration, and immigration control policies and laws prohibiting the entry of people living with hiv [ ] . the results also showed that between and , approximately % of dah were allocated to phc, and remaining % to hss. although there are no established norms or benchmarks on the balance between phc and hss allocations in dah, the high level task force on innovative international financing for health systems (hltf) proposed that approximately - % of the additional resources would be required for hss-that are broadly consistent with the above definitions-in order to achieve the millennium development goals (mdgs) for low-income countries [ ] . in terms of the mdgs, therefore, the balance between phc and hss in japan's dah could be roughly reasonable. however, in today's era of sdgs, the growing emphasis on social determinants of health makes it even more crucial that dah strengthens health system, including institutional capacity (effectiveness of surveillance systems and laboratory networks, etc.), administrative and financial systems, and human resources development [ ] . donor-recipient countries face the challenges posed by health transition, i.e., a double burden of morbidity, mortality, and associated health care costs from increasing non-communicable diseases (ncds) and continuing high communicable diseases [ ] . phc has played a successful role in the delivery of prevention and care interventions for communicable diseases, such as malaria, tuberculosis, and hiv/aids. however, it is imperative to expand the delivery of phc in countries undergoing health transition in terms of health promotion and disease prevention and treatment in response to ncds [ ] . with limited resources, several studies suggested the need to take a diagonal approach of hss to address ncds, rather than disease-specific, vertical programs [ , ] . hss has the potential to improve the delivery of phc in a cost-effective manner by dealing with the wide range of health problems encountered in health transition. hss are emerging important focus of some multilateral agencies, such as the world bank and the global fund (japan's major dah channels), as well as the gavi, the vaccine alliance. there is an increasing debate as to why donor countries, including japan, should invest more in ncds [ ] . a study demonstrated that recently only % or less of japan's dah went to ncds [ ] , whereas ncds accounted for - % of total disease burden in lowand lower-middle-income countries (lmics) [ ] . however, this does not imply that funds for infectious disease control should be used to scale-up to confront ncds through hss. between and , . - . % of japan's dah went to infectious diseases control including hiv/aids, which has aligned with disease burden in lmics to some extent (or lower), where infectious disease accounted for about - % of the total disease burden in lmics [ ] . importantly, japan's dah allocation should take full account of the health transition of dah-recipient countries and make the burden of disease an important criterion for prioritizing resource allocation [ ] . in the future, it will become increasingly important to promote prevention as well as treatment by focusing on hss in recipient countries. more effective health spending is needed in developing countries, and they should use all available resources. recognizing this need, sdg aims to strengthen domestic resource mobilization and improve domestic fiscal capacity for tax and other revenue collection [ ] . in addition, under the addis ababa action agenda, countries pledged to achieve the sdgs, largely using domestic resources [ ] . these are also recognized as common understanding to achieve uhc at the joint session of finance and health ministers at the osaka g summit this year as well as at tivad vii [ , ] . on the other hand, a recent study estimated that achieving uhc would require an increase in annual per capita health spending of more than usd by in lmics [ ] . more spending may be needed, especially as the country develops economically and prices rise. this figure is much larger than dah alone can cover. while taking into account the country's own priorities, it is the most important strategic challenge for donors to consider how dah can support the use and mobilization of domestic resources and how it can intervene in ways that reduce investment risks for the private sector [ ] . for multilateral aid, the global financing facility (gff) and global action plan for healthy lives and well-being for all is a new approach that leverages domestic resources as well as ongoing funding from private and public sources. japan is one of the donor countries of gff as of may . the first commitment, a pledge of million usd, to the gff by the government of japan was announced at the uhc forum [ ] . in addition, donors should be aware of the potential for the implementation of dah to impair the ability of dah-recipient countries to properly plan health budget disbursements, and should seek ways to avoid it. a study by the world bank and other institutions found that the costs of using parallel systems of dah and domestic resources were more than four times higher than relying solely on national financial systems and skills transfer [ ] . also, there is an evidence of negative correlation between dah and domestic resources; dah may constrain the domestic health budget and cause its significant portion substituted out of the health sector [ , ] . decision-making and implementation of dah should consider how financial flows in dah-recipient countries interact with each other. while oda system is well-known, many complexities are involved in its use. this study made use of dah on gross disbursements rather than commitments as disbursements are actual distributions of committed aid funds, while the commitments are amount the donor agreed to make available to. in some cases, disbursements could be more volatile than commitments, conditional on specific country events (e.g., political instability), and absorptive capacity during any one year [ ] . as noted in the previous study [ ] , it is difficult to draw a strong conclusion about the share of phc and hss for several reasons. first, there is a lack of global agreement on measurable indications for phc and hss. it also includes the lack of normative descriptions of the share of dah by donors for phc and hss. the method developed in the previous study (and used in this study) can be reproduced using oecd/crs data and may serve as a useful method to track future donor resources allocated to phc/hss. our estimates of dah are not necessarily comparable to those of the institute for health metrics and evaluation (ihme) at the university of washington, which also provides an alternative source of data on dah [ ] . ihme uses rather complicated mathematical procedures to classify aid based on a 'word search' of project/program content, rather than the long-established coding procedures followed by donors for the oecd/crs data base. ihme estimates tend to be relatively large in value than our estimates based on the oecd coding procedures. for example, in japan, a dah of was estimated to be million usd in this study, while ihme estimates was million usd (at constant price in ) [ ] . this may be because the ihme's estimation method using word search allows some consideration even in areas, such as 'agriculture' (crs code ), 'water and sanitation' ( ), and 'education' ( ) that the oecd coding procedures based on the crs code does not consider as 'health' ( + ). in addition, in the oecd coding, there are focus areas in the field of health, while ihme classifies health into focus areas. ihme also provides very important data, although the methods of estimation and classification are different. however, this study adopted the oecd coding procedures, whose categorization is more familiar and straightforward for policy makers and government officials in japan to understand. this study used only data from japan over a five-year period, and therefore does not provide long-term trends of dah or comparisons with other countries, or any consideration from the perspective of japan's relative position in global health diplomacy. this is our next research scope. with many major high-level health related meetings ahead, coming years will play a powerful role in reevaluating dah and shaping the future of dah for the world and japan. we hope that the results of this study, which provide an overview of dah in japan, will enhance the social debate for and contribute to the implementation of japan's dah in a more efficient and effective strategy. additional file : figure s . developing assistance for health channeled through multilateral agencies unicef: united nations children's fund; undp: united nations development programme; afdb: african development bank; asdb: asian development bank; iadb: inter-american development bank; global fund: the global fund to fight aids, tuberculosis and malaria; gavi: gavi, the vaccine alliance who: world health organization; unaids: joint united nations programme on hiv/aids; unfpa: united nations population fund; unicef: united nations children's fund; undp: united nations development programme; afdb: african development bank; asdb: asian development bank; iadb: inter-american development bank; global fund: the global fund to fight aids, tuberculosis and malaria; gavi: gavi, the vaccine alliance; jica: japan international cooperation agency. other un agencies include food and agriculture organization (fao), united nations relief and works agency for palestine refugees in the near east (unrwa), world food programme (wfp), etc. ngos include international planned parenthood federation, etc. others include global environment facility (gef), etc. table s . developing assistance for health channeled through multilateral agencies usd in million, %): (a) bilateral (loans), (b) bilateral (grants), (c) multilateral, (d) total. std: sexually transmitted disease. crs purpose code: basic health care = ; basic health infrastructure = ; basic nutrition = ; health education = ; health personnel development = malaria control = ; tuberculosis control = ; std control including hiv/aids = ; health policy and administrative management = ; medical education/training = usd in million, %): (a) bilateral (loans), (b) bilateral (grants), (c) multilateral, (d) total. phc: public health care; hss: health system strengthening. phc definition # = basic health care and infrastructure (crs purpose codes: , , , , ); phc definition # = reproductive health care and family planning ( , ); phc definition # = infectious disease control, including malaria and tuberculosis broader phc definition = definition # + # + # + # ; hss definition # = health policy, administration & management ( ); hss definition # = medical services hss definition # = population policy & administration abbreviations amr: antimicrobial resistance; au: african union; crs: creditor reporting system; dac: development assistance committee; dah: development assistance for health tuberculosis and malaria; hss: health system strengthening; ihme: institute for health metrics and evaluation; jica: japan international cooperation agency; lmics: low-and lower-middle-income countries; maff: ministry of agriculture, forestry and fisheries; meti: ministry of economy, trade and industry; mhlw: ministry of health, labour and welfare; mof: ministry of finance; mofa: ministry of foreign affairs; ncds: non-communicable diseases; oda: official development assistance sustainable development goals; std: sexually transmitted disease ticad: tokyo international conference on african development uhc: universal health coverage; who: world health organization references human security and universal health insurance tokyo: ministry of foreign affairs ministry of foreign affairs. yokohama declaration -advancing africa's development through people, technology and innovation. tokyo: ministry of foreign affairs japan's strategy for global health diplomacy: why it matters japan's 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affairs taro kono at keio university "mr. taro kono talks on oda tokyo: ministry of foreign affairs japan's official development assistance charter. tokyo: ministry of foreign affairs ministry of foreign affairs. cabinet decision on the development cooperation charter. tokyo: ministry of foreign affairs basic design for peace and health (global health cooperation). tokyo: ministry of foreign affairs japan's global health policy: challenges and challenges tokyo: institute for global health policy research organisation for economic cooperation and development how much donor financing for health is channelled to global versus country-specific aid functions? does donor proliferation in development aid for health affect health service delivery and population health? cross-country regression analysis from to countdown to : tracking donor assistance to maternal, newborn, and child health geobook: deflators: oecd oecd methodology for calculating imputed multilateral oda oecd international development statistics. paris: organisation for economic cooperation and development world health organization. declaration of alma-ata. geneva: world health organization world health organization. declaration of astana. geneva: world health organization donor and domestic financing of primary health care in low income countries universal health coverage (uhc) in africa: a framework for action protecting human security: proposals for the g ise-shima summit in japan signing of japanese oda loan agreement with the federal republic of nigeria assessed contributions by member states and associate members, showing a % increase in total assessed contribution financing for tokyo: ministry of foreign affairs g ise-shima vision for global health. tokyo: ministry of foreign affairs bilateral donors' interest vs. recipients' development motives in aid allocation: do all donors behave the same? global burden of disease health financing collaborator, network. past, present, and future of global 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intersectoral convergence: the case of the indian national rural health mission measuring progress from to and projecting attainment to of the health-related sustainable development goals for countries and territories: a systematic analysis for the global burden of disease study health transition in africa: practical policy proposals for primary care. b world health organ the 'diagonal' approach to global fund financing: a cure for the broader malaise of health systems? an assessment of interactions between global health initiatives and country health systems council on foreign relations. the emerging global health crisis: noncommunicable diseases in low-and middle-income countries. new york: council on foreign relations global, regional, and national disability-adjusted life-years (dalys) for diseases and injuries and healthy life expectancy (hale) for countries and territories, - : a systematic analysis for the global burden of disease study sustainable development goal resolution adopted by the general assembly on beating the drum in lower-income countries. oslo: global financing facility government of japan to invest us$ million in global financing facility to accelerate progress on universal health coverage understanding the costs and benefits of unharmonized and unaligned fm arrangements: pfm in health sector public financing of health in developing countries: a cross-national systematic analysis measuring the displacement and replacement of government health expenditure a review of health resource tracking in developing countries publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to express our gratitude to the staff of each ministry and agency who contributed to the data provision and coordination for the data analysis of japan tracker that the authors were in charge of. our estimates of dah were partially reported at japan tracker on august (https://japantracker.org/en/). japan tracker is the first data platform in japan to visualize the flows of japan's dah, launched in november under the industryacademia-government collaboration and support by the bill & melinda gates foundation. not applicable. all authors conceived of and designed the study and take responsibility for the integrity of the obtained data and accuracy of the data analysis. sn, hs, ms, hn, ks, sl, and ks acquired the data. sn and hs analyzed and interpreted the data. sn drafted the article. all authors made critical revisions to the manuscript for essential intellectual content and gave their final approval. this work was primarily funded by the bill & melinda gates foundation. the funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the views expressed in this paper are solely those of the authors. all data generated or analysed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord- - wbvdg r authors: takahashi, toru; maeda, ken; suzuki, tadaki; ishido, aki; shigeoka, toru; tominaga, takayuki; kamei, toshiaki; honda, masahiro; ninomiya, daisuke; sakai, takenori; senba, takanori; kaneyuki, shozo; sakaguchi, shota; satoh, akira; hosokawa, takanori; kawabe, yojiro; kurihara, shintaro; izumikawa, koichi; kohno, shigeru; azuma, taichi; suemori, koichiro; yasukawa, masaki; mizutani, tetsuya; omatsu, tsutomu; katayama, yukie; miyahara, masaharu; ijuin, masahito; doi, kazuko; okuda, masaru; umeki, kazunori; saito, tomoya; fukushima, kazuko; nakajima, kensuke; yoshikawa, tomoki; tani, hideki; fukushi, shuetsu; fukuma, aiko; ogata, momoko; shimojima, masayuki; nakajima, noriko; nagata, noriyo; katano, harutaka; fukumoto, hitomi; sato, yuko; hasegawa, hideki; yamagishi, takuya; oishi, kazunori; kurane, ichiro; morikawa, shigeru; saijo, masayuki title: the first identification and retrospective study of severe fever with thrombocytopenia syndrome in japan date: - - journal: j infect dis doi: . /infdis/jit sha: doc_id: cord_uid: wbvdg r background. severe fever with thrombocytopenia syndrome (sfts) is caused by sfts virus (sftsv), a novel bunyavirus reported to be endemic in central and northeastern china. this article describes the first identified patient with sfts and a retrospective study on sfts in japan. methods. virologic and pathologic examinations were performed on the patient's samples. laboratory diagnosis of sfts was made by isolation/genome amplification and/or the detection of anti-sftsv immunoglobulin g antibody in sera. physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of sfts. results. a female patient who died in received a diagnosis of sfts. ten additional patients with sfts were then retrospectively identified. all patients were aged ≥ years and lived in western japan. six cases were fatal. the ratio of males to females was : . sftsv was isolated from patients. phylogenetic analyses indicated that all of the japanese sftsv isolates formed a genotype independent to those from china. most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. conclusions. sfts has been endemic to japan, and sftsv has been circulating naturally within the country. severe fever with thrombocytopenia syndrome (sfts), an infectious disease with a high case-fatality rate, is caused by sfts virus (sftsv), a novel bunyavirus reported to be endemic to central and northeastern parts of china [ , ] . sftsv, which is classified into the genus phlebovirus and the family bunyaviridae, is suspected to be a tick-borne virus owing to evidence of its presence in species of ticks: haemaphysalis longicornis and rhipicephalus microplus [ , ] . approximately % of h. longicornis organisms collected from sheep, cattle, and dogs in shandong province tested positive for sftsv in virus genome amplification assays [ ] . a similar disease, which was named fever, thrombocytopenia and leukopenia syndrome (ftls), was independently reported to be caused by a novel virus, henan fever virus (hnfv) [ ] . despite the different names, "sfts" and "ftls" represent the same condition and "sftsv" and "hnfv" represent the same virus. the case-fatality rate of sfts is reported to be approximately % [ , ] . humanto-human transmission of sftsv was reported to occur through close contact with the blood and/or body secretions of infected patients [ ] [ ] [ ] [ ] [ ] . to our knowledge, there were no published reports of sfts outside of china before we performed the study described here. another tick-borne phlebovirus, the heartland virus, which was detected in missouri, is phylogenetically associated with sftsv, caused severe febrile illness with thrombocytopenia, leukopenia in the total blood cell count, and elevated levels of liver enzymes [ ] . we report the first identification of sfts in japan, which was detected in a previously healthy woman aged - years who died of multiple-organ failure in the autumn of , and findings from a subsequent retrospective study of sfts in japan. culture supernatants were subjected to viral rna extraction using high pure viral nucleic acid kit (roche diagnostics). complementary dna (cdna) was synthesized using super-script iii (invitrogen) with random primers and then randomly amplified using the illustra genomiphi v kit (ge healthcare life sciences). a cdna library was prepared using the nextera dna sample prep kit (illumina). a sequencing run for nucleotides was performed with miseq (illumina), using the miseq reagent sequencing kit (illumina). the assembled nucleotide sequences were used to determine homologous sequences by tblast at the national center for biotechnology information web site (available at: http://blast.ncbi.nlm.nih.gov/blast.cgi). the sftsv genome was detected by reverse-transcription polymerase chain reaction (rt-pcr) in total rna extracted from patient sera, using a high pure viral rna kit (roche applied science). reverse transcription was performed with ready-to-go rt-pcr beads (ge healthcare), using random nucleotide hexamers. pcr was performed to amplify sftsv-specific cdna fragments. pcr primer sets were designed to amplify the nucleoprotein (np) region of the sftsv genome (supplementary table ). the pcr conditions were as follows: cycle at °c for minutes followed by °c for minutes; cycles at °c for seconds, °c for seconds, and °c for seconds; followed by cycle at °c for minutes. vero cells were inoculated with rt-pcr-positive patient sera for virus isolation, cultured for - days, and examined for sftsv antigen detection by indirect immunofluorescence assay (ifa) with a polyclonal antibody raised against sftsv recombinant np (rnp; rabbit anti-sftsv rnp serum), which was produced as follows. sftsv rnp tagged with histidine-tag on the c-terminus was expressed in a baculovirus expression system, as previously described [ , ] . the np gene of sftsv strain hb (genbank accession no. nc_ ) was artificially synthesized. anti-sftsv rnp serum was raised in rabbits by immunization with the purified sftsv rnp, as previously described [ ] [ ] [ ] . the neutralizing antibody to sftsv chinese isolate hb strain [ , ] and japanese isolate yg (this study) was detected as reported previously, except for the target virus [ ] . with the exception of the antigen preparation, immunoglobulin g (igg) antibody titers to sftsv were determined by indirect ifa, using sftsv hb -infecting vero cells as previously described [ ] . pathologic studies with histopathologic, immunohistochemical, and in situ hybridization at-tailing (ish-at) methods histopathologic studies of formalin-fixed and paraffin-embedded specimens were performed using hematoxylin-eosin stain. immunohistochemical analysis was performed as previously described, with some modifications [ ] . the rabbit anti-sftsv rnp serum and the peroxidase-labeled, polymer-conjugated antirabbit immunoglobulin (envision/hrp, dako) were used in the immunohistochemical analysis as the primary and the secondary antibodies, respectively. normal rabbit serum and lymph nodes of necrotizing lymphadenitis without sftsv infection were used as negative controls for the antibody and tissue specimens, respectively. ish-at was used for detection of sftsv genomic rna (negative-strand rna) as reported previously, with the exception of the strand-specific oligonucleotide probes [ ] [ ] [ ] , which were designed for the s and l segments of the sftsv genome (supplementary table ). the sftsv copy number was determined by performing quantitative real-time rt-pcr analysis of rna samples extracted from paraffin-embedded sections as previously described, with some modifications [ ] . the amount of human β-actin messenger rna (mrna) in the dnase-treated rna extracted from each section was also determined and used as an internal reference for normalization [ ] . the primers and labeled probes are shown (supplementary table ). the culture supernatant from vero cells inoculated with sftsv isolated from the first patient was used for electron microscopic analysis. the samples were fixed with % glutaraldehyde. the fixed samples were negatively stained with % phosphotungstic acid and then observed using a jem- transmission electron microscope (jeol, tokyo, japan). the first diagnosis of sfts in japan was made public through an announcement from the ministry of health, labor, and welfare of japan on january . physicians were asked to volunteer information if they had treated patients who satisfied the following case definition: ( ) fever of > °c; ( ) gastrointestinal tract symptoms, such as nausea, vomiting, abdominal pain, diarrhea, and melena; ( ) thrombocytopenia, with < × platelets/l; ( ) leukopenia, with < × white blood cells/l; ( ) elevated levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase; ( ) absence of other causes; and ( ) death or admission to an intensive care unit because of the severity symptoms. information about patients was collected from their physicians. the retrospective recruitment of patients with suspected sfts was conducted from january to march . written informed consent was obtained from patients or responsible relatives. serum samples, which had been collected from the patients for future analyses to clarify the etiology and had been stored in each hospital by the respective physicians, used for the present study were sent to the department of virology at the national institute of infectious diseases (niid; tokyo, japan) for virologic analyses. clinical and laboratory data of the patients with sfts were also sent to the corresponding author (m. saijo) without information that made it possible to identify individuals. cdnas prepared from the patients' sera samples were used to determine sftsv genome sequences. the terminal sequences of sftsv genomes were determined by rapid amplification of cdna ends (race) at the ′ and ′ ends, performed by rt-pcr after ′ and ′ linker ligation using a dynaexpress mirna cloning kit, high efficient (biodynamics laboratory, tokyo, japan). purified rna from the culture supernatant of sftsv (yg )-infected vero cells was subjected to the ′ linker ligation according to the manufacturer's protocol. next, the ′ linker was ligated to the ′-phosphate end of the purified ′ linker ligated rna, according to the manufacturer's protocol, following rna purification by nucleospin rna clean-up xs (takara bio, shiga, japan). the ′ and ′ linker ligated rna was then reverse transcribed according to the manufacturer's protocol by super-script iii (invitrogen, carlsbad, ca), using either the ′ forward rt primer, which is the antiparallel sequence of the ′ linker sequence, or the pd(n) primer (random hexamer). rt-pcr was performed using q hot start high-fidelity dna polymerase (neb, ipswich, ma) and sftsv gene-specific primers (supplementary table ) with the ′ rt primer or the ′ primer, which is the antiparallel sequence of the ′ linker sequence. nucleotide sequences of each full segment of sftsv in patients' sera were aligned using muscle, an en suite program in the molecular evolutionary genetics analysis . software (mega team, japan). evolutionary distances were estimated using kimura's -parameter method, and phylogenetic trees were constructed using the neighbor-joining method. the robustness of the trees was tested using bootstrap replications. accession numbers of the nucleotide sequences of sftsv l-, m-, and s-segments are described in supplementary table . serum samples were used for virologic analysis after obtaining written informed consent from the patients themselves (for those who survived) or their responsible family members (for those who died). the clinical and laboratory data of the patients with sfts were sent to the corresponding author without personally identifying information. all of the protocols and procedures were approved by the research and ethics committees of the niid. the polyclonal antibody to sftsv rnp was produced by immunizing rabbits with purified sftsv rnp, with approval from the institutional animal care and use committee of the niid (no. ). a previously healthy woman aged - years who lived in the yamaguchi prefecture of japan was hospitalized with high fever, fatigue, vomiting, and melena in the autumn of . her body temperature was . °c. tick bite wounds were not observed anywhere on her skin. laboratory tests revealed a low platelet count of × platelets/l (normal range, - × platelets/l) and a low white blood cell count of . × cells/l (normal range, . - . × cells/l). serum levels of alanine aminotransferase, aspartate aminotransferase, and creatine kinase were abnormally high, while the c-reactive protein level was normal. a coagulation study revealed a prolonged activated partial thromboplastin time and a high d-dimer level. the patient's serum ferritin level of > µg/ l was extremely elevated (normal range, - µg/l). urinary analysis showed proteinuria and microhematuria. the blood culture was sterile. computed tomography of the chest and abdomen showed right axillary lymphadenopathy and bilateral renal swelling; however, there was no evidence of hepatosplenomegaly. bone marrow aspiration revealed mildly hypocellular marrow with an increase in levels of activated histiocytes and hemophagocytes. the patient's condition deteriorated rapidly on the day following admission with the appearance of macrohematuria and a massive amount of tarry stool. death occurred on the third day of hospitalization. serum was used for virus isolation using vero and felis catus whole fetus (fcwf- ) cells. cytopathic effect appeared in both cells within days. many dna fragments that were homologous to those of sftsv were detected in the culture supernatant of the cells inoculated in next-generation sequencing. dna amplified by conventional rt-pcr using either of primer pairs showed the expected sizes of or bp in agarose-electrophoresis (data not shown). the inoculated vero cells were tested for the presence of sftsv antigen in indirect ifa with rabbit anti-sftsv rnp serum and showed a similar positive reactivity ( figure a ). enveloped and spherical virions with approximate diameters of nm were detected by electron microscopy ( figure b) . the morphology of the virion is compatible with that of a bunyavirus. the virus isolated from the patient was named sftsv yg . autopsy findings included right axillary lymphadenopathy ( . × . cm; figure a) , bilateral renal swelling, mild retention of pericardial fluid ( ml), and hepatic steatosis. gastric ulceration was also observed in the pyloric region. severe necrotizing lymphadenitis with massive necrosis, the depletion of small lymphocytes, and severe infiltration of the swollen right axillary and right cervical lymph nodes by histiocytes and immunoblasts were observed ( figure b and c) . necrosis, which comprised nuclear debris and eosinophilic ghosts but not granulocytes, was distributed throughout the cortical area, the sinuses, and the capsule of the lymph node and had spread to the perinodal adipose tissue. no clusters of epithelioid histiocytes, stellate microabscesses, or granulomas were observed. there were no obvious intranuclear or intracytoplasmic viral inclusions. prominent hemophagocytosis was observed in these lymph nodes, the bone marrow, and the spleen ( figure d and e). the bone marrow was relatively hypocellular, with no reduction in the number of megakaryocytes. the liver showed mild microvesicular fatty changes in zone and mild inflammation, comprising lymphocytes and macrophages, around the portal tracts. the kidney showed subepithelial hemorrhage within the renal pelvis ( figure f ). significant hemophagocytosis were also observed in the mediastinal, hilar, and abdominal lymph nodes with no evidence of necrosis ( figure a, c, and e) . the findings in the remaining visceral organs were unremarkable. positive signals for sftsv np antigen were detected in the cytoplasm of blastic cells and necrotic regions in the cortical area of the right axillary lymph node ( figure b ). viral antigen-positive cells were also detected in the right cervical lymph nodes ( figure d ) but not in the mediastinal lymph nodes ( figure f ) , regardless of the level of immunoblast infiltration and hemophagocytosis ( figure e ). relatively few sftsv antigen-positive cells were detected in the bone marrow, adrenal glands, the liver, and the spleen ( figure g -j), with no notable cytopathic effects or necrosis. no antigenpositive cells were detected in the heart, lungs, kidneys, gastrointestinal tract, aorta, or iliopsoas muscle. the sftsv genome, sftsv genomic rna (negative-strand rna; figure a ), and mrna ( positive-strand rna; figure b ) were detected in the blastic cells in the right cervical lymph node by ish-at analysis [ ] [ ] [ ] , while no signals were detected using an irrelevant probe as a negative control ( figure c ). no signals showing necrotizing lymphadenitis without sftsv infection were detected in the lymph nodes (negative control; figure d ). sftsv rna was present, with high copy numbers, in the right axillary and cervical lymph node sections (supplementary table ). consistent with immunohistochemical analysis results, low copy numbers ( - copies) of sftsv rna were also detected in the bone marrow, the spleen, the liver, and the adrenal glands. a few copies (< ) were detected in other tissue sections that did not contain antigen-positive cells (as assessed by immunohistochemical analysis), suggesting that quantitative real-time rt-pcr detected cell-free circulating sftsv; a high viral load in the serum is characteristic of sfts infection [ ] . the number of sftsv rna copies/cell was calculated using the β-actin mrna copy number, estimated at copies/cell [ ] . the sftsv rna copy numbers in the right axillary and cervical lymph node sections were the highest among the tissues tested, while the bone marrow, spleen, and liver showed relatively lower copy numbers per cell (supplementary table ). serum samples collected from patients who were retrospectively suspected of having sfts were sent to the department of virology , niid ( figure ). the male-to-female ratio was : . the earliest infection dated back to ( figure b ). serum samples collected from patients (for patients, acute phase serum samples were not available for virologic analysis) were subjected to virus isolation and rt-pcr for amplification of the sftsv genome. serum samples collected from all patients were tested for igg and immunoglobulin m (igm) antibodies to sftsv in the indirect ifa. of the patients, men and women received a diagnosis of sfts: had positive results of virus isolation and genome amplification tests; had positive results of virus genome amplification testing and igm antibody to sftsv but had negative results of virus isolation analysis; and were positive for igg antibody to sftsv. in the present study, the patients who tested positive for sftsv on the basis of detection of igg to sfvsv were regarded as sfts positive because their symptoms were reminiscent of sfts and because reports of asymptomatic cases in china are quite rare [ , ] . for the purposes of the present study, the first patient was included with the retrospective cases, for a total of diagnosed cases. all of the patients in whom sfts was diagnosed were aged ≥ years ( figure a ) and came from western japan ( figure d ). disease onset occurred in all patients between the months of april and december ( figure c ). six of the cases were fatal. there was clear evidence of tick bite in cases. the clinical manifestations observed in sfts patients are summarized in table . all patients showed nonspecific febrile symptoms with gastrointestinal tract symptoms in the early phase of the disease. deterioration in consciousness, characterized by dysarthria, disorientation, and alteration in consciousness, was commonly observed. generalized convulsions were seen in the late stages of the disease in of the fatal cases. respiratory symptoms were rarely observed. superficial lymphadenopathy was detected in patients. hemorrhagic symptoms such as petechiae, purpura, melena, bloody vomit, gingival bleeding as a form of discharge, and excessive bleeding at the site of skin biopsy were observed in of the patients. blood urea nitrogen and creatinine levels were elevated in of patients. hematuria and proteinuria were observed in most patients. in all patients for whom bone marrow observation was performed, hemophagocytosis, with or without bone marrow cell dysplasia, was observed. the ferritin level was elevated in the blood of patients, including the in whom bone marrow examination was performed. abnormalities were observed in tests in all of the patients for coagulopathy, prothrombin time, activated partial thromboplastin time, fibrin/fibrinogen degradation products, fibrinogen, and/or d-dimer. the japanese sftsv strains were closely related to the sftsv chinese isolates but formed an independent cluster for each segment ( figure ) . no geographic or chronological relationship was found between the japanese and chinese strains. a and b) , right cervical (c and d), and the mediastinal (e and f ) lymph nodes. a, c, and e, infiltration by immunoblasts and prominent hemophagocytosis was generally observed in the right axillary, right cervical, mediastinal, hilar, and abdominal lymph nodes; however, necrosis was only observed in the right axillary and cervical lymph nodes. b, d, and f, viral antigen-positive cells were detected in the right axillary and cervical lymph nodes. positive signals for sftsv np neutralizing antibody response to sftsv sera collected from the surviving patients with sfts in the convalescent phase showed neutralizing activities to sftsv japanese isolate yg ( figure d ). the sera of these patients also showed similar degrees of neutralizing activities to sftsv hb . the clinical manifestations of japanese sfts were very similar to those of severe cases of chinese sfts [ , ] , but the casefatality rate ( of patients [ %]) was apparently higher than that in china, where an average of % of cases were fatal. it is worth noting that the characteristics of japanese sfts presented in this study were based on a limited number of patients and a strict case definition. to our knowledge, this may be the first report describing the pathologic findings of a patient with sfts. the local lymph nodes, which were the primary target organ in the first patient, served as the site of virus replication and showed marked pathologic changes, including massive necrosis. no neutrophil infiltration was observed in the patient. the spleen, liver, adrenal glands, and bone marrow contained a few sftsv-infected cells. however, no viral antigens were detected in hepatocytes or in the parenchymal cells of the adrenal glands. viruses belonging to the bunyaviridae family, which includes the rift valley fever virus, hantaan virus, and heartland virus [ ] , infect monocytes/macrophages [ , ] . these cells may control the spread (or containment) of viruses to cells within other organs. the pathologic and virologic observations suggest that sftsv replicated in the blastic cells within the lymph nodes and that replication took place predominantly in the local lymph nodes, not in the major organs. macrophages with phagocytosis of bone marrow cells were observed in all of the patients in whom bone marrow figure . detection of severe fever with thrombocytopenia syndrome virus (sftsv) rna in the right cervical lymph node by the in situ hybridization attailing method. a, sftsv genomic rna was detected in the right cervical lymph node by the in situ hybridization at-tailing method and a sense probe. sftsv genomic rna was detected in the cytoplasm of the blastic cells. b, the in situ hybridization at-tailing method with an anti-sense probe detected a few cells in the right cervical lymph node that were positive for sftsv messenger rna (mrna). sftsv mrna was also detected in the cytoplasm of blastic cells. c, no signals were detected in the right axillary lymph node by the in situ hybridization at-tailing method with an irrelevant probe (negative control). d, a sftsv sense probe detected no signals in lymph node sections showing necrotizing lymphadenitis without sftsv infection. examination was performed. furthermore, pathologic examination of the lymph nodes, bone marrow, and the spleen of the initial patient revealed marked hemophagocytosis, regardless of the presence of sftsv-infected cells ( figure d and e and figure a , c, and e). ferritin level was extremely elevated in the sera of all patients who were tested. abnormality in coagulopathy-associated indices was also observed in all patients. sftsv infections induced a cytokine storm, the level of which was associated with the severity of sfts [ ] . these results indicate that in addition to multiorgan dysfunction, hemophagocytosis and disseminated intravascular coagulation are major factors for poor prognosis. the mode of the natural sftsv lifecycle in japan should be clarified to enable better identification of risk factors for sftsv infection and to address strategies for reducing the risk of infections. further study is necessary to clarify the circulation of sftsv in nature in japan, in terms of tick species, percentages of sftsv positivity for each tick species, and the prevalence of sftsv-positive ticks, to better determine and evaluate the risk factors for sftsv infection in japan. japanese isolates formed a cluster that was independent from chinese isolates (figure ), indicating that sftsv has been circulating in japan naturally for some time. the earliest year for which we have evidence of sfts in patient sera samples was , and to our knowledge, the article by liu et al, which concerns sfts occurrence in china during , reports the oldest cases from china [ ] . it is also noteworthy that patients with sfts were reported in south korea in in conclusion, sftsv is prevalent in japan. japanese sftsv strains have characteristics similar to those of chinese isolates but an independent genotype, which indicates that sftsv has been present in japan for some time. metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus fever with thrombocytopenia associated with a novel bunyavirus in china the ecology, genetic diversity, and phylogeny of huaiyangshan virus in china isolation, identification and characterization of sfts bunyavirus from ticks collected on the surface of domestic animals 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immunodeficiency virus rna in formalin-fixed and paraffin-embedded tissues vascular endothelial growth factor messenger rna expression level is preserved in liver metastases compared with corresponding primary colorectal cancer detection of a novel bunyavirus associated with fever, thrombocytopenia and leukopenia syndrome in henan province, china, using real-time reverse transcription pcr severe fever with thrombocytopenia syndrome virus clinical and epidemiological study on severe fever with thrombocytopenia syndrome in yiyuan county, shandong province, china replication of hemorrhagic fever viruses in monocytic cells isolation of haemorrhagic fever with renal syndrome virus from leukocytes of rats and virus replication in cultures of rat and human macrophages host cytokine storm is associated with disease severity of severe fever with thrombocytopenia syndrome acknowledgments. we thank dr de-xin li and dr mi-fang liang, national institute for viral disease control and prevention, chinese center for disease control and prevention, beijing, prc, for providing us with the sftsv hb strain; dr roger hewson, virology, pathogenesis, and emerging disease, public health england-microbiology services, porton down, salisbury, united kingdom, for his critical comments; and the municipal officials who supported this work, in the prefectures in which patients with sfts were reported.financial support. this work was supported by the ministry of health, labor and welfare science research (grants in aid h -shinko-ippan- , h -shinko-ippan- , and h -shinko-shitei- ).potential conflicts of interest. all authors: no reported conflicts. key: cord- - cbeukpr authors: huang, michael c.; liou, ming-huan; iwaki, yoko title: the impact of r&d and innovation on global supply chain transition: gtap analysis on japan’s public r&d investment date: - - journal: j doi: . /s - - - sha: doc_id: cord_uid: cbeukpr policymaking for science, technology, and innovation (r&d) is stepping into a new era in the twenty-first century within a highly integrated production network, making it more challenging to capture the impact of r&d investment from an evidence-based approach. to unfold the paradox of the r&d spillover effect spared in the global supply chain, we use computable general equilibrium model with the gtap database v to analyze the impact of japan’s public r&d investment to the world focus on key sectors of global supply chain, namely chemical and pharmaceutical, electronic equipment, machinery, and transportation equipment to examine its output, external trades, and welfare. the productivity parameters triggered by public r&d investment are calibrated from the scirex policymaking intelligent assistance system—economic simulator (spias-e). the simulation results show significant increase in japan’s output and export for chemical and pharmaceutical, electronic equipment, and transportation equipment. the gdp growth was stimulated by . % and substantial welfare improvement by usd , million, while other countries such as malaysia and taiwan by . – . %. in contrast, the economic indicators of china reveal a negative impact, implying a structural change in the composition of the production network. it is notable to see a higher economic integration of oceania within the region through its vibrant production and trades. the study provides comprehensive global analysis on production networks and insights for evaluating the r&d investment spillover effects. the creation of the science, technology, and innovation (sti) knowledge platform initiated by public r&d investment could utilize the expansion of scientific facilities and related supportive equipment and human capital with the spillovers flow into entrepreneurship (kuroda ) . this concept urged the direction in japan's th basic plan for sti, emphasizing the evidence-based research and indicators to assist the government's policymaking system on sti and r&d investment. in the highly integrated global market, the production networks have naturally abolished tariff barriers for efficient manufacturing in the supply chain (fugazza and maur ) . nevertheless, the evidence-based research on other factors for manufacturing transition is scarce to provide a reference for the spillover effect that could attract more interest in this global supply chain analysis. according to the statistics bureau of japan (mic ), since , the r&d expenditure in japan has been steadily increased from to billion usd (fig. ) , whereas the majority is contributed by the private sector. on the other hand, the r&d expenditure share in gdp remains at the level of . %, which relatively higher than other average of oecd countries ( . %) except for south korea ( . %). however, the government-led r&d expenditure to gdp share has been at the low level of . %, lower than the average of oced ( . %) and most developed countries, such as korea ( . %), germany ( . %), and us ( . %), respectively (meti ) . comparing the volume of r&d expenditure, the scale of public investment is much smaller than private sectors; nevertheless, the public r&d investment in basic science could generate more spillover effect to facilitate the productivity in multi-sector. such r&d investment mostly presents in the form of the competitive research fund granted to research institutes and universities. unlike the outcome from private r&d investment usually turns to protected patent to ensure the profitability, public-funded research outcome could be publicized and shared with the society after a specific period (usually -year); thus, the policy cost-effectiveness could be examined more systematically (kuroda et al. ) . the technology also accelerates the access of medicine through marketing on cyber platform seamlessly with affordability (biswas ) ; the connectivity of medical service through electronic equipment in distant prescription may also speed up the application of information communication technology (ict) and the pharmaceutical industry along with the global deregulation. it is inevitable that the demand for electronic equipment, machinery, and transport equipment would increase substantially, stressing the government to react appropriately to take plausible measures and science, technology, and innovation (sti) policy to improve economic growth and welfare. since the s, the foreign direct investment (fdi) outflow from japan and other newly industrialized economies (nies) in asia have contributed to regional production networks with substantial share global demand for electronic equipment, transport equipment, and machinery. the mushroomed free trade agreements in the s also speeded up their independence. therefore, the supply chain disruptions caused by natural disasters could make a significant impact on other countries without physical damage in cases of great earthquakes in taiwan ( ), japan ( ), and the floods in thailand ( ). japan's th science and technology basic plan (cabinet office ) advocates that japan should aspire to transfer from information society to "society . ", which infers a human-centered society that balances economic advancement with the resolution of social problems by a system that highly integrates cyberspace and physical space. such transition requires substantial input for ict products for domestic infrastructure as implementation for more efficient and effective actualization of productivity in smart manufacturing and smart city development. to accommodate such transition, the domestic production may be stimulated again and such public investment for r&d and innovation could also serve a positive shock with spillover effect to accelerate the production transition among the global supply chains. the growing complexity in the global supply chain in logistics and intermediate inputs has made it challenging for a business to reduce the vulnerability of disruption (christopher and peck ), while the determination of production networks is the fractionalization between specialized components of manufacturing and exchange in the global supply chain (baldwin ) . such a framework is continuously transiting intra-or inter-regionally from the competitiveness in the product innovation of the increasing capital intensity and upgrade, mostly in the electronic equipment, for their usage for communication and city renovation in transaction and transportation (navarro et al. ) . the platform economy is contributed significantly by the rapidly spreading e-commerce since the s with the support of ict products such as personal computers and the internet. with the increasing popularity of the smartphone in the s, the shared economy rephrased the business landscape worldwide with robust applications such as virtual shops, restaurants, and miscellaneous that highly depended on internet service with intensive input of electronic equipment. such development does change not only the way of life but also stimulate the creation of innovation from a considerable small unit (evans and gawer ) . while the significantly positive impact of r&d investment on gdp growth and e-commerce have been testified (anvari, and norouzi ) , it would be more desirable to see its spillover effect among sectors and region globally. for emerging economies, manufacturing sectors require stable investment to build a smart society system. while impact assessment of possible mega natural disasters on asian supply chains (huang and masuda ) and innovative financing framework for asean countries (huang et al. forthcoming) have been widely discussed, there remain gaps for the spillover impact of r&d investment. the research aims to provide an evidence-based approach to japan's public r&d investment to increase productivity and its impact on the integrated production networks. as the impact of the trade war between the us and china does not limit to the large economy but also to the emerging market (carvalho, azevedo, and massuquetti ) , to stabilize competitive production network is of importance than risk hedging. furthermore, along with the new age after the coronavirus shock, the research expects to shed light on unfolding the potential collaboration. confronting the critics on government r&d intervention may distort the market. empirical studies in the korean pharmaceutical industry provide a contrasting approach that government r&d expenditure repairs the market failure for the smaller scale of venture firms (choi and lee ) . the timing for investment in r&d cluster formation also matters for welfare optimization before a considerable long time for technology to be commercialized from the laboratory. therefore, government's role in long-term decision-making foresight for r&d investment (lee and yang ) is critical with demonstration effect to the industrial development. also, government r&d expenditure could generate higher labor productivity growth with the flexibility (dawid and hellmann ; coccia ) ; this evidence suggests the vibrant role initiated by the government to provide essential support in the target sector. the innovation network serves significant factors for pharmaceutical sectors for new product development (mazzola et al. ) . the partnership and the flow of supply chain may receive more attention in the emerging market, where actions of small and mediansized enterprises (smes) toward innovation are also highly correlated with supply chain clusters (ren et al. ) . in such circumstances of uncertainty, the government's decision making may significantly affect the cluster's move and coordination following an evidencebased method to determine the specific sector for r&d investment. while flexibility is desirable, industries that could actively respond would be more potential and resilient. the government expenditure on r&d also shows the leading effect on private investment (sussex et al. ) . such demonstrate behavior could result in a greater return, and the accumulated capital would have a direct impact on productivity. nevertheless, the spillover effect between firms does not show as significant as internal r&d (isaksson et al. ) , implying the importance of public r&d investment, which generates a high return in information technology could come with intertemporal knowledge spillovers (miyagawa and hisa ) . the pharmaceutical industry operates with high risks for its costly r&d input; however, such risk could be pooled through public facilities under proper coordination between research institute, education system, and the private sectors. japan's pharmaceutical industry was considered at a smaller scale compared with the us, but its financial condition has fewer constraints (mahlich and roediger-schluga ) . its competitiveness in the deregulation accelerated by the comprehensive progressive trans-pacific partnership (cptpp) may be potentially strengthened (kawasaki et al. ). after the burst of the bubble economy in japan in the early s, the r&d capital is estimated more important than physical capital (suzuki and chida ) , whereas it remains difficult to capture the impact and contribution of the r&d to productivity. as japan went through the economic stagflation, the cost-effectiveness of government expenditure became rather tightened due to the shrinking population in the super-aging society. empirical evidence shows that the importance of public r&d investment in educational institutes such as the university system for cutting-edge research (becker ) . such investment provides the most spillover for the high-skilled human capital trained in the public order could also contribute to the productivity of the private sector. the evidence-based research thus becomes vital to allocate budget for r&d and innovative investment. r&d, in many aspects, could foster the firm's production and marketing activities to meet the requirements. the intangible r&d investment contains the features of sunk cost, spillover, scalable, and synergies (haskel and westlake ) . such a measure of productivity improvement should be decomposed more specifically to generalize the r&d contribution (chan et al. ). however, the survey data of the firm-level will not satisfy the inquiry of r&d effectiveness comprehensively. to capture the spillover effect of public r&d investment, it would be indispensable to investigate the structural change of industry from the input-output table (kuroda et al. ) . the evolution of intermediate input implies the technology improvement; for instance, in , less coal and iron input required for steel production than in , representing the increase in productivity. similar cases could also be found in the automobile sector, which needs more input of semiconductors and ic chips because of the development of ict products. the scirex policy intelligence assistance system (spias) was constructed, affiliated with an economic simulator (spias-e), which provides a -year projection of productivity parameters contributed by public r&d investment. such a system is based on the timeseries data and demographic trend sourced from japan's input-output tables since for recursive computable general equilibrium (cge) simulation. the r&d policy option also derives the specification of tangible and intangible capital stock (kuroda et al. ) , and its application on technology improvement for renewable energy in japan (huang and kim ) . despite the sophisticated r&d foresight demonstration, given the constraint of spias-e only for japan's analysis, it is desirable to connect the system for a global-scale impact. the paper proceeds as follows; the methodology section introduces the model structure and selected formula for the analysis. the data and scenarios section explains the data source, research scope, parameter setting, and calibration. in the section of simulation results, key economic indicators such as output, price, and the external trade are demonstrated in figures, along with the welfare analysis on gdp and welfare, followed by the concluding remarks and future perspectives. the computable general equilibrium (cge) model has a long history in analyzing national and global trade income with the global trade analysis project (gtap) model. since its establishment in the s, the complexity of the gtap model continuously grows with more sectors and regions involved (mensbrugghe ). the framework provides clear policy implications through key indicators such as the changes of output, prices, trade volume, as well as the impact analysis in the optimal level of the implementation of subsidy and tariff sectorally and simultaneously. we apply a cge model with the gtap database version to examine japan's public r&d investment and its spillover effect on other important players in the global supply chain. the gtap database v consists of sectors and regions, accounting for worlds % gdp and % population as of the base year of (aguiar et al. ). the composite of japan's manufacturing and population began to have structural change with higher input of electronic equipment in communication, energy, and agriculture, resulting in more economic integration with the global supply chain. in this section, following table , we introduce the parameters, variables, and formula used for the simulations based on hertel ( ) . the gtap model is widely used for trade analysis with informative implications for sectors, deficits, and welfare. the composite function of factor production, demand, output, and consumption could be derived as: composite factor production the government behavior in the model is unable to save but to impose direct taxes on the household to fill the gap between its consumption expenses x g i,r,t and its other revenues from production tax t z j,r,t , import tariffs t m i,r,t , and export taxes t e i,r,t . the function is thereby derived as: direct tax where t d r = direct tax revenue; t z j,r = production tax revenue; t f h,j,r = factor input tax revenue; t e i,r,s = export tax revenue; t m i,r,s = import tariff revenue.p q i,r,t denotes the price of the armington's ( ) composite good. based on the suggestions from the national account system (un ), the r&d activities are accumulated in the capital stock, rather than in the intermediate good. government investment is derived in the form of providing capital-use subsidy on the sectoral capital stock, for it is set as an immobile factor, so that the investment could be limited to the selected sector at the investment determined. labor is set as mobile factor within region, so it could flow to other sector for profit optimization. factor input tax factor tax rates further, the energy goods demand for the household x p i,r are the function of the goods production function. the coefficient parameter of ax i,j,r denotes for the intermediate input coefficient, which shows the productivity improvement triggered by r&d investment the external trade functions derive as follows: import flow function composite exports supply function composite import demand function finally, the welfare function is determined by the utility function associate with expenditure function (cost minimization expenditure attaining a utility level), implying the consumption level variation from the output price changes. utility function: the welfare (wf) indicates the utility difference (uu ) before and (uu ) after the policy intervention as equivalent variation (ev). to analyze the key sectors of the global supply chain, we aggregate regions from the base year of to interpret the impact on the economic indicators specifically (table ) , the breakdown details could refer to "appendix" ( table ). the sectors and regions are categorized based on their representativeness in the supply chain. because of the trade gravity, we tend to use a single country in asia to review its impact, while aggregations were made for other regions for simplicity. for the r&d investment scenario, following the definition and suggestion of sna (un ), the r&d investment is used for policy simulations as a capital-use subsidy to increase capital accumulation in the capital stock. according to kuroda, ikeuchi, hara, et al. ( ) , the productivity change is assumed to be actualized after a -year r&d investment for a % more public r&d expenditure on basic science. based on the statistics of , the public r&d investment was categorized into five fields. we provide the policy shock of a total usd , million ( -year amount in present value), equivalent to . % of japan's total capital stock as a capital-use investment ( table ). the improvement rates are calibrated from spias-e simulator on changes in the price of capital goods (psk). in table , we aggregate sectors from sectors classified in gtap database, and sectors classified in spias-e (table ) to accommodate this study. the scenario shows that productivity increases in some sectors such as agriculture (agr) by %, chemicals and pharmaceuticals (chp) by . %, and energy (eng) by . %. nevertheless, with the consideration of shrinking population parameter setting in spias-e (kuroda et al. ), sectors such as metal and steel (met), electronic equipment (eep), service (srv) could not gain productivity improvement despite the substantial increase in we conduct a -year public r&d investment in a static simulation. japan's output shows an increase in most of the sectors, especially for eng ( . %), chp ( . %), teq ( . %), agr ( . %) and eep ( . %) (fig. ) . in comparison, there are only little growth for mch ( . %) and decrease for trs (− . %) and srv (− . %) partially due to the shrinking population. interestingly, the output of eep increases substantially despite lower productivity. the overall technology installation for "society . " promoted in japan since could explain the massive demand in all manufacturing, transportation, and service sectors. regarding the price change, significant increases are found in met ( . %) and agr ( . %), while chp shows a decrease of . %, indicating the technology advancement for this sector. when looking from a global perspective (fig. ) , we focus on four sectors with high interdependence on regional networks, namely chemical and pharmaceutical (chp), electronic equipment (eep), machinery (mch), and transportation equipment (teq). the productivity increase has led the different consequences. for chp, indonesia, vietnam, the rest of asia, oceania, and the rest of world show growth of - %, while other regions show a decrease, implying a less regional integration for the chp sector. for eep, taiwan, oceania, malaysia, indonesia, and thailand also show a vibrant increase by - %, indicating their growing integration. on the other hand, korea, china, vietnam, the rest of asean, and europe show a decrease. the change could refer that the production networks caused by the productivity growth in japan could substitute the output from china. the output growth of mch for japan is at a relatively low level of . %, while taiwan, indonesia, malaysia, oceania, and the rest of world have higher output growth by - %, indicating a transition of mch production networks. teq sectors have been japan's competitive sectors; the output growth of % shows a strong supply ability, whereas other regions only show little change, while the rest of asean and europe show a decrease of - %. it is interesting to see significant growth from oceania, especially in eep. although the volume is relatively smaller than other products, the drastic growth shows that oceania is playing a more important role in the production networks. the price changes imply the affordability of improved technology. the public r&d expenditure in japan has caused a price decrease . % in chp sector, while other key sectors range steadily from to %. in other regions, output price of teq has increased by - % in korea, taiwan, malaysia, thailand, and vietnam, indicating that the difficulty for these regions to accommodate the technology advancement in teq. however, higher prices in electronic equipment still stimulate the regional output, especially in taiwan, malaysia, and thailand. the price change is rather steady for oceania with a higher potential to have production integration with japan. in other regions with distance such as usmca and europe, the change of output and price are very small. overall speaking, japan's public r&d investment could help deepen the production networks with oceania, taiwan, malaysia, indonesia, thailand, whereas the impact with korea and china is insignificant. to further explain the composite of trade structure, we demonstrate an external trade transition with the change rate of trade volume (fig. ) . the productivity increase in japan would strengthen its export in the target sectors of chp ( . %), teq ( . %), mch ( . %), and eep ( . %). oceania also shows the same trend. other export growth could be found in taiwan, and indonesia also for eep. however, the export in most regions shows a decrease of - %, especially in chp. regarding the import change, the decrease in almost all regions indicates that the domestic demand does not change significantly, but the composite of trading partner may change, which requires further look into the breakdown. taiwan shows a slight increase in electronic equipment imports may indicate that their role in the production network. such transition infers that the productivity improvement in japan could satisfy its output and global market expansion. the breakdowns of trade volume could provide more details to identify the change of trading partners (fig. ) . for the chp, the trade volume with japan in almost all regions increases by - %. the trade volume of oceania with other regions has a great growth of - % in europe, usmca, malaysia, the rest of asean, and the rest of the world, implying that oceania and japan could have complementary relations in the chp. consistent with the fluctuations of the output, a significant increase in trade volume in the eep sector indicates the high correlation of production networks between japan, taiwan, malaysia, and oceania for - % (fig. ) . notably, the trade volume of eep for intercontinental relation has higher demand from the asia-pacific region, such as europe and usmca. with worthy of attention, the trade volume of china, south korea, and europe with all the regions also drops substantially by - %, implying that the r&d investment in japan might lead a switch on the map for the global supply chain. japan's productivity growth in the mch sector strengthens its trade volume with all the regions by - %, and the domestic demand could also be satisfied by itself (fig. ) . similar to the results in electronic equipment, the substantial decrease in trade volume in mch from china, europe, and the rest of asean indicate that the structural change of the demand. if advanced machinery equipment could be accessed from japan at affordable prices, the market share would reflect rapidly. notably, the increase in south korea, china, and europe shows an ambition to secure their capacity in global supply chains because mch serves the base for all manufacturing. after the great east japan earthquake and the thai flood in , the global supply chain for teq already occurred a structural change to hedge the disruption risk from a single country. comparing with other sectors, the trade volume of teq with japan still shows a significant increase in all regions by - % (fig. ) . the demand for teq in south korea, china, the rest of asean, and europe is still increasing, and therefore the supply of japan may provide the most cost-effective component assisting the industry. such transition also enables japan to accelerate the teq for the next generation, such as a more affordable hybrid engine and the auto-driving system to maintain its competitiveness and production and application. we analyze the welfare from dimensions of gdp growth and the social benefit change, which is illustrated in the form of equivalent variations (ev) after the policy shock (fig. ) . the public r&d investment in japan with productivity parameters could increase japan's gdp growth by . % thanks to the substantial growth in output and export. it is notable that the gdp grows in taiwan ( . %), malaysia ( . %), and vietnam ( . %), implying the spillover effect on the regional production network. in contrast, south korea, china, europe, and the rest of asean show a gdp decrease by . - %, inferring the output and import substitute in the new structure of the supply chain led by japan. the determination of social benefit relies on the output price and household consumption under policy intervention. the productivity improvement triggered by japan's r&d expenditure substantially decreases the output price of target sectors, making greater affordability both domestically and globally. the social benefit of japan shows a substantial increase of , million usd; however, the fiscal costs caused by r&d expenditure may worsen japan's deficit with a financial burden. regions with gdp growth also suffer a bit worse-off social benefit due to the higher output prices in south korea, taiwan, oceania, and asean states for the similarity of the export-orientated sector in the regional production network. among all, china, usmca and europe fall significantly in both gdp and social benefit, indicating the higher level of regional integration and diversification of "world factory" from one single country to multiple sources. evaluating the r&d impact on the supply chain has been an important aim for the government in sti policymaking in an evidence-based approach. we applied a cge model with the gtap v database on four key sectors of chemical & pharmaceutical, electronic equipment, machinery, and transport equipment to examine their spillover effect on global supply chain and economic impact. the calibration of capital price changes sourced from spias-e has enabled us to capture the productivity change and its spillover effect under the public r&d investment in basic science. such methodology is innovative to unfold the projection of r&d impact comprehensively on a global scale. the simulation results show that japan's output and export on target sectors could substantially increase, contributing gdp growth by . %, and substantial social benefit improvement. moreover, other countries in the region, such as malaysia, indonesia, thailand, oceania, and taiwan, will also be benefited from output and gdp growth. the supply chain integration shows the highest integration in the electronic equipment sector, and the structural change of trade reveals for china and oceania in the trade volume breakdown. oceania shows a significant increase in output and trades with japan as a new vibrant role in the production network. in contrast, the decreased output and trades of china imply the replacement of its position in the global supply chain. for achieving the target of society . in japan under the circumstance of super-aging society and shrinking population, the advancement of productivity would be a critical issue for sti policy making for public r&d investment. the simulation results in this study provided quantified consequences in chemical and pharmaceutical, electronic, and transportation equipment sectors, indicating the massive demand for japan's medical service, smart vehicles and other infrastructure. the notable import of japan's product also implies the r&d lead gap and its competitiveness, providing other partner countries to set best r&d investment strategy in response to japan for collaboration. the research provides broad policy implications of public r&d investment in a globalscale analysis with economic indicators and welfare change interpretation. these insights could provide an informative reference for government resource allocation toward the transition of markets and production networks. although the gtap model demonstrates a straight-forward policy intervention assessment, the assumption of -year public r&d investment and its actualization and addition on overall capital stock may oversimplify the issue due to the intertemporal time-lag in innovation application. therefore, a recursive model would be recommended to fill the gap of the dynamic approach. these preliminary results could serve as evidence to raise awareness of countries in the production network for constructing a multi-region r&d investment database. moreover, the parameters of productivity improvement are only limited in japan due to the single source of spias-e. in the real world, r&d and innovation are taking place in all regions simultaneously. the r&d data infrastructure for many other countries is a desirable input to form a clearer picture of inclusive growth expects to contribute to economic integration and resilience for the global supply chain. singapore s_asia mongolia; rest of asia; rest of southeast asia; bangladesh; india; nepal; pakistan; sri lanka; rest of south asia oceania australia; new zealand; rest of oceania usmca canada; usa; mexico europe austria; belgium; cyprus rest of efta; albania; bulgaria; belarus; croatia; romania rest of eastern europe; rest of europe row rest of north america; argentina; bolivia; brazil; chile; colombia; ecuador; paraguay rest of south america; costa rica; guatemala; honduras el salvador; rest of central america; dominican republic; jamaica iran islamic republic of; israel saudi arabia; turkey; united arab emirates; rest of western asia rest of north africa; benin; burkina faso; cameroon rest of western africa; central africa; south central africa; ethiopia; kenya; madagascar; malawi; mauritius; mozambique; rwanda rest of eastern africa; botswana; namibia; south africa; rest of south african customs the gtap data base: version the impact of e-commerce and r&d on economic development in some selected countries table aggregation of sectors from spias-e classification sector original classification in spias-e agr agriculture (main products agriculture (intra-firm r&d activity) mining (intra-firm r&d activity) energy manufacturing (main products); energy manufacturing (intra-firm ict activity); energy manufacturing (intra-firm r&d activity) food (intra-firm ict activity) food (intra-firm r&d activity) synthetic (intra-firm ict activity) synthetic (intra-firm r&d activity) pulp, paper (intra-firm ict activity) pulp, paper (intra-firm r&d activity); miscellaneous manufacturing (main products); miscellaneous manufacturing (intra-firm ict activity); miscellaneous manufacturing material (intra-firm ict activity) material (intra-firm r&d activity) public, non-profit); r&d materials (industry) chp chemical (main products) chemical (intra-firm r&d activity) eep fibre-optical cable fibre-optical cable (intra-firm ict activity) fibre-optical cable (intra-firm r&d activity); semiconductor manufacturing (main products); semiconductor manufacturing (intra-firm ict activity); semiconductor manufacturing (intra-firm r&d activity); communication devices (main products) communication devices (intra-firm r&d activity); computing equipment computing equipment (intra-firm ict activity); computing equipment (intra-firm r&d activity); semiconductor devices (main products); semiconductor devices (intra-firm ict activity) semiconductor devices (intra-firm r&d activity) electronic component (intra-firm ict activity); electronic component (intra-firm r&d activity) software (intra-firm r&d activity) information management (main products); information management (intra-firm r&d activity) internet (intra-firm r&d activity) machinery (intra-firm ict activity) machinery (intra-firm r&d activity); electronic devices (main products); electronic devices (intra-firm ict activity) electronic devices (intra-firm r&d activity) robot (intra-firm ict activity) precise machinery (main products); precise machinery (intrafirm ict activity); precise machinery (intra-firm r&d activity) teq heavy machinery, transportation equipment (main products); heavy machinery, transportation equipment (intra-firm ict activity); heavy machinery, transportation equipment (intra-firm r&d activity) trs transportation transportation (intrafirm r&d activity) srv construction (main products) construction (intra-firm ict activity) construction (intra-firm r&d activity) communication (intra-firm ict activity) communication (intra-firm r&d activity) commerce (main products) commerce (intra-firm ict activity) commerce (intra-firm r&d activity) medical, welfare services (main products) r&d life science (public, non-profit) r&d information communication (public, non-profit) industry); miscellaneous service (main products); miscellaneous service (intra-firm r&d activity) 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intangible knowledge stock government r&d investment decision-making in the energy sector: lcoe foresight model reveals what regression analysis cannot the determinants of pharmaceutical r&d expenditures: evidence from japan supply chain of innovation and new product development the trend of japan's total r&d expenditure, meti, the government of japan the summary of the investigation on science and technology, mic, the government of japan estimates of intangible investment by industry and productivity growth in japan the effect of ict use and capability on knowledge-based cities search scope and innovation performance of emerging-market firms quantifying the economic impact of government and charity funding of medical research on private research and development funding in the united kingdom contribution of r&d capital to differences in tobin's q among japanese manufacturing firms: evidence from an investment-based asset pricing model united nation chapter -modeling the global economy-forward-looking scenarios for agriculture see tables , and . key: cord- -k l unc authors: lu, li; lankala, srinivas; gong, yuan; feng, xuefeng; chang, briankle g. title: forum: covid- dispatches date: - - journal: cult stud crit methodol doi: . / sha: doc_id: cord_uid: k l unc covid- pandemic is the first truly global crisis in the digital age. with death count worldwide reaching , merely months after its first outbreak in china in late december and . million cases reported in countries and territories as of july , this ongoing pandemic has spread far beyond domain of world health problem to become an unprecedented challenge facing humanity at every level. in addition to causing social and economic disruptions on a scale unseen before, it has turned the world into a site of biopolitical agon where science and reason are forced to betray their impotence against cultish thinking in the planetary endgame depicted in so many dystopian science fictions. it is in this context that this forum offers a set of modest reflections on the current impacts incurred by the covid- virus. blending ethnographic observations with theory-driven reflections, the five authors address issues made manifest by the crisis across different regions, while keeping their sight on the sociopolitical problems plaguing our life both individually and collectively. taken together, they provide a grounded documentary for the archive that the covid- virus is making us to construct. the apparition of these faces in the crowd. -ezra pound, "in a station of the metro" the french word envoi is polysemic, defined as dispatch, the action of sending, something that is sent, a poetic dedication or dedication of a literary work, and the marking of the beginning of a process. this article is a dispatch from hubei, china, based on the author's -month stay in his hometown qianjiang, a small city in the middle of hubei, during the coronavirus pandemic. firsthand observations sent from the epicenter give us a clear picture of what the coronavirus has done. moreover, this article argues that the coronavirus marks a spectral moment in which a repressed trauma returns. there have been fierce debates on the origins of the coronavirus and the political, economic, and social significances of the pandemic. popular representations of the coronavirus which isolate, stigmatize, and terrify the other are symptoms of a returning trauma, which is caused by bodily memories of being victims in past disasters. a derridean reading of the envoi highlights the inherent failure of sending: what is sent can always be held up by a malfunctioning in the process of the sending or postal system, and the meaning of the trauma is lost. this traumatic failure results in a repetition in representation and the return of what is sent to the writer/sender. proposing a supplement, this article foregrounds bodily knowledge acquired through social and political trauma by virtue of fear of the coronavirus. this fear of what is familiar reminds us of the feeling of the uncanny. according to freud and derrida, the uncanny is related to the spectral working of a hidden desire that repeatedly returns as a haunting body, representation, and history. this line of thinking helps us to better understand conflicting representations of the coronavirus. the coronavirus is a ghost. this is not merely a metaphorical proposition; this is accurate in the sense that the coronavirus instantiates our phantasms, fears, and desires toward ghosts. in this regard, derrida's specters of marx provides us with a basic framework for understanding the coronavirus as a ghost. the first teaching of derrida is that ghosts do not come at just any time but in spectral moments that do not belong to time. by pointing precisely to the c scxxx . / cultural studies <span class="symbol" cstyle="symbol">↔</span> critical methodologiesli et al. present or now-time, derrida ( ) regards spectral time as "a disjointed now that always risks maintaining nothing together in the assured conjunction of some context whose border would still be determinable" (p. ). second, a ghost is a phenomenon in the game of repetition and difference. neither exclusively situated in life nor in death, neither visible nor invisible, a ghost is "the frequency of a certain visibility. but the visibility of the invisible. and visibility, by its essence, is not seen, which is why it remains epekeina tes ousias, beyond the phenomenon or beyond being" (derrida, , p. ) . third, to "make oneself fear" is essentially ineluctable in the experience of a ghost. one becomes frightened of a ghost "on the condition that one can never distinguish between the future-to-come and the comingback of a specter" (derrida, , p. ) . in other words, what one fears is not the ghost, but the fear, imagination, and one's subject inspired by the ghost. finally, "a ghost never dies, it remains always to come and to come-back" (derrida, , p. ) . whatever repression the dead may suffer, the return of the dead is anticipated, and "this being-with specters would also be, not only but also, a politics of memory, of inheritance, and of generations" (derrida, , p. xviii) . in light of derrida's framework on specters, once the coronavirus finds a host, it starts to live a ghostly life. the coronavirus pandemic irrupted during a time of turmoil. as the chinese president xi jinping has expressed, the world is experiencing profound shifts unseen in a century. while the trend of globalization is markedly receding, nationalism, popularism, and isolationism are on the rise. the eulogic discussions of "chimerica," a popular term coined by the british epidemiologist neil ferguson in , are being replaced by the theories and practices of the china-u.s. decoupling. the trade dispute between china and the united states puts an end to the chinese ideal of great harmony in the world. in addition, as his campaign slogan "america first" shows, donald trump epitomizes the idea of american exceptionalism. in traditional chinese thinking, famine, natural disasters, and plague happen when the political order or legitimacy are out of joint. during this disjointed moment, a plague was anticipated, even fabricated before it came. according to a widely circulated story in the we-media during the height of the coronavirus, wang yongyan, an academician specializing in chinese medicine at the chinese academy of engineering, predicted half a year ago that a plague would come after the dongzhi (winter solstice), one of the chinese solar periods. in addition, he predicted that the plague would last until next spring. in hindsight, rumors about a new virus were spreading right after the dongzhi. or, simply put, divination went hand in hand with the plague during a time of disjointing, disjunction, or disproportion. while scientists are still trying to track down patient zero, conspiracy theories about the origins of coronavirus have been spreading. bat soup and biological warfare are on the top of the list of suspected criminals. like ghosts, the coronavirus takes shape in the game of visibility and invisibility. in this game, ways of seeing determine how a virus can be understood. approximately made up of . microns, the coronavirus can only be seen under an electron microscope, made visible with the help of scientific equipment and representations. in contrast, a poet like ezra pound sees the invisible through his gifted imagination. his imaginative inspiration and aesthetic reflection allow his keen observation to become a line of poetic beauty and philosophical complexity. through this form of observation, an invisible apparition becomes visible in the faces of the crowd. similarly, the depiction of a fictional killer-virus called wuhan- in dean koontz's novel the eyes of darkness, re-gained popularity among those who regard it as an imaginative depiction of the coronavirus. in line with this imaginative depiction, mr. wang, well-trained in traditional chinese medicine, claimed that his prediction was based on his reading of the xiang (image) of the sky, earth, plants, animals, and human beings. visible to the naked eye, xiang functions as the visible traces from which an invisible plague becomes visible to an expert in traditional chinese medicine. in other words, with scientific support, talent, and training, people are able "to see this invisibility, to see without seeing, thus to think the body without body of this invisible visibility" (derrida, , p. ) . in this way, the ghostly nature of the coronavirus lies in the different frequencies of its visibility. however, despite our faith in being able to depict, and make distinctions between, the invisible and the visible, the way of seeing the coronavirus, especially in this time of turmoil, is politically conditioned and manipulated. when i took a night bullet train to wuhan with my family for vacation, it felt like an ordinary chinese family reunion trip during the spring festival: carriages packed with passengers, luggage, excitement, anxiety, and weariness in the air. one of the reasons for the peaceful atmosphere was that china and the united states had signed a trade agreement a few days before, sending a false message to the world that rationality and peace would return. one thing was markedly noticeable on the train: most passengers wore a facial mask for fear of an officially unidentified but unofficially sarslike virus. to my surprise, a line of masked faces was greeted at the exit by the smiling faces of relatives or friends, the indifferent faces of railroad workers, and the shrewd faces of barkers at the hankou railway station. this lack of consistency indicates that aspects of the coronavirus were kept secret. furthermore, this scenario at the station reminded me of a horrifying scene in the film the cassandra crossing, an eye-opening disaster thriller for my generation directed by george p. cosmatos. in this harrowing film, an international express carrying a virus-infected terrorist approaches a station at night. when the train reached the station, the passengers, who were kept from the truth, were confronted with members of the u.s. army in white biological hazard protective suits lined up on both sides of the platform. in both cases, the dynamic of the visibility and the invisibility of a virus was of political significance. the facial masks and the protective suits were used not only to protect people from a virus but also to make the secret of the virus both visible and invisible. in other words, political manipulation complicates the ways that a virus is seen and how the coronavirus, in particular, is seen as a political ghost. the coronavirus pandemic frightened people because it looked like the return of a specter, namely sars. because of its fatality and residua, sars remains an unresolved trauma for many chinese. at the early stages of the coronavirus pandemic, what was most frightening was its assumed high fatality rate. similarly, the short notice given for the lockdown of wuhan, a huge city of more than million residents, sent a clear message to everyone that the novel coronavirus was the grim reaper. corona, the brand of the first car i owned and of the beer i had on my first visit to a mexican restaurant, was colored by images of a fearful virus, deserted streets, calm officials on tv news channels, and panicking crowds in wuhan hospitals caught on video by the we-media. unlike the countries who proposed or actually enacted herd immunity, the chinese authorities imposed very tough immunity measures, a lesson learned from the sars pandemic, when highways, the railway station, and docks in my hometown were closed overnight. nursing homes were under quarantine; no visitors were allowed in. local authorities advised avoiding public gatherings, including public square dancing and playing majiang. the most popular forms of social activity, especially for retired people, were no longer available. after the initial panic, it was discovered that the fatality rate of the coronavirus was much lower than sars. according to the world health organization, the sars mortality rate worldwide was about %. in early february, the chinese authorities claimed that the coronavirus mortality rate in wuhan was about %. subsequently, what elicited fear in the population was the future-to-come, particularly in the form of social unrest. on one hand, stricter quarantine measures were implemented: all roads were quickly blocked with cranes or tankers or stones; vehicles' use was not allowed, unless a special permit was issued; all grocery shops, markets, restaurants, and hotels were shut down; residents were not allowed to exit their residential areas except for grocery shopping at an arranged supermarket. in addition, central and local authorities watched closely for other concerns, such as food shortages and the inflation of prices. thanks partly to its rich agricultural products in a land of fish and rice, the impact of the coronavirus on food supply and prices did not affect my hometown. however, under the restrictions put in place, my hometown looked like a ghost town, and the uncertainty of the future frightened people of all social strata. in fact, what people fear most is that the coronavirus will never die and will come back again and again, either in the form of a future-to-come or a return of the dead. regardless, despite the medical or political ambition to eradicate the coronavirus, we might have to accept the fact that the virus will co-exist with us forever. for instance, the coronavirus has been mutating, and the way the coronavirus replicates itself in the cells of other organisms is ubiquitous. this mechanism of repetition and difference functions both literally and metaphorically. on one hand, the coronavirus reproduces itself through difference. merely a collection of genetic materials that seems to think with/like a human once it infects its host, the virus induces a feeling of the uncanny, a topic to which i will return later. in addition, news sources reported that infected patients tested positive again after they had been released from the hospital. robert redfield, director of the u.s. centers for disease control and prevention, admitted that some deaths from coronavirus have been discovered posthumously (cnn, ) . in other cases, the coronavirus acted like a whimsical tyrant who inadvertently signed a death sentence. for example, the only cases of death in my residential area was an old couple who lived in an apartment very close to that of my parents. they got infected by their son and daughter who came back from wuhan. what remained a mystery was that the son and the daughter had stayed with their parents for more than days, much longer than the latent period of coronavirus. days after they were hospitalized, they died one after another. on the other hand, the coronavirus reproduces difference in its host organisms. the neighborhood my mother lives in is an acquittance community and an aging society. cadres and volunteers from the neighborhood committee have diligently attended to the needs of the old. aware of the higher fatality rate of the old, an ageist exhortation to quarantine was broadcast repeatedly through a portable loudspeaker placed at the gate of the neighborhood committee building. as stigmatized targets, senior residents were susceptible to the emotion of shame and, for this reason, chose to stay at home. the use of broadcasts and the instigation of shame illustrates how the coronavirus (re)produces, moderates, and polices the line between the public and private spheres. the coronavirus also changed the affective, moral, and power economy of the family. the spring festival is supposed to be the perfect time for a temporary family reunion of joy and harmony. when the lockdown continued longer than everyone expected, generational conflicts broke out. in extreme cases, the political infected families while they were trying to contend with the coronavirus during quarantine. for example, fang fang, a veteran chinese writer who lived in wuhan, posted her thoughts on life in quarantine on her or her friend's weibo account. those posts were later collected and published under the title wuhan diaries. public opinion on those posts varied and eventually led to a political debate between left-wing and right-wing netizens, eventually affecting family members who conflicted in their attitudes toward the wuhan diaries. along with the coronavirus, the memory of personal, generational, and political traumas returned. sars, the cultural revolution, natural disasters, and national humilities were recurring themes in representations of the coronavirus. the suffering and trauma in the epicenter deserve an envoi/dedication, and efforts have been made to achieve this goal, such as daily national and international coverage, fang fang's wuhan diaries, and we-media postings. in these kinds of representation, a rhetoric of "suffering as sublime" is usually at play. in addition, stigmatizing the suffering of others, or blaming the other for one's suffering, is another kind of dedication. both kinds of representations of the coronavirus attempt to take the moral higher ground by attempting to fix the coronavirus as a mere object awaiting to be represented. no matter what position the representation takes toward the coronavirus and its significances, the will to truth turns a dedication quickly into a testimony and even a perjury. a virus is an infectious agent that replicates only within a host organism. for the host, a coronavirus is a deadly stranger and an intimate family member at the same time. familiar, frightening, and secretive, the coronavirus reminds us of the uncanny, as discussed by freud. in his pioneering study, freud focused on the unsettling psychological state of the uncanny. distinct from the feeling of fear, the uncanny is a kind of terrifying feeling that is associated with something known and familiar. after an etymological investigation of the german words heimliche/unheimliche, and a close reading of hoffmann's story "the sand-man," freud ( ) unearthed the origins of the uncanny: "it may be true that the uncanny [unheimlich] is something which is secretly familiar [heimlich-heimisch], which has undergone repression and then returned from it, and that everything that is uncanny fulfills this condition" (p. ). he also associates the feeling of the uncanny "with the omnipotence of thoughts, with the prompt fulfillment of wishes, with secret injurious powers and with the return of the dead" (freud, , p. ) . following the lead of freud, derrida worked on the concept of the uncanny to engage with marx's concepts of repetition, specter, and fear. refuting the claims that the tenants of marxism have died, derrida emphasizes the strange familiarity of the specter of marxism in the age of advanced capitalism. as derrida insists, the specter of marxism will continue to return from the future to visit us, to live with us, and to alert us. similarly, derrida ( ) interprets the uncanny through the concept of absolute hospitality, in which "one may deem strange, strangely familiar and inhospitable at the same time (unheimlich, uncanny)" (p. ). remaining structurally open to future interpretation, the uncanny in derrida's account presupposes a materialism without substance, a messianic without messianism. derrida's understanding of the uncanny is critical to my reading of the coronavirus as a ghostwriter of envoi. as a ghostwriter, the coronavirus is a ghost who writes from the future. as a stranger and a family member, it writes with and in the place of the host. by writing an envoi, a kind of writing haunted by failure and repetition, the coronavirus makes itself visible and frightening in a spectral moment. however, the envoi is not exclusively governed by a ghostly logic that is followed by and instantiated through the coronavirus. in critiquing the tendency to unearth an ultimate truth, eve kosofsky sedgwick & frank ( ) regard affects as a possible way out of the binary opposition of truth and falsehood in representation. by invoking the power of the performativity of shame, they highlight the negative affects neglected by identity politics, dismissed and stigmatized: without positive affect, there can be no shame: only a scene that offers you enjoyment or engages your interest can make you blush. similarly, only something you thought might delight or satisfy can disgust. both these affects produce bodily knowledges. (sedgwick & frank, , p. ) in their view, shame is neither subversive nor mandatory; it works with other affects, drives, and representations to adapt the body to its situation. foregrounding bodily knowledge acquired through trauma commits us to thinking differently about representation and the envoi in question. fear of the coronavirus is not only the fear of a returning trauma as a ghostly logic in representation. more importantly, the coronavirus writes itself and writes about bodily memories of trauma in a constant play of materialization: inscribing fear in itself and on the body of the host permanently. with the end of the lockdown in hubei, the coronavirus pandemic is almost over in china. however, the coronavirus has been sending, and will keep sending, its fearful envoi. the enduring sign of the coronavirus pandemic for indians was not related to medicine or public health. it was the unprecedented exodus of migrant workers from metropolitan centers to their native rural districts, sometimes hundreds of miles away (mukhopadhyay & naik, ; petersen & chaurasia, ) . the scale of this migration was vast and is still being understood. it certainly provokes disturbing questions about urbanity and the fragility of a political compact that kept people in their place through calibrated deprivation (dahdah et al., ) . but for our purposes here, i will explore the ways in which it underscores the varying effects of the pandemic on different classes of people and the diversity of its signification. the virus in india is both a medical event to be dealt with through appropriate public health measures and a mediated discourse that has developed its own ramifications and responses. i argue that both forms of the virus have had tragic and miserable consequences, but on different classes and groups of people. like the televised persian gulf war of that jean baudrillard found to be a distinct and distorted signifier of the actual fighting on the ground, the virus itself is not the same phenomenon once it is transformed into a signifier for other meanings and purposes. the novel coronavirus later named covid- emerged in the public consciousness as a distinct problem with the rapid rise in infections in several indian states by february . in march, the government of india mandated an immediate "lockdown" of the entire country. this new term burned itself into the national consciousness and its many vernaculars almost instantly, as its meaning became physically apparent. it involved the physical arrest of people wherever they happened to be at the moment, and the prohibition of all commerce, traffic, and circulation. it was announced with a -day notice period by the prime minister, in an eerie echo of a similar announcement in of the withdrawal of paper currency. that tragic farce had laid a historical precedent for this second tragedy to come. as a deeply iniquitous society and economy were forced to a halt, the effect was expectedly unequal. metropolitan indian citizens soon learned to cope with the new hardships of "work-from-home," homeschooling, online classes and meetings, and such social-media-driven innovations as cooking and cleaning without domestic servants and entertaining themselves in their houses and apartments. the government also encouraged the adoption of derivative coping mechanisms as soon as they were observed in other countries: applauding medical workers from the safe confines of apartment balconies and terraces; singing, chanting and clanging metal plates and dishes with utensils in cacophonous, solidarity of the gated classes; lighting lamps and candles; and waving mobile phone flashlights at appointed times (krishnan, ) . however, the actual effect of the virus became inseparable from the effect of the "lockdown." the sudden impoverishment of the majority of the country's population led to starvation, medical neglect, and a national panic. while invisible to the citizens in its first few weeks, it became impossible to ignore, when workers across indian cities started to simply walk back to their native villages. their exit from cities also emphasized the fragility of urban belonging: that in a crisis, indian cities were fundamentally empty shells, drawing people not through cosmopolitan attractions or civic rewards but by rural misery. at this point, the virus was still largely a media phenomenon, while the "lockdown" was what had directly affected most indians: the sudden disappearance of work, wages, commerce, and circulation magnified the precarity of urban existence. the largely informal national economy quickly unraveled in a crisis. this crisis was exacerbated by the role of the virus in continuing the ideological and political discourses of the chaotic period immediately preceding the lockdown. the use of the virus to carry out "politics by other means" can be seen in other polities as well, but its entanglement with indian politics is particularly useful as a means to understand the virus as a set of signifying practices. the context of this political use of the virus as a signifier is also inseparable from the highly mediatized nature of indian politics and society. the virus emerged as a discursive phenomenon in india at a crucial juncture in a national conflict over changes to the country's citizenship laws. with the rise to national power of the ruling rashtriya swayamsevak sangh (rss, or the national volunteers organization-a fascist group founded in ), india's national government had been attempting since to achieve its political goal of abolishing its secular and liberal constitution through a steady dismantling of public institutions (roy, ) . this conflict worsened in with the re-election of the rss-controlled government headed by the current prime minister, and the consequent repeal of laws that had hitherto guaranteed the autonomy of the occupied territory of kashmir. this was followed by a critical change to citizenship laws to specifically exclude muslims from gaining indian citizenship and institute a new "citizens' register" to determine afresh the legal status of all residents. with reports of the parallel construction of detention camps outside major cities, the fascist inspiration and ominous intent of the new laws became clearer and more immediate. protests and political resistance to the new measures emerged across the country, and were met with violent responses from the police and rss groups. matters had reached a head when the nationwide lockdown was suddenly imposed. except in a few indian states such as kerala, with still functioning local health systems, the lockdown did not involve any public initiative to test or prevent the spread of the virus. instead, in keeping with the ruling ideology of our time, citizens were mandated to protect themselves, on pain of being brutalized by the police if they failed. in this chaotic sauve qui peut scenario, the rhetoric of basic preventive measures took on ominous ideological connotations depending on who you were and where you lived. as it became clear that only access to clean running water, adequate space, and a home to live in would guarantee the efficacy of the public health guidelines, medical advice became meaningless for much of the country's population, especially the inhabitants of vast informal urban settlements in the metropolitan cities. in effect, a dual situation emerged: a parallel virus had infected the classes who lived in gated urban communities and formal neighborhoods and who followed its progress in daily primetime news trackers. positive cases, testing ratios, death rates, and other numbers soon flew across television and website screens in macabre charts, graphs, and complex animations, as breathless studio anchors enthusiastically tracked the competitive fatalities across states, regions, cities, and countries. as the formal state and civil society response to the virus grew more and more into a media discourse, its actual effect on the population was determined by existing social conditions and ideological practices than by the ideals of public health. in the early period of its spread, the illusion of its control was maintained through the interpellation of the mass television audience as ideal national subjects. in a series of televised speeches, the prime minister exhorted citizens to planned acts of mass discipline, such as the applause, noise-making, and lamp-lighting exercises mentioned earlier. it took several costly weeks for the citizens to realize that this national son-et-lumière had only served to deafen and obscure a different and more real virus that had silently spread illness and death among urban populations who did not have houses or apartments with balconies. a starved public health system soon proved inadequate and unprepared. because this real crisis was not mediated or televised, there was no appropriate or meaningful response to it. the easy congruity of the eagerly adopted virus prevention measures with the practice of caste-based rituals of discrimination was not lost on most indians (george, ) . this fortunate coincidence enabled the easy normalization of virus prevention as a legitimization of existing hierarchical practices. the convenient prescription of social distancing appeared to keep the privileged class of wealthy and respectably middle-class white-collar workers as far away as possible from the physical contact or proximity of their social inferiors. the pandemic thus seemed tailor-made for defenders of hindu caste hierarchies, a righteous and suitably scientific legitimation of social discrimination. the fantasy of caste purity would have remained an abhorrent social remnant if it had not become part of state policy in the last few years. but in the context of the stateled legitimation of religious hierarchies and the consequent onslaught on emancipatory laws, this entanglement of the virus with caste and with the violent hate crimes against muslims acquired a dangerous dimension. it is this distrust of and disgust with a compromised public health system that drove so many indians streaming out of cities and into the relative safety of their impoverished rural communities. the alienation of muslims as a national other has been a part of the basic doctrine of india's current ruling group ever since its founders, awed by the nazi policy of extermination, adopted a similar goal for the erasure of non-hindu communities in india. the mass protests and popular uprising against the rss's attempts to irrevocably alter the basic structure of the country's republican constitution had reached a tipping point when the covid- epidemic was suddenly deemed emergent enough to impose an unprecedented "lockdown," in effect a de facto police state across the country. the imposition of the lockdown allowed police to destroy protest sites, detain protestors, and unleash a reign of terror across indian cities. caught in the initial crossfire were members of an apolitical muslim religious group, the tablighi jamaat, whose convention in delhi had been interrupted by the lockdown. jamaat members trapped in the organization's premises by the curfew were found to be infected with the virus. the consequent media narrative of the discovery of the infection among the jamaatis veered into the fantastical, with nightly news anchors debating the strategies of a "corona jihad" that was to be waged by militant muslims using the virus as a weapon (perrigo, ) . this dog-whistle narrative of muslim bodies as unclean spreaders of a foreign disease dovetails with similar narrative frames used to portray hindus from laboring and working castes as well. the manufacture of conspiracies surrounding the coronavirus can be seen across the world and is not unique to india. a disturbingly large proportion of americans, for example, appear to believe that the virus has been manufactured to enable mind-control through vaccination and g cellular signals by a ruling elite (fisher, ) . on rare occasions, these conspiracies do spiral out into real effects such as the bombing of cellular towers in britain and the anti-vaccination movement in the united states. in india, however, the covert encouragement of such theories by the state itself, to legitimize the hatred toward muslims, exacerbates and normalizes the rumors as mainstream prime time news which is then amplified and shared through an organized social media campaign (ellis- petersen & rahman, ) . the vilification of the muslim other serves two purposes, one of furthering the state's broader agenda of religious and caste purity, and the other more immediate goal of providing a scapegoat for the inescapable rise in infections and deaths due to the virus and the inability of the state and society to understand the crisis. the brutal police crackdown that accompanied the lockdown and the violence of its imposition across the country were a small reminder of the routinization of the "lockdown" as a way of life in the occupied valley of kashmir, part of the only muslim-majority state in the indian union. the effects of the police state as a normalized entity have been multiplied since the abrogation in of constitutional laws guaranteeing the region's autonomy, even if such laws were honored more in the breach in preceding decades (zia, ) . the uncanny resemblance of a public health curfew to a military occupation is not coincidental, but the result of the colonial origins of both, and of the state institutions they represent. the symbiotic existence of caste-based discrimination, the extermination of a religious minority, and the colonial occupation of an entire province within the same body politic is made possible by the continuous interpellation of the mass of people to become national citizen-subjects. this call to obedience, broadcast daily through primetime television and magnified through the near-mandatory use of mobile phones, is the only sign of a nation-state that is otherwise absent in the real world. the failure to stop the spread of the real virus is obscured as the interpellated citizen is urged, cajoled, and threatened to participate in the simulacral fight against a mediated virus in a purely semiotic realm. the washing of hands without the precious reality of running water, the maintenance of "social distance" in the absence of space, the exhortation to "work from home" for a population that is not housed, and the discourses of online socialization and commerce are all much more than signs of mere denial: they are the components of this new semiotic space, enabling the call to national belonging in a new domain, bereft of its mooring in the world. from a broader historical perspective, the coronavirus epidemic does not appear to have affected indians as much as the far greater fatalities caused by more prosaic diseases, hunger, and the increasingly toxic air and water (rukmini, ) . what has caused the greatest pain and panic is the response to the epidemic. this response has been not to the virus itself, but to a simulacral virus that appears to occupy the same space and shares the same name as covid- , but which is a mere signifier, pointing to other, older evils. like baudrillard's hyperreal war, it has surpassed the real virus itself and has come to occupy its place. it cannot be wished away or prevented with a vaccine, it needs a response in kind: of new counter-signs and counter-discourses. a catastrophic pandemic unseen in a century, the current raging of covid- around the globe has undoubtedly produced a unique symbolic site for global, regional, and national imaginations. as the earliest epicenter of this infectious illness, china has witnessed the proliferation of discourses about the evolution of the pandemic on various media platforms, through which the chinese public has the rare chance to reflect on important issues regarding identity construction, social reformation, and nation building. while much attention has been paid to the stigmatization of china in euro-american politics, media, and everyday whisper that label the natural coronavirus as a cultural and ethnic fault (fu, ) , what has been overlooked is how china has portrayed other countries in this global health crisis, especially those surrounding nation-states in the same geopolitical area. east asia, or the sinosphere in the broader sense, with the collective memory of fighting sars in , is thought to have responded to covid- more efficiently than many western countries (salmon, ) . how, then, is the east asian encounter with covid- depicted in the chinese public discourse? how does such depiction envisage china's relations with neighboring countries and its position in the area? in this essay, i discuss the ways in which the coronavirus pandemic has been appropriated by the chinese public for a (re)imagination of east asia. by exploring the evolving representations of its neighboring countries throughout the epidemic on chinese media platforms including weibo, wechat, and zhihu, i argue that the talk of the regional responses to covid- envisions a china-centered union of selected east asian countries in parallel with the historical tributary system of the sinosphere. through the expression of the nostalgia for imperial china, the discursive reconstruction of the east asian identity is a ratification of china's contemporary ambition to reclaim its geopolitical dominance. synchronized with the rapid transmission of the coronavirus in china and east asia between january and march , the chinese public in this early phase drew close attention to the unfolding of the epidemic in its nearby countries, and japan and south korea in particular. with the disease breakout involving diamond princess (japan) and shincheonji church of jesus (south korea) frequently making news headlines, the discussions of how those countries responded to covid- flourished online, which, in combination with the continuous debates over china's own pandemic threat management, contributed to the imagination of the covid- rampancy as a regional challenge that china and its neighbors faced together. central to the discursive formation of this imagined community was the celebration of the incessant interaction and cooperation between china and some east asian countries to combat the virus collectively. in the wake of the outbreak when china was threatened by the crumbling of its health care system, the countries under the spotlight-japan and south korea-were widely appraised for the sympathetic and supportive approaches they took to help china overcome the severe shortage of medical resources. the media reports of japanese and south korean governments leading the international aids to china (gong, ) were echoed by numerous warm anecdotes on social media championing the heartfelt support from their people. perhaps the most well-known story of this kind, a japanese institution wrote a chinese-language verse on the boxes of masks it donated to the province of hubei: "rivers low, mountains high; the same moon in the sky" (trans. zhao, ) ("山川异域，风 月同天"), which immediately went viral online because of its signification of the long-lasting friendship between china and japan. according to account of the expedition to the east by the great master (唐大和上东征传) written by omi no mifune (淡海三船) (see wong, ) , this sentence was from an ancient poem written on the edges of the buddhist robes japanese missions (遣唐使) brought to tang china as the tribute from prince nagaya (長屋王). given its profound roots in the history of japanese envoys to imperial china learning from the chinese culture and civilization, this verse went beyond re-fostering the traditional sino-japanese solidarity. analogizing japan's mask donation with ancient japanese envoys' gifts, it also evoked the retrospective commemoration of the hierarchy between china and japan in history which almost vanishes in the modern era. therefore, the popularity of this verse may indicate the aspiration for the reoccurrence of such bi-lateral relations. indeed, this was only one example of the ubiquitous imaginary of the pan-east asian cooperation and exchange of goods and information as a modern emulation of the tributary system through which imperial china maintained its diplomatic and trade relations to neighboring countries and consolidated its dominance in the region for over a millennium. after china started to keep the pandemic under control and resume the production of medical supplies, this metaphor was further perpetuated in an attempt to accentuate that china's supplies of medical goods and anti-epidemic lessons to nearby countries drastically outnumbered what it was initially given. on weibo, china's return of masks and respirators to its neighbors was often explicitly compared with the "vassals' gifts" chinese emperors assigned to tributary states in posts like this: tribute is both the highest form of alliance and an advanced way of investment, but (this time) it is based on masks! recently, xinwu district in wuxi, jiangsu province donated , to toyokawa, aichi prefecture in japan in return for the , masks, protective clothing and other anti-epidemic materials toyokawa donated to xinwu district in february. (weibo source, march , ) this nostalgic use of metaphor implies a crucial undertone of sinocentrism of the public imagination of the community comprising china and bordering countries fighting against the coronavirus. the tracing of the origin of east asian solidarity to the past is suggestive of the ambition of the present. the chinese public not only fantasizes about a reunion of china, japan, and south korea for covid- but more importantly yearns for the recovery of their nation's leadership and centrality in this battle. as the coronavirus expands rampantly to the rest of the world from march onward, chinese media coverage quickly catches up with the shift of the epicenters from east asia to europe and north america and reformulates the pandemic as a global health crisis. against the depiction of how covid- created chaos, helplessness, and dysfunction in western societies stands the stark contrast of east asia as a safer zone where the outbreaks have been largely contained with success. with the similar control of cases less than , , japan and south korea remain at the heart of this imagined safe zone in company with china even though the reality has seen even fewer confirmed cases in other parts of asia as well as the recent resurgence of virus spreading in all these three countries. this rhetoric is in concert with the prevalence of online deliberations about why east asia as an area has performed better than other parts of the globe in the containment of the virus. at the core of these discourses lies the construction of an east/west binary which frames the global responses to the pandemic into a competition in which "we" (the east/ east asia) have triumphed "them" (the west/euro-america). although china and neighboring countries diverge in the official approaches to handle the pandemic, their relative efficiency in virus containment in comparison with the west is considered to be guided uniformly by the cultural values they share as part of the "confucius east." in particular, collectivism-the principles of prioritizing community interests to personal interests, pursuing social harmony, compliance to authority, avoid causing inconvenience to others-has been glorified as the main drive for the people in east asia to more effectively cope with the governmental strategies in contact tracing, testing, social distancing, and mask wearing. similarly, the regional cooperation in the pandemic management is regarded as a manifestation of these values. for example, the reflections on how south korea has set a model of disease control using mass tracing and testing tend to recognize the smooth uptake of this procedure facilitated by koreans' collectivist mind-set that downplays individual privacy and complies with the data-mining measures to track and publicize their locations, activities, and close contacts. meanwhile, other popular discussions blame the religiosity of the shincheonji church members whose gatherings caused the initial covid outbreak in south korea, which is reflected from the titles of zhihu posts that describe the diffusion of the virus through "hallelujah" such as "the occupation of south korea by covid- , everything has to start from 'hallealia'" and "south korean cult hallelujah devastated the country." apparently, these titles have no intention to mask the underlying tone mocking at the role of christianity in the acceleration, not mitigation, of disease spreading, which further serves as a foil to the power of confucianism to help south korea navigate away from the disaster. in fact, satire targeting at christianity represents the broader criticism of western cultural values in hindering the efficacious enforcement of restrictive and surveilling measures against the coronavirus. the east asian identity is thus reaffirmed through the clashes between the eastern and western civilizations. however, it is worth noting that the narratives about the east asian conquest of covid- are again permeated with the metaphor of the tributary system delineating china as the leader and role model in this imagined "safe zone." not only does the attribution of the regional success to confucianism call up the historical chinese centrality in the sinosphere but the emphasis on china's ability to offer lessons and instructions from its early experience for its neighbors to benefit from also ratifies the restoration of the "teacher/student" relation between imperial china and pre-modern japan and korea. far from a total reenactment of the historical sinosphere, this chinese imaginary of east asia engages with a purposeful selective process that amplifies china's solidarity with some east asian countries but simultaneously mutes others in the same region. as remarked earlier, a majority of the online narratives about the cooperative responses to covid- in east asia revolves around china, japan, and south korea, with less frequent inclusion of singapore as well as occasional reference to such countries as mongolia and myanmar. this emphasis on forming a coalition with japan and south korea is compatible with china's longterm agenda of promoting and dominating the china-japan-south korea union (中日韩一体化), which was recently reiterated by the three governments' consensus to speed up the negotiation of the free trade zone (中日韩自贸 区) (wang, ) . in this sense, the covid- crisis has offered a discursive site for the chinese state to rebuild this trilateral bond and remodel its significant neighbors whose national images, due to the respective disputes around diaoyu islands and thaad (terminal high altitude area defense), have been negative in china for almost a decade. while the china-japan-south korea triangle is romanticized in connection with other small countries, the alienation of some confucius societies from this imagined "cooperative" east asia is quite striking, especially given the outstanding results some of them have produced in the prevention of disease transmission. the first excluded category includes taiwan, hong kong, and macau-the territories outside the mainland in the great china area. whereas macau is often forgotten by the media as it has always been, both taiwan and hong kong are widely criticized and mocked for their attempt to politicize the pandemic as a weapon to confront beijing and increase international recognition. the second group pertains to north korea and vietnam-the authoritarian states that have close political and ideological bonds with china. for instance, north korea has been constantly questioned and satirized because of the lack of transparency in the disclosure of its epidemic circumstances. vietnam's outstanding handling of the virus which led to only confirmed cases and death was nearly silenced in the mainstream media coverage. in the unusual reference to vietnam in some zhihu conversations, vietnam's success was rarely celebrated but considered as a "threat" to china's leadership in containing the pandemic in the area. the trivialization and exclusion of these countries/ regions from the chinese imagination of east asia as a collective force fighting against covid- is not unexpected. in the first place, the negative attitudes toward them (except macau) reflects a backlash against the restrictive, noncooperative methods those governments have enforced to block the virus from mainland china (e.g., full border closure; ban on exports of medical supplies), which signifies their resistance to be incorporated into the modern tributary system chinese people have aspired. yet for taiwan and hong kong, this exclusion repeats the endeavor of chinese official propaganda to erase the distinction between them and the mainland and disavow their political autonomy. instead of being completely out of the picture, their responses to the coronavirus are mainly discussed as part of the chinese experience to consolidate the national identity. for north korea and vietnam, the negative impression may partly result from china's ongoing diplomatic conflicts with them in recent years regarding the south china sea and denuclearization, respectively. nevertheless, the shaking of the "socialist brotherhood" on the matter of covid- also implies the reluctance of the chinese public to articulate a regional identity around the axis of a shared political regime. in fact, assimilating itself with ideological and political allies is likely to obscure the focus of this imaginary on china's historical and cultural alignment with japan and south korea. as covid- begins to shift both the scholarly and media focus on an international scale to reconsidering the dark sides of globalization (chan & haines, ) and mourning for the disruption of european union (trofimov & pancevski, ) , china's reversed agenda of imagining a regional union is stunningly intriguing. on one hand, the eagerness to build solidarity with east asian countries represented by japan and south korea might be a strategy to react to the racialization of covid- as a "chinese virus" and the demonization of china as a "public enemy" and "trouble maker" in the euro-american political and media agenda (viala-gaudefroy & lindaman, ). by articulating china's resemblance (and collaboration) with the bordering democratic capitalist states (rather than the "socialist brothers") in the "confucius-inspired" success of halting the virus, the public discourse strives to construct a collective identity of the east so as to brush off china's label of the other imposed by the western imagination. ironically, this consolidation of the eastern identity also serves as a repercussion to otherize the west as the loser to the coronavirus. on the other hand, the rise of this east asian imaginary centering around china's historical and cultural bonds with japan and south korea has far-reaching implications for china's geopolitical strategies beyond the covid- pandemic and the realm of public health. rested upon the trope of the imperial tributary system, this imagination reflects how the chinese public discourse echoes the state ambition to recuperate the historical dominance of china in the sinosphere, which is part of chinese communist party's long-term project of "the great revival of the chinese nation" (中华民族伟大复兴), or in xi jinping's term, the "chinese dream" (中国梦). incorporating japan and south koreathe most important american allies in east asia-into the imagined tributary network might serve the specific purpose of weakening the u.s. hegemony in the region (see ikenberry, ) , whereas the tactic exclusion of north korea and vietnam indicates the indifference of many chinese to the state's political and ideological "comrades" (whose traditional alliance with china has often proven itself unstable and delusionary in the changeable economic and political dynamics in east asia). more importantly, this selective reimagination of the eastern union expresses the chinese public's nostalgic ideal of the nation's revival, which dreams of a return to the middle kingdom, the empire that reunites and leads east asia through culture and history. during the year , which is anticipated to be the warmest year in human history, we failed to stop the rampant spread of a coronavirus called covid- and its disastrous impact on societies and individual lives. unlike its "cousin" sars, which broke out in early and vanished into thin air largely because of rising temperatures, the current respiratory epidemic has yet to show any sign of amelioration with the arrival of summer. news photos have shown audiences an incredibly bleak, bizarre, and somewhat surreal picture of life during the pandemic. streets are evacuated. stores are closed. public services are paralyzed. modernized cities have become empty and ghostly quiet. only scattered people equipped with medical face masks walk anxiously in this futurist, apocalyptic scene. to use timothy morton's concept, the covid- pandemic has become a "hyperobject," a phenomenon that possesses an ahuman time scale and an extremely diffused quality in occupying space. in such a space-time reconfiguration, or, in plain language, during this type of disaster, humankind becomes an obsolete idea, as humans no longer play a meaningful role in the space-times created by and for "hyperobjects." unfortunately, such a concept bares relevance in light of the uncontrollable proliferation of the coronavirus across the globe at this juncture. worse still, some epidemiologists warn that a new round of outbreak will likely occur soon in the coming fall. a possible scenario could repeat the conditions after the / fukushima daiichi nuclear disaster, when breathing with face masks, people eventually became accustomed to a state of emergency as the conditions for living and dying in the anthropocene. "[p]oison has become a normal feature of daily life, the second nature we have to inhabit" (berardi, , p. ) . while one can attribute the deterioration of nature to neoliberalism and its disastrous governance, this essay, rather, speculates on what foregrounds the involutional relationship between humans and the earth beyond the "nature-culture" divide. whether one is willing to admit or not, viruses are neither creation ex nihilo nor culturally and politically constructed representation. instead, they are beings that have always been part of earth's composition. in a prophetic book, the natural contract, the late philosopher michel serres ( ) describes the evolution of the earth's composition. in ancient law and modern science, nature was treated as an objective reference point, because it had no subject. existing objectively "out there," the earth was a space that did not depend on humans but only acted passively in relation to causality. yet, witnessing the ecological crisis arising in the th century, humans realized that the earth has been affected by our behavior and is now behaving like an aberrant subject! in recent scholarship, this subject has been referred to as gaia, the capricious goddess of the earth (see latour, , p. ) . the earth is full of action and so is covid- . as described in news reports, the coronavirus looks for and hijacks its hosts; it finds easy purchase on, and takes control of, human bodies; it kills many, but not all, of its hosts so as to keep moving, spreading, replicating, and surviving. it would be impossible to talk about the virus without referring to those actions. cited by the washington post, a virologist came up with a vivid analogy for viruses by comparing them with destructive burglars. "they break into your home, eat your food, use your furniture and have , babies" (kaplan et al., ) . as the word "object" refers to entities that are inanimate and subject to chains of causality, viruses, in this sense, hardly fit into this definition. for instance, covid- remains mostly enigmatic, not least because it is considered strikingly sneaky-"the virus doesn't really want to kill us. it's good for them, good for their population, if you're walking around being perfectly healthy," said another virologist in the same washington post article (kaplan et al., ) . besides doing things such as breaking-into, eating, and having-babies, the virus is further endowed with intentions-it does not want to kill us! however, the coronavirus should not be mistaken for a subject, especially a subject-agent, which is historically associated with liberal humanism since the enlightenment and which is deeply rooted in the "nature-culture" divide, an ontological regime referred to by latour as "the modern constitution" (see latour, b) . the idea of the subject as a product of euro-american modernity is indivisible from its aim to achieve individual sovereignty and autonomy. in a politico-legal sense, bounded individualism is the most evolved form of this idea in the wake of the global expansion of capitalism. faced with an unprecedentedly active earth in the late th century, nonetheless, this anthropocentric conception of the subject-agent has been confronting exponential challenges, among which the current coronavirus pandemic constitutes the latest one. to be clear, the term "subject" is a mismatch for covid- , not because it is agentless and incapable of doing the same things that humankind does. the contrary is true: the state of being of the virus-what it is-can unfold only through its actions and long after its performances. at stake for the virus and humans is that there are "no pre-constituted subjects and objects, and no single sources, unitary actors, or final ends" (haraway, , p. ) far from being a de-animated object, or an anthropomorphized subject, gaia, the increasingly "rioting" earth, is a collective of actions that distributes agency in heterogeneous and surprising ways. as a result, "we must not believe in advance that we know whether we are talking about subjects or objects, men or gods, animals, atoms, or texts" (latour, a, p. ) , and also viruses until their actions are captured, and rendered into shapeswhether the shape of a human or of a virus. the story of the human-centered history is being replaced by an explosion of narratives about the increasingly animated and animating earth. however, the dualism of the subject versus the object, unfortunately, is still perniciously conserved in the mainstream reaction to the covid- pandemic. when societies are forced to act on the pandemic, the virus is almost exclusively treated as an object subject to the chain of causality. this tendency is clearly reflected in the mobilization of wartime rhetoric and discourses in conjunction with governments' anti-epidemic measures. for instance, when visiting wuhan right after its lockdown, sun chunlan, china's vice premier, warned that the country was facing "wartime conditions." likewise, only month later, president donald trump declared a national state of emergency over the coronavirus outbreak in the united states. in this antagonistic discourse, contending with the virus, a not-yet-tamed and potentially threatening other, is framed as a relationship between humans and their enemies. for those who believe humans and only humans make history, a self-proclaimed war on the virus is unavoidable! peace, accordingly, is only imaginable to be reached, or more precisely restored, to an already existing order, established primarily for humans. mobilized to describe the relationship between covid- and humans, "war" is a terrible and even dangerous choice in terminology, due to its undertone of human exceptionalism. in fact, nearly % of the cells in a human body is "part of a vast community of companion species, particularly bacteria and viruses" (smart & smart, , kindle ) . unfortunately, most humans have yet to learn the meaning of living and becoming-with these beings who are made by and making humans at the same time. in her book staying with the trouble: making kin in the chthulucene, donna haraway ( ) invites readers to contemplate our troubling present, the chthulucene, an emerging regime of naturecultures, as opposed to the "nature-culture" divide. contrasting to the discourse of the anthropocene and the capitalocene, both of which are conceived as human-induced condition, the "chthulucene" is, first and foremost, concerned with earth beings who live in "manifold forms and manifold names in all the airs, waters, and places of earth"-they are monsters which "demonstrate and perform the material meaningfulness of earth processes" (haraway, , p. ) . the vicious coronavirus is evidently one of these monsters. despite the havoc it is creating in the present, the epidemic is a manifestation of the biotic and abiotic powers inherent in earthly actors and is part of "ongoing multispecies stories . . . in times that remain at stake" (haraway, , p. ) . as implied by the title, one of the valuable lessons of haraway's book is that, for humans in particular, there might be no better option other than to stay with troubles, of which humans are never innocent. staying with the troubles demands caring for all the threads that bind us together and make our existence possible in the first place-humans are made by countless earth beings and vice versa. it also means that we are required to weave unexpected and even dangerous connections with others, in haraway's ( ) words, making kin as oddkin "in unexpected collaborations and combinations . . . we become-with each other or not at all" (p. ). this insight is particularly useful for thinking about viruses. because viruses have "no cellular machinery of their own, they become intertwined with ours. their proteins are our proteins" (kaplan et al., ) . in this sense, the evolution of humans and viruses is inseparable from the process of involution of the two into one. in other words, becomingwith means that, by definition, a "we" always precedes an "i," a "you," or a "they." the so-called "asymptomatics" provide an excellent example of this point. asymptomatics refer to those who test positive for covid- but, confusingly, do not suffer from illness or show any symptom of the disease. asymptomatic infections or carriers are possibly greater in number than those with symptoms. at this point, it is impossible to decide which of the two types is more typical of covid- infections, because, as a researcher at the university of oxford says, "there is not a single reliable study to determine the number of asymptomatics" (shukman, ) . in the same news report, neil hall, a biomedical expert, suggests considering asymptomatic cases of the coronavirus as the "dark matter" of the epidemic, as invisible and not-yet identified dark matter is believed to make up most of the matter in the universe. despite the fact that no conclusion has been reached about the enigmatic phenomenon of asymptomatics, the differences that manifest among patients reveal that the virus, and the particular cases of infection, should be examined as specific units. in other words, between the virus and humans, the specificity of an encounter matters. unlikely to be autopoietic systems that reproduce autonomous units, the virus and an infected body constitute a collectively produced, sympoietic system that does not have self-defined spatial or temporal boundaries. in these cases, and from a non-anthropocentric, philosophical point of view, the idea of bounded individualism has to be discarded for good. beyond the divide between the subject and the object, what emerges are ontologically heterogeneous practitioners who are involved in each other's lives. besides evolution, living also relies on involution. without any intention of "romanticizing" covid- and the current pandemic, staying with the trouble, as articulated by haraway ( ) , is "to make kin in lines of inventive connection as a practice of learning to live and die well with each other in a thick present" (p. ). the coronavirus does not happen as a matter of fact, which "passively" waits to be discovered, investigated, tamed, or neutralized by "active" humans. what we call the covid- pandemic manifests itself as a differentiating and relational effect because it matters by bringing into being various relations between humans, and between humans and their oddkin. in this view, science is only one practice among many others to capture the efficacy of its mattering. in addition to biomedical measures, a more critical question for the coronavirus crisis is "what method does the matter demand" (thompson, , p. ) ? proposed by haraway for living in the chthulucene, the string figure might also serve as an appropriate method and image for the pandemic, characterized by its exceptional contagiousness and interactivity. consisting of "passing on and receiving, making and unmaking, picking up threads and dropping them," the string figure is all about "becoming-with each other in surprising relays" (haraway, , p. ) . crucial to this method is that it does not guarantee what is obtained turns out to be good in the end, because living itself has become so dangerous in this very thick present-agencies are distributed, conflicting, and entangled in a myriad of practitioners, human and non-human alike. in this pandemic, we are all playing the game of string figures with our oddkin. it is not beneficial to judge in advance who is a subject and who is an object, or which one is active and which one passive, as all participants might be capable of something that matters in one way or another. for example, one thing that the respiratory disease teaches us is that not only breathing matters but also the manner how one breathes matters to others. life and death happen inside specific connections and their mattering in mundane, and even fleeting, encounters. making covid- matter requires us to reanimate "what is coming into states of matter and mattering in bodies, stories, acts, and events" (stewart, , p. ) , in other words, in the vicissitudes of our ordinary lives. for the future of this thick present, one key is to stop imagining the crisis of the coronavirus as something wholly predicated on effective vaccines and scientific solutions. instead, humans must learn to connect and also care for threads, some of which are obvious, some elusive, some vicious and dangerous, and some fictional. we may need to discard terms such as "overcoming" or "solution," and turn to terms like "participation" concerning all that we are uncertain of but have to live and become with, together, in the "metamorphic zone" called the earth (latour, , p. ). what does it mean, the plague? it is life, that is all. the most abundant biological entities on earth, viruses are forever and everywhere. suspended between living and being dead, they are simply there, a slimy strip of ribonucleic acid (rna), as biologists tell us. poorer in life than tardigrades, incapable of movement, and having no logistic of their own, they ride on and feed off others to replicate themselves, to become the viruses that they are. as smart schoolchildren know, they are transmissible and must be so transmitted as to go viral, to become the viruses as we know them. dependent entirely on carriers, that is, exploiting others' hospitality, without which they have no life (but also no death either), viruses exemplify transmissibility. they live and thrive, as it were, only if their hosts are susceptible, in motion, and in contact and they die or die down when susceptible hosts are either unavailable or no longer hospitable. defined, that is, made finite, by transmissibility, and yet transcending its barren finitude through parasitism, viruses exist and operate like pure media, self-generating and selfgenerated by being entirely coterminous with the channel through which they flow and multiply. interpolating and encoding themselves in the metabolic cycle of others, thereby reproducing themselves passive-actively, they mediate by colonizing others and, in so doing, mediate themselves by proxy, going about so energetically and indiscriminately as to cause the demise and thus thwarting unwittingly their own propagation. if viruses communicate anything, if their shadowy occupation of host bodies sends any message, it is their very own communicability, their ability to disseminate themselves over a large population with effort less than minimal. although all over creation and in abundance, most of the viruses cause us no harm and we pay them little attention, even though they populate our body and capitalize on its resources. they become a matter of grave concern when they infect us, when they not only put themselves inside (in-ficere) our body and stain its normal functioning, but also threaten to afflict as many people as their "infectivity" attacks. more dangerous and less tamable than most microbes, viruses invade our body and compromise it at the cellular level. they do not just make us sick; they bring about plague. once seen as a cause of infection, viruses accrue significance and take on the label "pathogens." to refer to viruses as pathogens implies that they are "medicalized," that they not only enter into a relation with humans who regard them as toxic and virulent, but are also seen as a problem to be addressed in a methodical, systematic, that is, "scientific," manner. it is through this medicalization that viruses are individuated and identified as a distinct biological entity and, having been so captured and given a name-for example, h n , mers-cov, sars-cov- , covid- , and the like-by what might be called the "clinical gaze" and its taxonomic procedures, they enter into sciences and become a focus of medical research, made all the more pressing if and when they create public health crisis. more than one hundred years after martinus beijerinck gave the name contagium virum fluidum (contagious living fluid) to the incitant of tobacco mosaic first discovered by adolf mayer and dimitri ivanovsky, viruses are now actively collected, classified, and manipulated by scientists in highly restricted spaces called laboratories, most of which, like the viruses housed carefully therein, are hidden from public eyes. while slimy poisons were once thought to be sent down by god to punish us for our sins, we now see viruses not only as an object of scientific investigation but also as a medical challenge that nature poses to us as biological creatures on earth. like birds, bats, and rats, we are all equal opportunity hosts to killer germs. not all viruses are fully pathogenic, but pathogenic viruses are ever ready to go viral when the conditions are ripe. however, although viral infection may break out and spill over, it does not mean that there is a pandemic. "pandemics," as virologists tell us, "begin when a brandnew virus infects a human who also at that point is able to transmit the virus to other humans" (buettner, ) . two points should be noted without delay. first, pandemics are not created by transmission of viruses from some source to humans, but from humans to humans. breakouts of viral infections among members of a primate community deep in the amazon rainforests, for example, may be large scale and may disturb ecological balance alarming to conservationists, but they do not for all that count as pandemics in the sense that the term is properly used. viruses might infect one or more individuals, but humans are responsible for creating the conditions that transform infections to outbreaks and outbreaks into pandemics. pandemics, in other words, are not natural or biological phenomena; they name a human crisis, a contagious malady plaguing humans who are both agents and patients at the same time. contagious diseases are disastrous to all, locked, as we are, in the same bubble in which microbes live and grow, but pandemics are decidedly more pernicious in that we become, often unknowingly, the source and the cause of our own infestation. second, pandemics are "declared." as is the case with catastrophic events in history, like wars, famines, or mass cultural anomaly as bizarre as the chinese sorcery scare of , whose duration and identity result from an act of punctuation and sense-making entirely sociopolitical in nature, pandemics too begins with a performative act that announces their beginning and, having made them to begin in this way, determines when they reach their end, even though the viruses and their carriers may still be with(in) us (see kuhn, ) . naming not microbial activities in nature but a crisis for humans, pandemics are events made real, public, and urgent, as just said, by a performative-a speech act, to be exact-whose authority in pronouncing their beginning and end depends on the very force that makes the declaration authoritative and forceful in the first place. brought into being by discourse and public communications, pandemics are social constructions; they signal a state of emergency-appearing, first, as physical ailments on the part of individuals, subsequently identified and ratified by medical and scientific community as a real health problem, and finally materialized by authoritative broadcast and public acknowledgment, thereupon becoming a public policy issue to be addressed by political leadership, all these over a determinate territory. once established as such, a pandemic individualizes a collective paroxysm, making it a public enemy by giving it a face, a name, a certain life span in the social calendar, without which the havocs wreaked by the virus would not be the crisis its name designates and invokes. it is in this declarative nature of pandemics that we can see how viruses, once medicalized and publicly acknowledged, are inevitably entangled with science, history, culture, and politics. socially constructed by a decision, by a cut or break into regular time, they mark a "zone of exception," a temporal heterotopic, as it were, where we, individually and collectively, stand to one another as equal subjects to illness, unfreedom, and death in the unending drama of man against nature and its hostile elements. viruses are viruses are viruses. they have no political content; operating according to the laws of physics, chemistry, and biology, they come and go on their own rules and on their own times, as nature dictates. in sharp contrast, pandemics are biopolitical phenomena; they are moments of discontinuity or rupture in social order, shot through from start to finish with forces and factors that shape culture, history, and economy, which in turn determine what they mean and how they come about and come to pass. moreover, and importantly, a pandemic is not a single, monolithic event; it is a series of localized epidemics, each with its own point of origin, its own history, its own epidemiological pattern and impacts. further still, all these factors crisscross one another in a complex, nonlinear fashion, amassing multiple agents and stakeholders in such a critical fashion that the language of war is often used by the authority in charge to quell the infectious assault. pandemics force social changes precisely because the changes they incur invite resistance. it is for this reason, perhaps for this reason alone, that pandemics inevitably appear as a site of social contestations, politicizing and politicized by the heterogeneous constructions barely betrayed by the name of a single virus. it is for this reason too that pandemics assert themselves as a sign of generalized cultural and economic strife, a symptom of social struggle underlying the health terror that a viral breakout unfailingly induces. covid- is a novel virus, novel in that scientists do not fully understand how it afflicts the body and therefore cannot predict its epidemiological paths. to control its spread, we have no choice but to employ methods developed from past experiences, such as quarantine/isolation, social distancing, face coverings, and contact tracing, to name a few now well-known. because viruses are infectious, to control its spread is, understandably, to separate and to isolate. this means that people be kept away from one another. instead of gathering or being together, we make ourselves scare; better yet, we isolate ourselves, even if begrudgingly. more than that, the injunction of isolation leads straightaway to insulation in that the ultimate, foolproof means of isolation is to literally atomize ourselves, to turn ourselves into windowless monads. indeed, all the mitigation measures we hear about of late-quarantine, mask wearing, hand washing, and social distancing-are in reality anti-social measures. don't reach out and don't touch anyone! cover up your face! just as social distancing-a contradiction in terms of sorts-means keeping physical distance, and just as mask wearing reduces mutual recognition based on simple vision to its unnatural minimum, (self-)isolation and quarantine all but eliminate human contact of all kinds. when the plagues struck, we were all lepers; when covid- strikes, we are all windowless monads. pandemics are born of communicable diseases, yet for this reason, they force us to be incommunicable. flattening individuals and bringing to a halt exchange and commerce of every kind, they turn a society into one that is against society. if there is a history of pandemics, it is a history of anti-social history. neither alive nor dead, neither this nor that, viruses are by nature improper. never proper, that is, never being (of) themselves, they appropriate-always ready to make others their own. they are pure media, as suggested earlier. viruses are pure because they mediate unconditionally. however, inasmuch as unconditional mediation performed by viruses leads to the demise of their host, upon whom they depend for their parasitic reproduction, viruses end up annihilating themselves by their very nature; they are always already their own collateral casualties. rendering themselves nil by simply being and subsisting as themselves, pure media are no (longer) media. unconditional mediation ends all mediations. by bringing society to go against itself, viruses commit suicide, so to speak, by killing their host, by the unconditional abuse of others' hospitality. and, alas, weat least some of us-are spared. covid- is a new virus. but, unlike the known flu viruses, or h n , sars, and the like, covid- is considered "novel," not the least because, as indicated earlier, it frustrates scientists' understanding. "it has been like nothing else on earth," says an infectious-disease expert, who falls victim to the virus; "i knew i had the disease; it couldn't have been anything else," but "i don't understand what's happening in my body" (yong, ) . there are many things, inanimate or living, on earth that are like nothing we know so far, and there are many things happing in our body that we do not understand at all. covid- can justifiably be called "novel," but isn't every virus novel in its own way and at some moment in time? isn't being novel the normal course of event in life and in life sciences as well? "there is novelty here," remarks a prominent epidemiologist karl friston upon leaving a lab meeting about covid- , but he quickly adds, "so this, from my point of view, is just an average day" (kosner, ) . being novel is the very characteristic of all viruses and many other things in nature as well. the novelty of covid- may not be as novel as we think. what is possibly novel about covid- is the fact that it gives us the first pandemic in our truly globalized age. the global village, in which we now live, is so hyper-connected-not only by technology but also through affluence, commerce, and global travel-that an infectant can travel from one city to another as fast as jet streams flow. connectivity translates qualitative diversity into measurable multiplicity, reducing distance and difference for the formation of the common, which in turn strengthens connectivity. to be alive, as few would disagree, is to be connected, literally and in every other sense. but this means that we must live in and with the risks that global connectivity brings to us. to be connected brings with it the possibility of being stranded in harm's way. as covid- makes clear, "connectivity is the killer" (kosner, ) . after all, life depends on maintaining boundaries and keeping differences. deadly viruses are deadly because they breach them. as infection rate rises, so does anxiety. and bleak scenes spread as wide as the virus goes. deserted streets, boardedup stores, closed factories, shot-down public transportations; remote learning, work-from-home; stock markets crashed . . . and, worse yet, "i just lost my job." individual solation leads quickly to desolation across the board. and economy bears the brunt of a colossal coronal attack. shortly after covid- spread out of wuhan, china, to europe in january , stories about the economic plight began to top the list of topics in public forums and news media. the future we face seems to lie in one of the two choices: to die from hunger or to die from the disease (餓死 或病死), as the expressions go in chinese media. it is not for no reason that a policy brief released in june by the united nations on the impact of the pandemic is given the title "the world of work cannot and should not look the same after this crisis" (guterres, ) . the address on the launch of this brief, given by the secretary-general antónio guterres ( ) , begins as follows: the covid- pandemic has turned the world of work upside down. every worker, every business and every corner of the globe has been affected. hundreds of millions of jobs have been lost . . . many small and medium-sized enterprises-the engine of the global economy-may not survive. after painting a depressing picture of the future and explaining how difficult it will be for the world economy to return to "normal," guterres's ( ) address makes a hardly perceptible turn when he says "let's not forgot the pre-covid- world was far from normal." it seems then that, rather than shattering the world of work as we know it, the covid- pandemic simply exposes in higher resolution the "tremendous shortcomings, fragilities and fault lines" that have been eroding society and economy from the bottom-up for decades. the pre-covid- world, in which we thought we lived a normal life, is not as normal as we think (see guterres, ) . to save the economy under siege is to "return to normal" as soon as possible, so cry the bureaucrats and journalists alike. but what is "normal" in this case? what does "being normal" mean exactly? is the world, old or new, ever normal? there are norms regulating life, but has there ever been a "normal life" as such? the so-called normal life, a life before covid- , to which we pray to return, is in truth one of recollection, a romantic one at that, as the un policy brief readily admits. just as a viral infection may display more than one symptom on the part of its victims, embody more than one single illness, and create more than one single public health challenge, life, as it is actually lived, is hardly reducible to one normal life. in fact, the socalled normal life is the one that brought us the pandemic in the first place. to live is to live normally; to return to normal is what living is all about. the so-called new normal is both new and not so new, which is to say, it is neither really new nor really normal. perhaps the world has never been and will never be normal, whatever our idea of "being normal" means. if a pandemic can turn the world upside down, it is because life has been turning and turning again. and anything that returns cannot be entirely new. humans have been haunted by viruses since time immemorial. from the prehistoric pandemics in northeastern china , years ago, uncovered at sites now called hamin mangha and miaozigou, to the justinian plague ( - ad) that may have helped to bring down feudalism, or the small pox outbreak that finally toppled the aztec empire before hernán cortés returned to the region in the spring of , viral infections have tormented the lands and their people over millennia. traveling with host animals and humans, viruses had gone global long before globalization became a fact. there are known pandemics in the last years, all displaying the same pattern of spiking in seasonal waves after the initial attacks. covid- , and some of its coronal cousins, will undoubtedly expand the list. to those who are living through its assault, the impacts brought about by covid- are more or less clear and more or less measurable. but what is the meaning of covid- when the current pandemic is over? will it be remembered? if so, in what way and to what extent? if the history of pandemics has taught us anything, it is that history tends to repeat itself, that viral outbreaks are an ineliminable part of the natural history, in which humans are a part and in which no "zone of being" is free from viral infection. recall the spanish flu of , the worst pandemic during the last two centuries. it is estimated to have wiped out million people worldwide, meanwhile infecting million, a third of the world's population at the time. however, despite its short distance of mere one hundred years from us, few people today know much about it, and still fewer are able to understand or feel the impact it had at that time. its centenary a short time ago passed noiselessly, certainly not for lack of stories or records. like the many plagues before it, the spanish flu, it seems, never quite made itself into what reinhart koselleck ( ) calls the "the space of experience" (p. ). failing to make its way into collective memory, it is also helpless in figuring into our "horizon of expectation" (koselleck, , p. ) . if the spanish flu faded largely from memory, all the woes caused by covid- are, likewise, likely to dissipate in time, regardless of how we feel and say about it now. there was a pre-coronavirus world, and there will be a post-coronavirus world, but viruses, known or novel, will outlast our worlds. viruses are everywhere and forever. so, plagues will never disappear for good (camus, , p. ) . but what then does it mean, the pandemic? it is life, that is all. a troubled memory, fading, under the vast indifference of the sky. until the gate of oran closes again. assembled in this forum, the five short essays provide some modest reflections on the coronavirus pandemic and its still unfolding consequences. committed to a variety of disciplinary perspectives and interests, the authors did not set out by pursuing any preset direction or common agenda supposedly carried out collectively in our intellectual labor. rather, what unifies the diverse inquiries in these essays is the shared awareness about the confusion in the public discourse that constantly fails to distinguish a coronavirus called covid- from the covid- pandemic, or as briankle reminds us in his essay, from "a series of localized epidemics." this alertness constitutes a common ground in addressing specific issues or phenomena in these essays. this forum is anything but comprehensive. if it can contribute to the discussion of the crisis, it is most likely because all the essays refuse to bind the pandemic exclusively with the coronavirus and to position the virus and humanity in rigid opposition to each other. in her multispecies ethnography, anna tsing tells a marvelous story about matsutake mushrooms. "when hiroshima was destroyed by an atomic bomb in ," says she, "it is said, the first living thing to emerge from the blasted landscape was a matsutake mushroom" (tsing, , p. ) . when human history temporarily comes to a halt in disasters, matsutake, and also viruses in our case, may well survive and continue to thrive with their own stories. histories are being made every day by humans and non-humans alike; however, the future for those histories to converge has still yet to come. as demonstrated in the essays gathered here, while governments and the public are desperate to frame the virus in their own social and political narratives, the virus also works hard to inscribe its historicity on the earth and humans too. if a message must be dispatched out to all at this juncture, it is that for a future of collaborative survival, the stake of living together has nothing to do with harmony and conquest, but is derived from "disturbance-based ecologies" (tsing, , p. ) , that is, plagues. plagues are life, that is all. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. gong yuan https://orcid.org/ - - - xuefeng feng https://orcid.org/ - - - x notes . in a trilogy of contemporary essays, baudrillard ( ) argued that the war in the desert was a new phenomenon, because it was defined and shaped by its discursive aspect as a form of television programming, regardless of what happened on the ground. . that earlier announcement called "demonetisation" led to a vast contraction of commerce and an immiseration of a majority of the population which has still not recovered, and is now widely considered an unnecessary exercise of personal whimsy. for an analysis of the economic consequences, see ghosh et al. ( ) . . the decline of indian agriculture is not adequately discussed in the celebration of urban growth. its effects are however inescapable and directly lead to the growth of informal settlements in cities (balakrishnan, ; jaffrelot & thakker, ) . . mediatization, or the analysis of events with their mediated construction as the starting point, is a phenomenon that has grown in importance across cultural contexts, as media theorists attempt to understand the increasing influence of media forms on culture, especially with the virtualization of human interaction and the redefinition of community through the use of social media and mobile communication. for a fuller discussion, see couldry and hepp ( ) . discontents: an indian history ( ), jayal ( ) traces the current shift in the discourse of indian citizenship from an egalitarian rights-based model to a new regime predicated on religious and cultural identity, given shape through the concomitant technocratic frames of transactional welfare and biometric identity. . four short essays provide further context and narrate the response: shankar et al. ( ) . . partha chatterjee's ( chatterjee's ( , insightful categorization of indian society separates the distinct ontological domain of a small formal "civil society" that includes rights-bearing citizens, from the vast undifferentiated mass of the population that constitutes "political society" and which forms the actual locus of democratic practice. . the current indian government has dramatically increased the acquisition and use of big data in governance, including a reliance on biometric identification for access to welfare programs and the use of mobile phones for access to services: part of the government's covid- response was in the form of a mandatory mobile application that purported to use location tracking to show active virus infections in the user's vicinity. an analysis of its invasive nature can be found in a working paper by deb ( ). . societies always declared war on problems they cannot solve: war on drugs, war on poverty, and the like. it is no surprise to see donald trump refers to covid- as an "invisible enemy" and calls himself a war-time president in his speeches on the covid- pandemic. similar examples abound across the board throughout history. it is widely recognized by epidemiologists today that the model developed by john snow based on the cholera outbreaks between and in england is too linear to be of any use in contemporary pandemics. like the global climate instability or financial market volatility, pandemics are nonlinear phenomena, displaying a high degree of uncertainty that defies simple causal explanation. on this, see kosner ( ) . . it is therefore not surprising that we observe donald trump repeatedly refers to covid- as wuhan virus or kung flu in reaction to his rise and fall in poll and public opinion as he tries to find scapegoat, in this case, china, for his sorry failure in handling the crisis. a virus is always more than a virus when it enters the body politic. india's brutally uneven development patterns are mapped in routes migrant workers are taking home. scroll the gulf war did not take place bifo covid- : straight answers from top epidemiologist who predicted the pandemic the plague. vintage diseasescape: coping with coronavirus, mobility, and politics. global-e the politics of the governed: reflections on popular politics in most of the world lineages of political society: studies in postcolonial democracy cdc director says some coronavirus-related deaths have been found posthumously conceptualizing mediatization: contexts, traditions, arguments the covid- crisis in india: a nascent humanitarian tragedy. books & ideas public policy imperatives for contact tracing in india specters of marx: the state of the debt, the work of mourning and the new international coronavirus conspiracy theories targeting muslims spread in india. the guardian why coronavirus conspiracy theories flourish, and why it matters. the new york times the standard edition of the complete psychological works of sigmund freud as coronavirus spreads, so does racism and xenophobia covid- lockdown and the pandemic of caste demonetisation decoded: a critique of india's currency experiment chinese-japanese-s. korean relations evolve to meet the challenge of covid- . global times the world of work cannot and should not look the same after this crisis the companion species manifesto staying with the trouble: making kin in the chthulucene american hegemony and east asian order by revealing magnitude of migrant worker phenomenon, covid- points to rural distress. the indian express reconfiguring citizenship in contemporary india the coronavirus isn't alive. that's why it's so hard to kill sediments of time: on possible histories karl friston takes on the pandemic with the brain's arsenal high on talk, low on substance: modi's speech showed india is ill-prepared for covid. the caravan soulstealers: the chinese sorcery scare of the pasteurization of france we have never been modern agency at the time of the anthropocene migrant workers distrust a state that does not take them into account it was already dangerous to be muslim in india. then came the coronavirus india racked by greatest exodus since partition due to coronavirus. the guardian india: portents of an ending: modi, the rss and the rise of the hindu far right. the nation how covid- compares against other killer diseases in india why east beat west on covid- : east asia has handled and contained the pandemic far better than the west on nearly all metrics shame in the cybernetic fold: reading silvan tomkins the natural contract the demagogue and his labyrinth. the baffler coronavirus: the mystery of asymptomatic "silent spreaders posthumanism: anthropological insights mattering compositions labyrinth of linkages: cinema, anthropology, and the essayistic impulse coronavirus crisis threatens to split an already fractured eu the mushroom at the end of the world: on the possibility of life in capitalist ruins donald trump's "chinese virus south korea agree to continue push for free-trade deal despite ongoing tensions across region. south china morning post buddhist pilgrim-monks as agents of cultural and artistic transmission: the international buddhist art style in east asia, ca covid- can last several months the popularity of "rivers low, mountains high; the same moon in the sky" and its english translation the hindu rashtra comes of age. contending modernities li lu is an associate professor of literary theory at the school of chinese language and literature and a research associate at the center for literary theory at beijing normal university. he teaches courses on marxian aesthetics, critical theory, and translation theories.srinivas lankala teaches in the department of communication at the english and foreign languages university, hyderabad. he works in the areas of semiotics and critical theory, and engages with questions of politics, history, and nationalism. key: cord- -pqy bikp authors: hayes, adrian c.; jupp, james; tsuya, noriko o.; brandon, peter title: book reviews date: journal: j popul res (canberra) doi: . /bf sha: doc_id: cord_uid: pqy bikp nan the common picture of the health of populations around the world today is confusing. on the one hand, death rates in most developing countries have declined dramatically over the past years and people live longer; on the other hand, infectious diseases once thought to have been conquered are making a comeback and hiv/aids could become the worst epidemic in history. in this book mcmichael argues that the way to make sense of this paradox is to use a broad human ecological perspective on population health. he gives a masterful and engaging account of the 'long history of the changing patterns of human ecology and disease' from the pleistocene to the dawn of the twenty-first century. patterns of health and disease are examined as the product of ever-changing interactions between human biology and the social and physical environments. new patterns of disease emerging today may reflect the fact that humans are stressing ecological life support systems beyond the limits of their tolerance. in the space of this review i can only mention a few of the many topics mcmichael skilfully weaves into the story. biology establishes some fundamental parameters for population health. mcmichael's first three chapters discuss relevant aspects of human evolution. human and chimpanzee genuses diverged in africa - million years ago as the cooling of the pliocene produced an ecological niche 'for an ape able to survive mostly out of the forest' (p. ). then the enhanced cooling of the pleistocene (beginning about million years ago) led to rapid speciation of the homo genus. it is now generally agreed that a succession of homo species migrated out of africa, with extensive periods of co-existence among different homo species. homo sapiens and homo neanderthalis, for instance, shared much of western eurasia for thousands of years before the latter species became extinct around , years ago. since then homo sapiens has been the sole survivor of the homo genus -and, as mcmichael poignantly points out, if the current anthropogenic decline of species continues, it could become the sole representative of the whole great ape family. many distinctive characteristics of human biology were established as adaptations to environmental conditions of the pleistocene. as conditions changed, some of those adaptive responses became no longer so 'adaptive', and the resulting imbalances between biology and environment can manifest themselves as a tendency to disease. one example discussed by mcmichael is adaptations in insulin metabolism. insulin is an 'ancient vertebrate hormone' (p. ) that plays a vital role in the way the human body processes ingested carbohydrates and fats and converts them into energy for cell metabolism. the australopithecines (our pre-homo ancestors, including the celebrated 'lucy') appear to have been mostly vegetarian. as the tropical vegetation thinned during the global cooling of the pliocene, however, 'the survival benefit gained from supplementary high-quality meat protein would have increased ' (p. ) . certainly the hominines (species of the homo genus) emerging during 'the even cooler pleistocene … took to meat-scavenging and hunting … the product of an evolutionary branch that invested in cognition rather than mastication ' (pp. - ) . we would expect natural selection to have a corresponding effect on the way the body uses insulin. mcmichael, drawing on the work of j.v. neel and others, hypothesizes that 'meat-dependent hunter-gatherers may have reached the end of the pleistocene, around , years ago, with a reduced insulin sensitivity by comparison with their australopithecine ancestors ' (p. ) . this sets the stage for mcmichael to advance a 'provisional thesis' (p. ) to account for the prevalence pattern of 'type ii' (non-insulin-dependent) diabetes in the world today. the prevalence of type ii among major population groups varies by more than a factor of ten, and the variation is still striking even after controlling for obesity. europeans have low rates. assuming that reduced insulin sensitivity was the 'background' genotypic condition , years ago mcmichael speculates that a few neolithic populations might have diverged from this condition and developed a more insulin sensitive metabolism. moreover those few populations could well have included the proto-europeans, who were after all among the first to develop settled agriculture, implying a diet with more carbohydrates again. middle eastern farmers domesticated not only plants, but also goats, sheep, and later cattle. 'from around , - , years ago these agrarian communities began consuming milk and milk products. the result, today, is that genetically based tolerance of lactose (milk sugar) is much more prevalent in european than in most non-european populations' (p. ). genetic adaptation of populations to diets with differing glycaemic loads could help account (in conjunction with dietary changes and obesity levels) for their different levels of glucose intolerance and frank diabetes. mcmichael's line of reasoning takes more twists and turns than i can repeat here, and lack of direct confirmatory evidence means much of it remains speculative. as the late stephen jay gould ( : ) remarked, paleoanthropology has 'more minds at work than bones to study'. but his account shows how mismatches between biology and environment can affect population health. 'patterns of health and disease are the product of antecedent biological evolution interacting with current social and physicalenvironmental conditions' (p. ). other selection pressures he discusses, aside from food availability, are climate (leading to adaptations in skin pigmentation and the immune system) and infection (chapter includes a fascinating account of the co-evolution of parasite and host in the case of malaria). chapters - explore how a succession of changes in human ecology during the period of recorded history have affected patterns of health and disease by virtue of their impact on the co-evolutionary interplay between microbes and humans. this interplay is ubiquitous wherever there are human populations and is older than 'history' itself. when early humans became meat-eaters they exposed themselves to various animal parasites; when they migrated out of africa they encountered unfamiliar microbes. in these chapters, however, mcmichael emphasizes three broad historical transitions. the first corresponds to the rise of settled agriculture and the concentration of population in the early civilizations of the middle east, egypt, south asia, east asia and central and south america. this created a new web of relationships among animals, humans and microbes, facilitating the migration of microbes from animal to human populations. 'smallpox arose via a mutant pox virus from cattle. measles is thought to have come from the virus that causes dis-temper in dogs, leprosy from water buffalo, the common cold from horses, and so on' (p. ). most of the well-known infectious 'crowd' diseases appear to have developed during this transition; although the 'leap' from animal species to humans can still occur today, of course, as hiv and sars attest: writing before the recent sars outbreak mcmichael notes: 'in southern china, the intimate pig/duck farming culture creates a particularly efficient environment in which multiple strains of avian viruses infect pigs. the pigs act as "mixing vessels", yielding new recombinant-dna strains of virus which may then infect the pig-tending humans ' (pp. - ) . each of the ancient civilizations acquired its own repertoire of locally evolving infectious diseases. the second transition corresponds to the era of fluctuating contact among the eurasian civilizations through trade and warfare from around bc to ad . mcmichael builds on mcneill's ( ) thesis that this contact resulted in the transmission and swapping of microbes, leading to episodic epidemics followed by periods of gradual re-equilibration between the infectious agent and human host population. the complex aetiology of the black death in mid-fourteenth century europe, for example, seems to include an outbreak of bubonic plague in china during the s. 'after many turbulent centuries, this transcontinental pooling resulted in an uneasy eurasian equilibration of at least some of the major infectious diseases' (p. ). the third great historical transition refers to europe's exploration and conquest of distant lands, and (using language reminiscent of diamond ) the export of 'its lethal, empire-winning, germs to the americas and later to the south pacific, australia and africa' (p. ). the large aztec and inca populations had no herds of wild animals they could domesticate and remained relatively free of 'crowd' infectious diseases until they were decimated by smallpox, measles and influenza introduced by europeans. falciparum malaria and yellow fever were brought to the americas in the seventeenth century by the trans-atlantic slave-trade. in similar fashion the aborigines of australia suffered deadly epidemics following the arrival of europeans on that continent. and within years of captain cook's first visit to hawaii in the native population declined from around , to less than , . mcmichael points out that the third transition was more a 'dissemination' of microbes that had co-evolved with the eurasian population to other parts of the world than an 'exchange'; and he is sceptical that syphilis was really introduced to europeans from the amerindians, as is often claimed. in any event the third transition represents another major process re-equilibrating the balance between microbes and humans, this time across transoceanic populations. are we experiencing a fourth major historical transition today? around many experts thought that tuberculosis, cholera and malaria would soon be conquered; it was time 'to close the book on infectious diseases', declared the us surgeon-general (p. ). but now these diseases are increasing again, and a host of other diseases or their pathogens have been newly identified; they include lyme disease, hepatitis c and e, human herpes viruses and , ebola virus and legionnaires' disease. something 'unusual' seems to be happening to patterns of infectious diseases (p. ). to explain this mcmichael suggests we must look at contemporary human-induced social-environmental changes which provide new opportunities for microbes to invade and colonize the human body: worldwide urbanization, intravenous drug use, changing sexual practices, changes in medical practice (blood transfusion, organ transplants), intensive food production, poverty and inequality, irrigation, deforestation, eutrophication of rivers, and so on. in the last three chapters mcmichael draws together these themes in a more prospective view, detailing the risks to population health inherent in the way we are currently 'depleting or disrupting many of the ecological and geophysical systems that provide lifesupport' (p. ). chapter includes a discussion of the health impact of climate change. as in his earlier work ( ) mcmichael adds his voice, with eloquence and authority, to the imperative of establishing more sustainable ways of living. mcmichael's book is impressive in the way it marshals research findings from diverse fields, and even more important for the way it clarifies our contemporary situation. however, in telling the story about population health from a social ecological perspective his narrative is ahead of his analytics. he criticizes the conventional epidemiological approach (taking the individual as the unit of analysis) for its reductionism, but gives no systematic exposition of his own more holistic conceptual framework detailing precisely how it is 'some important health-determining factors operate essentially at the population level' (p. ). similarly his conceptualization of anthropogenic environmental change (building on peter vitousek and others) is pretty all-embracing and not yet structured to help us focus on those parts or processes most relevant for population health. the persuasive force of this book comes more from the weight of its examples of social-ecological processes influencing patterns of health and disease in populations than from any systematic theory describing these relationships. mcmichael is clearly aware of these limitations; we hope he can address them in future work. a more explicit analytics will facilitate a more felicitous telling of the story. experts and advanced students from any number of population-and healthrelated disciplines will find this book useful and stimulating. demographers will especially like the way it clearly positions 'population' centre stage; and the book's central thesis is certainly a boost to those of us who like to see demography in a strategic position at the intersection of the human and natural sciences. for those working in family planning, there is a challenging section on the health consequences of modern reproductive interventions ( pp. - ). institutions which have supported this fine work deserve some credit too. mcmichael was professor of epidemiology at the london school of hygiene and tropical medicine when he wrote this book; he is now with the national centre for epidemiology and population health (nceph) at the australian national university. the disciplinary structure of modern universities is not always conducive to bold synthetic research like mcmichael's. this book shows how important it can be: in fact the future health of human populations could well depend on it. the basic proposition of this interesting and timely book is that 'irregular migration occurs because states make rules about who can legally cross their borders. under conditions of globalization, these rules promote transnational economic activity, but limit who can work and stay ' (p. ) . in other words, there is a contradiction between increasing globalization and a declining willingness to accept mobility of labour as opposed to capital and goods. this contradiction is especially apparent in the european union and the united kingdom, with which much of this book is concerned. britain, france and the netherlands had a long tradition of free entry for imperial subjects, which they eventually found politically embarrassing. they now have the practice of free entry for european union subjects, which they may also find embarrassing as the eu extends to eastern europe and even turkey. with its focus on britain, this study sees irregulars as mainly those who arrive through legal means but then work, which they are forbidden to do. peoplesmuggling, which has become so controversial, is not central to their analysis. because the majority entering britain do not need visas, it is easy to get in. because there is no national identity card, it is easier to work illegally than in most european states. serious criminal activity is a matter for the police, who are relatively indifferent to breaches of immigration rules. the basic problem remains that britain, like several other european states, does not really have an immigration program. those who wish to remain permanently and to become citizens must either be closely related to those already there, or make a successful asylum application, or gain access to the very restricted working-visa or business-visa system. only recently, and since the publication of this study, has the government moved towards developing a coherent system allowing a quota of permanent residents; it has done so in response to widespread hysteria, worked up by the populist media, against asylum seekers. one weakness of this study is that it concentrates on economic motivations; yet the recent surge of asylum seekers, as in australia, includes many who are genuinely fleeing wars in afghanistan and iraq. the british situation is made more acute by many from collapsing african states, or balkan romanies, making well-based claims of ethnic persecution. these latter will probably have full access to britain once their states have joined the european union. at the core of this study is an analysis in depth of irregular migrants living and working in london. the groups chosen are brazilians, who have no strong historic connections with england; poles, who have stronger links; and kurdish turks, the only ones who can make plausible claims to be asylum seekers. other than some of the kurds -who are highly politicized -the irregulars simply come to make more money than at home, have no real desire to settle permanently and are ready to work for low wages and below their level of qualification. one interesting feature of these irregulars, which may well apply generally, is that they are young and well educated. they may come from poor countries but they are not peasants or labourers. what little we know about asylum seekers suggests that they are also well educated and skilled. the difference is that they have few places left to go, whereas many of the openly 'economic irregulars' have every intention of returning home in due course. because they are relatively well-qualified, many of those studied here have access to the variety of non-government organizations which have sprung up to help them and represent them to the authorities. they are certainly not voiceless even if inclined to avoid direct contact with government for fear of deportation. because london has a long history of immigrant labour in service industries, there does not seem to be much concern with the present situation. as host to millions of tourists it needs a mobile labour force, and it does not have a serious unemployment problem except in some declining enclaves in the east end. many of those interviewed have worked illegally for years and have rarely encountered any attempt to discourage or remove them. there is a contrast between this liberal approach to casual labour and the much stricter attempts to discourage permanent settlement. however, the flood of asylum seekers in recent years is changing this. the book's publication was too early to account for the recent moves in britain towards a regime reminiscent of that in place in australia. as presented here the british authorities do not really mind people coming to work in low-wage industries because they do not expect them to settle. london is now so cosmopolitan that racial tensions are less politically significant than in some smaller cities which have recently experienced violent reactions. as is regrettably common in texts published in europe and north america, there are only passing references to australia. this reflects our isolation, which also makes the 'dilemmas' of irregular migration less pressing than for states with long land boundaries or short sea crossings. however, australia does have a complex, planned and longstanding immigration program, with some principles and practices extending back for over a century. this is in contrast to the united kingdom situation studied at the heart of the book. some issues which are of interest to australian scholars and addressed by this book include: will irregular immigration increase despite increasingly rigorous attempts to prevent it? does the admission of large numbers of students, tourists and working-holiday temporary migrants make this more likely to happen? is flexibility needed in assessing asylum seekers as against 'economic migrants'? how can immigration policy be insulated against populist agitations and political expedience? can sydney hope to be a 'world city' without a degree of 'irregular migration' to supply transient labour in services? does our expanding relationship with the island states of the south pacific make regularization of immigration on a more generous basis more likely? does it really matter if there are thousands of 'irregular migrants' working in the economy? how can people-smuggling, trafficking in women, drug-smuggling and terrorism best be controlled without draconian restrictions on legitimate movement? this is an interesting and useful book. its tendency to see all movement as essentially motivated by economic inequality is of prime importance, even if that is only part of the story. globalization without liberal migration regimes, on this view, could be oppressive. but are the rich societies ready to modify this? this study ends on the note that 'globalisation has consisted in the development of the world economy under conditions most favourable for capital and the first world countries, and under terms that discriminated against the developing countries in the liberalisation of trade. … for these reasons, it is doubtful whether an international regime for managing migration, would be a step towards international justice' (p. ). australian national university this volume offers anthropological accounts of the evolution, production, and consumption of various social policies in contemporary japan. consisting of nine chapters, it is a collection of papers prepared originally for an international conference. chapter , by goodman, discusses the evolution of applied anthropology, and explains what anthropology can offer to the studies of social policy. the anthropology of social policy is not only the study of meanings ascribed to slogans and symbols associated with the policy but also that of its production and consumption. as goodman argues, social policies not only emerge from a particular socio-cultural context, but they also provide important clues to how social values are constructed and altered. chapter , by bestor, attempts to sketch a 'cultural biography' of civil society in contemporary japan. it first traces the postwar development of the notion of civil society; it next examines the multiple uses of related terms such as civil society, volunteerism, npo, and ngo; and then provides an example of the development of civil society by describing a sudden rise in volunteer activities and the enactment of the npo law following the hanshin earthquake in . instead of devoting many pages to explanations of the meanings of different terms, the chapter would have been more interesting if it were focused more on the processes that gave rise to volunteerism and the ensuing legislation by illustrating the interaction between volunteer activities and the policy responses. in chapter , roberts analyses the problems associated with low fertility, by examining programs within the angel plan -the comprehensive policy to increase the rapidly falling birthrate -and public discourse associated with its implementation. roberts demonstrates that the policy initiatives are not consistent with, therefore not effective in altering, the prevailing gender division of labour. by identifying some of the voices influencing the production and implementation of the policy, she also illustrates the nature of changing social norms and the roles of policy in bringing about such changes. although some demographic facts are misrepresented, the chapter nevertheless offers vivid information regarding how the plan is produced and consumed, therefore furthering our understanding of the importance and difficulties of balancing work and family in contemporary japanese society. in chapter , stevens and lee analyse how two sets of government policies, those of the ministry of health and welfare and the others of the ministry of justice, influence the provision of maternal and child healthcare for foreigners in japan. they argue that legal and cultural stresses, exerted by the way the policies determine the eligibility and provisional guidance for healthcare, make it difficult for foreign women to have the care they need, and that in turn leads to higher-risk pregnancies. public policies and laws are influenced by the perception of ethnicity, and the lack of policies to deal specifically with health problems of foreign mothers suggests a dual reality of health and welfare in different segments of the population. however, their arguments are not fully supported by empirical evidence, according to which the mothers who suffer higher health risks are those from developing countries in southeast asia whereas the risks for other foreign mothers are similar to, or significantly lower than, those for japanese mothers. providing that foreigners comprise vastly different groups, it seems necessary to account for their differences in socio-economic status, demographic features, and legal status before we determine whether foreign mothers indeed suffer higher health risks. chapter , by ben-ari, examines the interrelation between the organizational features of japanese preschools and the way by which they are predicated on notions of normal development of children. based on participant observations, visits to childcare institutions, and secondary analyses of administrative documents used by these institutions, ben-ari shows how organizational arrangements of institutions of early childhood education are related to the ways that children are socialized according to a uniform set of ideas that the state deems normal and ideal. this chapter is, like chapter , an example of practical anthropology at its best. it offers useful and interesting information based on two primary methods of applied anthropology: field work and content analysis of documents. in chapter , goodman analyses the 'discovery' of child abuse in japan and the development of social policy to deal with the problem. his view is that the upsurge of reported cases of child abuse in the s was largely a result of the campaigns by the media and child-welfare institutions. mothers are found to be the main abusers of children, and these groups viewed the increasing child abuse as a consequence of nuclearization of the family, combined with the prevalent myth of motherhood. goodman shows that through the implementations of regulations and policies, this discovery altered the relationship between the state and parents by giving greater powers to authorities to intervene in what had previously been seen as the exclusive and private domain of the family. chapter , by thang, offers an overview of programs to promote interaction between the elderly and school-aged children and teenagers in japan. through an attempt to interpret intergenerational interactions in the context of the japanesestyle welfare society, thang seeks to show the needs for and significance of intergenerational interaction programs. although we cannot deny the need for programs to promote intergenerational interactions, i cannot help feeling that such programs are supplementary in an array of policies designed to support and care for the elderly. given the rapidity of population ageing and the pervasiveness of its socio-economic, demographic, and political consequences, more attention should be paid to how these programs are related to mainstream elderly policies such as the golden plan and the long-term care insurance, and to how these policies and programs together alter the perceptions and treatment of the elderly in contemporary japan. in chapter , tsuji examines the evolution of death-related policies in japan over the last years. tsuji shows that mortuary practice is shaped by interplay among policies, individual actors, and traditions, being conditioned by wider socio-economic and demographic changes. though it is a little too long on explanations of prewar development, i found the chapter interesting and informative, illustrating skilfully that since meiji the government has used death policies to exert control over the family and individuals, and that mortuary practices have changed in recent decades in the face of constraints driven by low fertility. chapter , by mackie, discusses some of the ways in which social policy affects individuals differently. it argues that the archetypal citizen in the contemporary japanese political system is a male, heterosexual, able-bodied, fertile, white-collar worker. this in turn suggests that our understanding of citizenship can be broadened if we view citizens as embodied individuals whose positions are situated somewhere in a spectrum, shaped and constrained by legal and institutional structures. while the typology of citizens provided by this chapter is useful in understanding how citizenship is defined by the society, it falls short of explaining how changes in social policy are related to changes in the notion of citizenship in contemporary japan. there are general weaknesses in the volume, stemming largely from the very nature of the enterprise. in all, however, the volume provides useful insights into how problems and policy issues shared by many industrialized countries are tackled in japan. thus it will be of interest not only to japan specialists but also to those who are interested in social policy in industrialized societies in general. is an ambitious book. the first book in a series on the australian economy and society, it provides informative analyses about education, work, and welfare in australia over the last decades of the twentieth century. this important work on contemporary australia creatively presents data on shared and divergent attitudes among australians, social trends over time, comparisons among social groups, and comparisons with other nations. the book is distinctive in using large, representative samples of australians and persons from other nations to make conclusions and inferences. three principal sources of data are used: the international social science surveys/australia, the international social survey program, and the international survey of economic attitudes. combined, the three surveys yield a sample numbering over , individuals. the surveys are nationally representative and, depending upon the topic under investigation, permit the authors to make generalizations to the populations as a whole. with the improvements in measurement theory and survey questionnaire design, the authors can provide authoritative statements about people's attitudes and behaviours within countries and compare australians' attitudes and behaviours to those of persons in other nations. with some exceptions, the nations with which australia is most often compared are english-speaking or european. two chapters that provide interesting applications of the comparative analysis approach using survey data are chapter , 'conflict between the unemployed and workers in nations' and chapter , 'participation in the labour force'. the tabulations in each chapter develop the authors' stories as eloquently as the accompanying narratives. furthermore, the large amounts of data collected from the surveys permit the authors to exploit more advanced multivariate techniques. throughout the book, various multivariate modelling strategies are used to better understand relationships between such topics as education and earnings; job complexity and earnings; upbringing and attitudes about trade unions; and risky lifestyles and ideals about societal responsibility for diseases. obviously, in the interests of science and effective policy development, debate should surround the methods used and the conclusions reached. notwithstanding the need for such debate, the book carefully documents its methods and justifies the statistical approaches taken. readers concerned about empirical social science will welcome the multivariate models featured in the book, especially since most of its predecessors, which aimed for the same grand scale and scope, often omitted multivariate models. that omission was rarely made by choice, but was usually due to data constraints. attention to methods, creative use of surveys, and strategically placing findings in national and international contexts produce some fascinating findings in the book. some of the more notable findings include workers' desires for job security, even if it means less pay; stronger support for trade unionism among public service employees than among blue-collar workers; australians' desires to see the influence of unions no further weakened after many years of sustained government efforts to do so; a tension between australians' feeling sorry for their fellow australians who have suffered from diseases as outcomes of risky health behaviours on the one hand, yet also feeling one must take responsibility for one's own life-style choices on the other hand. and, lastly, a finding that australians do more complex jobs than their counterparts in five european nations, but that complexity is not the most important determinant of salaries. rather, the apparent determinants of australian salaries are status and maleness. the book accomplishes its goal of clearly and concisely presenting its findings and interpreting what they mean for contemporary australian life. topic vi, 'retirement', which contains three chapters, is a good example of the clarity and compactness of the writing style and presentation of facts. however, readers may sometimes find the book too dense and the huge number of graphs, figures, statistics, replicated survey questions, and technical notes distracting instead of illuminating. perhaps the commendable effort to document rigorous scientific analyses is occasionally counterproductive. overall, the work is essential reading and it offers researchers, policymakers, and others concerned about australia's future a myriad of social and economic data in one source. if this book is indicative of future volumes in the series, then the series will have great utility. guns, germs and steel: the fates of human societies planetary overload: global environmental change and the health of the human species plagues and people key: cord- -w cg b authors: nan title: spotlight on osaka date: - - journal: nature doi: . /nj sha: doc_id: cord_uid: w cg b nan spotlight on osaka n orthern osaka is rapidly becoming one of the world's most successful biomedical clusters through active collaboration and the pursuit of research that is attractive to young scientists, says renowned immunologist tadamitsu kishimoto. following approvals in japan last year, japan's first antibody drug, tocilizumab (sold as actemra in japan), this year also received government approval in europe for its application to be extended as an immunosuppressive agent. it can now be used to treat rheumatoid arthritis and articular inflammation in addition to the rare castleman's disease. the drug is a humanized monoclonal antibody that inhibits the receptor of interleukin (il)- , a protein known for its involvement in immune and inflammatory responses. tocilizumab is now expected to become a highly popular treatment for rheumatoid arthritis. the development of tocilizumab is perhaps the best example reflecting the underlying strengths of medicinal and biological research and the businessoriented mindset in northern osaka, the site of one of asia's largest biomedical clusters. soon after il- was discovered by immunologist tadamitsu kishimoto at osaka university in the mid- s, it was swiftly combined with the technological prowess of a pharmaceutical company. their persistent work led to the successful market introduction of the drug two decades later. "osaka embraces a culture that allows unconventional thinking, which has led to a bloom in numerous achievements of translational research. and it still does, " says kishimoto, a professor of the graduate school of frontier biosciences at osaka university. "in the early s, for example, many people thought small molecules would be the mainstream of drug development, but we looked to large molecules like antibodies, " says the native of osaka who is also one of the university's former presidents. northern osaka has been a centre of life sciences research dating back to the early nineteenth century, when the predecessor of osaka university's faculty of medicine -the school known as tekijuku -was first established. the university has since become a global frontrunner in research on proteins, microbiology and immunology. the region's pharmaceutical tradition in fact started much earlier: as early as the seventeenth century, a district known as doshomachi began to flourish with the arrival of hundreds of merchandisers from around japan to participate in the burgeoning pharmaceutical trade. the merchants included the predecessors of takeda pharmaceutical and other major modern companies. despite the recent challenging conditions of the global economy, the biomedical cluster in northern osaka has progressed largely unimpeded thanks to government-sponsored economic stimulus packages, which have greatly increased financial support for promoting those research fields in which japan has been particularly competitive. the northern osaka cluster is also accelerating its unified efforts towards translating research achievements into practical uses, kishimoto says. the need for a practical focus was recognized as far back as , when the senri life science foundation was first established. the foundation provides research grants, organizes seminars and supported the creation of the biomedical cluster. the saito life science park was opened in as the core of the research cluster. the osaka prefectural government also established the osaka bio headquarters in the cluster in to coordinate various activities and facilitate progress on life sciences strategies. kishimoto now heads the foundation and the bio headquarters, among his other public roles. many of the significant advances at the biomedical cluster are the products of impressive work by young researchers, kishimoto says. as an example, naoki hosen at the graduate school of medicine at osaka university, supported by the foundation through the japanese government's knowledge cluster initiative ( nd stage), is developing therapeutic antibodies targeting antigens specific to cancer stem cells. in another example, ken ishii at the university's research institute for microbial diseases is working on an influenza vaccine adjuvant that is activated by innate immune signalling pathways. "the most important thing is not money, but the fostering of the next generation of researchers, " kishimoto says. "our biomedical cluster can provide young researchers with a promising career pathway if they come and work here for a few years. " just kilometres from downtown osaka, the northern osaka biomedical cluster is a concentration of the world's foremost academics and japan's leading pharmaceutical companies. the well-balanced combination of academics and industry is key to driving product innovation and research success through active collaborations. the cluster centres around the saito life science park. several of japan's top biomedical research institutes are located within five kilometres of the park, including the suita campus of osaka university, the osaka university hospital, the national institute of biomedical innovation (nibio), the national cardiovascular center (ncvc), the osaka bioscience institute and the senri life sciences center. the tradition of modern academic research in osaka is derived from tekijuku, a school that taught western studies and medicine in the nineteenth century and which became the predecessor of osaka university's faculty of medicine. many of the scientific activities undertaken in the biomedical cluster are highly evaluated internationally. the institutes in northern osaka play a critical role in a number of large-scale national projects. for example, last year the japanese government included four projects from osaka university, nibio and the ncvc in its five-year super-tokku initiative. the program provides a platform for the swift evaluation of pharmaceuticals and medical equipment through the flexible use of public funding in order to accelerate the research and development of advanced medicine and medical equipment in japan. the cluster lies south of saito and encompasses the district of dosho-machi, where more than pharmaceutical companies have been established. five of japan's top ten pharmaceutical companies are headquartered in this district and neighbouring areas. osaka is also known as a home for electronics companies such as panasonic and sharp, as well as a number of manufacturing companies involved in the development of advanced medical equipment. these companies receive dedicated support from the osaka pharmaceutical manufacturers association and the osaka medical instruments association. in the biomedical cluster, industry, academics and policymakers coordinate effectively to promote biomedical activities. the senri life science foundation plays a key role in these activities, and also takes charge of the regional administration of the japanese government's knowledge cluster initiative ( nd stage). in , the osaka prefectural government commissioned the establishment of the osaka bio headquarters within the cluster to coordinate industry-academic collaborations more effectively. in its most recent move, the bio headquarters in collaboration with some of the top brains in osaka developed the biomedical strategy , which set the goal of making osaka the world's fifth most influential biomedical city over the next decade. to this end, osaka is ready to increase support for venture companies, accelerate deregulation and speed up clinical trials. saito life science park -leaping ahead how to better join forces s et among the green hills of northern ibaraki city and southern minoh city in osaka, the saito life science park has established itself as a leading hub of life sciences research in japan. the -hectare site currently hosts six major research institutes, including nibio, as well as three commercialization incubator facilities with capacity to support start-ups and laboratories. occupants are eligible for a range of publically funded subsidies and tax incentives. more than researchers and staff working in the park benefit from its close location to osaka university and the ncvc, providing a basis for close research collaboration. anges mg is one of the innovative biopharmaceutical companies headquartered at the saito bio-incubator, and is currently developing two gene therapy medicines as mainstay projects. another company, soiken, is developing various biomarkers and assay systems as well as implementing clinical trials for the development of drugs and functional food. both companies are using research outcomes from osaka university and are recognized as frontrunners in japan's biotech industry. the bio-incubator is not restricted solely to japanese companies: it also houses a japanese subsidiary of san diego-based anticancer. the life science park opened in as the first part of osaka's large-scale urban redevelopment plan to create the international culture park, an area that has been nicknamed saito. next year, development will commence in the central area, which will also focus on life sciences research and the innovation industry, with completion scheduled for fiscal . the life science park offers not only a superb research environment, but also convenience -it is located about minutes by train from downtown osaka. the grand opening of the park in was marked by the inauguration of an extension of osaka's monorail service to the area. m atchmaking is a common practice undertaken at biotech events worldwide to introduce laboratory seeds to companies -but the effectiveness of such introductions for creating real outcomes is often questioned. the osaka chamber of commerce and industry (occi) has proved to be one of the few successful coordinators of such introductions. since , the occi has organized the forum for the industrialization of next-generation medical systems, an open platform for the joint development of medical instruments for clinical applications. every month, doctors and scientists make proposals for collaborations based on their ongoing research. if companies participating in the forum become interested in a proposal, the occi sets up working groups or individual meetings to promote collaboration. so far, working groups have been established, covering % of presenters, and a quarter of the presenters have subsequently gone on to launch a joint development project in collaboration with a commercialization company. in one example, a laboratory of fumio miyazaki at osaka university and the company daiken medical are currently working to develop a robot capable of holding and manipulating an endoscope during surgery, which could one day replace a human assistant. the forum is the most successful activity in the industry in japan and has become so popular that there is now a waiting list for making presentations. behind the high success rate of the forum is the comprehensive hands-on support of the occi's coordinators and administrative staff, who assist in many ways, from advising presentation topics, to offering follow-up support before and after the forum. participation in the forum is not limited to osaka or the greater kansai area, and the number of participants has been on the rise, now totalling research organizations and companies. the occi is looking to partner with overseas clusters and biomedical associations, and the first such move was made recently with the formation of the biobusiness alliance of minnesota. in osaka, globally recognized institutes like the ncvc are situated beside the accumulation of medicalrelated companies. by setting research institutes and medical companies as centripetal forces, the occi continues to contribute matchmaking capabilities to create a globally competitive industrial cluster in osaka. osaka's collaboration focus has led to the creation of one of the more ambitious projects to come out of the biomedical cluster -the pharmaceutical innovation value chain, which is run by the non-profit organization biogrid center kansai in osaka. the value chain allows stakeholders in any stage of drug development to participate in the creation of innovative drugs. the latest innovation to emerge from the biomedical cluster is the protein mall kansai, which was inaugurated in may this year. the protein mall acts like a catalyst: it searches for member and non-member companies interested in collaboration, and shares information among them to spur interaction. it also provides expert advice on protein-related business and potential collaboration. all the services supporting participation are provided free of charge. the protein mall is at present considering supporting technology for a protein synthesis developed by the institute for protein research at osaka university, and for a process of human protein production using vegetable chloroplasts developed by a collaboration between the nara institute of science and technology and the institute for virus research, kyoto university. "a unique protein production method and a unique protein itself would also be interesting to members, " says katsube. whereas some institutions are preoccupied with history and traditions, osaka university, or handai as it is colloquially known, prefers to talk about the future and seems to take delight in a tradition of being ahead of the times. 'live locally, grow globally' is the kind of motto you'd expect from osaka university. tracing its roots back to the tekijuku school established by the legendary pioneer of western medicine koan ogata in , osaka imperial university was formally established in as the sixth of japan's seven imperial universities and the only one to be built largely with private funds. from this start, the university has grown rapidly into a world-class educational institution of over , students and staff with formal links to universities in countries around the world. these unique beginnings -a combination of longstanding commercial roots and an early pedigree in medicine -have produced a seat of learning that thrives on continuous development, expansion and amalgamation with other local institutions, most recently with the osaka university of foreign studies in . the university was an early adopter of an interdisciplinary approach to learning when it created the school of human sciences in . they also recognized before most the importance of academic and industry partnerships and established the institute of scientific and industrial research way back in at a time when conventional wisdom viewed such liaisons with suspicion. now in the sixth year of its latest mediumterm plan, the university is looking to the next phase of development in its research capabilities under three themes stressing 'fundamentals' , 'curiosity' and 'responsibility' . and as one might expect, cooperative research with industrial partners features heavily. this translates into the concept of 'industry on campus' , which will see parts of the largest of the three sites of osaka university redeveloped as a cluster of high-tech facilities including the nanotech incubator, the technological alliance institute and a centre devoted to imaging, photonics and photovoltaics research, all in time for the university's th anniversary in . these facilities will provide an environment where academia and industry can work together on basic research as well as projects of commercial interest. whilst key initiatives are being implemented in high-tech science and technology, it is probably in the field of medicine more than any other that the strength of osaka university is most keenly felt. from its inception, the school of medicine has been a key driver in the growth of the university as a whole, as well as being at the forefront of many important developments in japanese clinical medicine and basic medical research. early on it established a reputation as a centre of excellence in the treatment of infectious diseases, notably for smallpox and tuberculosis, in the process becoming the site for the first popularization of smallpox vaccinations and bcg inoculations in japan. the osaka university school of medicine subsequently became known as the leading institute for transplant surgery in japan, beginning with the first kidney live locally, grow globally whilst noting strength in organ transplantation, hirano is quick to point to other areas at which the university excels -immunotherapy and translational research aimed at bringing the potential benefits of treatments such as cancer vaccines and stem cell therapies out of the lab and into the clinic. the university also boasts state-of-the-art medical imaging resources including a magnetic resonance imaging facility powered by an . tesla magnet -currently the world's largest -as well as a number of advanced positron emission tomography sources allowing lab animal experiments to be performed in real time. hirano is convinced that the university's achievements spring from a solid base of all-round excellence in general medicine and basic research. however, maintaining this strong foundation is not without its problems. "these days fewer and fewer medical students are going into basic medical research after graduation, " observes hirano, himself an osaka university phd, with regret, "and the result is a shortage of talent in those areas. " to tackle this problem, the school implemented a number of changes to the undergraduate curriculum to make it easier for students to gain experience in areas of medical-related research outside the school, and also created an elective course allowing students to carry out medical research for a few hours each week outside of normal class times. introduced in april of this year, the 'after five' option already has a healthy take-up rate, and enquiries from prospective future students for the course in future years are growing steadily. it is a typically forward-looking, flexible approach to a challenging problem. but that's the way they like to do things at handai. in , the japanese government announced the recipients of its world premier international (wpi) research center initiative program. the wpi program focuses government financial support on five priority projects to create elite research institutes that attract the world's top scientists. the ifrec at osaka university is one such institute, selected for its mission to "unveil the whole picture of the dynamic immune system" using a systems biology approach. immunology has long been one of osaka university's greatest scientific strengths, beginning with former presidents yuichi yamamura and tadamitsu kishimoto and now exemplified by the director of the ifrec, shizuo akira, who is a pioneer of innate immunity and currently the world's most cited scientist in the field. to take immunology to its next level, the ifrec seeks to move beyond the test tube where immune cells work in isolation and observe these cells live in the body, leading to a better understanding of the dynamics driving immune cell communication and cooperation. akira explains, "in vitro experiments only allow snapshots of immune cells. we want to capture the movement of cells dynamically. therefore, we need to develop new research techniques to understand immune responses in the body. " this means the ifrec will be the leader of a new systems immunology field that combines immunology, imaging and informatics. in addition to gathering several of the world's top immunologists, the ifrec has assembled an extraordinary imaging team led by one of the world's leading researchers in bio-imaging, toshio yanagida. the large number of high-impact publications produced by akira and yanagida and their impressive list of accomplishments and awards made the ifrec an obvious choice for the japanese government when it considered where to commit at least million yen annually for ten years or more. "our selection for one of these highly competitive programs was recognition of our research and the potential to achieve the goals of this challenging project, " says akira. just assembling a multidisciplinary team with international standing, however, is not enough. in order to accomplish its stated goals, the ifrec is also changing the way scientific research is undertaken in japan. this means more independence for young principal investigators, and emphasis on collaborative efforts -an absolute necessity for bringing together disparate fields like immunology and imaging, and researchers with different educational and cultural backgrounds. as akira notes, "we have around people involved in this program, % of whom are from overseas, including % of the principal investigators, with backgrounds ranging from engineering and physics to medicine. " one of the more recent principal investigators to join the ifrec is diego miranda-saavedra, who previously worked on developing bioinformatics techniques to study transcriptional networks in blood cell development. "i felt like i could accomplish more in ten years here than i could anywhere else. i saw that the ifrec is made up of a very enthusiastic and collaborative community of outstanding scientists, and that the science done here is not only world-class, but also worldleading. i quickly understood that the ifrec's goal is to answer very important and difficult questions in immunity and that this is achievable with the amazing setup here. my lab at the ifrec consists of a bioinformatics core with access to supercomputing facilities and wet bench space to validate our in silico predictions. " atsushi kumanogoh was attracted to the centre by its strong emphasis on imaging, which he believes will significantly advance his team's work on semaphorins and their role in the cross-talk between the nervous system and the immune system. having already isolated the first semaphorin (sema d) by subtractive cdna cloning in , kumanogoh has seen his research benefit from the imaging facilities at the ifrec. "our images offer a deeper insight into how semaphorins regulate cellular migration in the nervous and immune systems, " kumanogoh says. it is this unique environment that allows the ifrec to pursue its ambitious projects. "at present, we are not able to predict the outcome when a certain pathogen invades the body, " explains akira. ultimately, the ifrec seeks to do just that by visualizing how the body reacts when a pathogen invades. "by integrating immunology and imaging, we seek to understand the dynamic interactions of immune cells and their activation. this will allow us to manipulate the immune system, leading to the development of vaccines. " whole body imaging of the immune system a chimeric mouse generated from embryonic mouse cells and genetically modified embryonic stem cells. such mice are essential for generating knockout mice -important tools for immunology studies at the ifrec. advertiser retains sole responsibility for content |naturejobs| september such advances rely on allowing researchers to focus on their science. "a genuine international environment is critical for this world-class centre, " says takao kodama, administrative director of the ifrec. this means not only aiding researchers in their grant applications and other matters that relate to their science, but also facilitating a smooth transition for them and their families when they arrive to live in japan. this is one reason why cevayir coban brought her family to osaka to work at the ifrec, where she now leads a project investigating innate immune responses to malaria. "malaria affects millions of people each year and kills mostly children and pregnant women, yet no vaccine is available to prevent it. " almost immediately, she was able to form collaborative projects with imaging and informatics groups to create a new generation of drugs and vaccines to combat malaria. while immunology is at the forefront of this project, yanagida sees other significant benefits. "i am interested in the dynamics of living cells, " says yanagida, who as an engineer takes a strong interest in the design of the immune system. "an immune cell is an amazingly efficient machine superior to anything we can engineer. unravelling how living cells handle large amounts of information and function in tandem has the potential to allow us to design machines that use far less energy. " it is this component of the ifrec that should appeal to researchers without a direct background in immunology. "for me, the immune system is a model to better understand how cells function and how to build better machines, " adds yanagida. "the ifrec may be an immunology centre, but that most certainly does not mean it should only consist of immunologists. in fact, if we are to achieve our stated goals, then it can't. " the imaging team is aggressively applying new combinations of various imaging and labelling techniques, like multiphoton-excited fluorescent microscopy and nano-environmentsensitive probes, to conduct single-molecule imaging, nanometry tracking and magnetic resonance imaging of immunological systems. this gives physicists and engineers an unusually direct role in systems-based immunology research. this also applies to bioinformatics. "it doesn't matter if you have been working in another computational field. if you're at the top at what you do, then you can make a positive contribution here, " says daron standley, leader of the structural bioinformatics lab. "my team consists of people from backgrounds in bio-engineering, computer science and even financial modelling. when recruiting someone, we're not concerned with what you did at your last job. we are more concerned with finding smart people who have a strong desire to work with a variety of scientists in other fields in order to develop a more complete view of immune function. " it is this notion of building a collaborative immunology centre, made up of top scientists with diverse backgrounds, that will help the ifrec to attain its ambitious goals. through state-of-the-art facilities, guaranteed investment and an exceptional group of scientists, the ifrec is creating the necessary environment to take immunology to a new level. in vivo two-photon microscopy of bone tissue. microscopic blood vessels in the bone marrow of a transgenic mouse visualized by masaru ishii's group at the ifrec. - yamadaoka, suita, osaka - , japan the rimd was set up in after a donation from gendo yamaguchi, an osaka merchant. "we conduct research on infectious diseases, immunology and cell biology, " says hitoshi kikutani, director of the institute. "we also have a brother institute at osaka university, the research foundation for microbial diseases, known as biken. it is japan's largest manufacturer of vaccines and a major exporter as well. biken is currently developing a vaccine to fight the h n pandemic flu virus. " profits from the sales of vaccines are used to fund research at the rimd. "we accept graduate students for multidisciplinary research on microbiology, oncology and molecular biology, " says kikutani. "we have more than research staff here. " rimd researchers have made major breakthroughs, such as the discovery of cell fusion, which led to the development of monoclonal anti-bodies -important tools in biochemistry, molecular biology and medicine. the rimd's most notable research achievements include the discovery of vibrio parahaemolyticus, a bacterium that causes food poisoning, and the development of a vaccine for varicella (chickenpox), which is now manufactured by biken and licensed worldwide. "these discoveries were possible because this institute has been a core facility within osaka university from the outset, " says kikutani. "we have many young researchers from all over the world, and from backgrounds ranging from medicine to chemistry and other physical sciences. " with funding from the japanese government, several national universities including the rimd are maintaining research centres overseas to investigate emerging and re-emerging infectious diseases in asia and africa. an example is the thailand-japan research collaboration center on emerging and re-emerging infections (rcc-eri). "we established the rcc-eri in bangkok in , " says kikutani. "the rimd is focused on disseminating information to prevent epidemics, developing medicines and vaccines, and extending our network with other asian countries. " the rcc-eri, set up in partnership with thailand's national institute of health, is equipped with state-of-the-art facilities including 'bio-safety level ' laboratories for conducting research on hiv/aids, avian influenza and other zoonotic infections, and intestinal infections. "the international network of overseas research centres supported by the japanese government is similar to the network maintained by the centers for disease control and prevention in the us or the pasteur institute in france, " says kikutani. the rcc-eri supports ten japanese researchers in bangkok, who conduct research as well as train local staff. toshihiro horii at the rimd is developing a pioneering vaccine for malaria -known as the 'neglected disease' . "malaria is a huge burden for the human race, " says horii. "about % of the world's population lives in malaria-endemic areas. there is increasing urgency for a malaria vaccine and anti-malarial drugs. " horii has developed a vaccine based on serine repeat antigen (sera), a protein produced by the plasmodium falciparum parasite responsible for malaria. "an epidemiology survey conducted by us in uganda showed that people with high concentrations of antibodies against sera did not show symptoms of malaria, " says horii. "i have developed artificial sera in the form of the se vaccine, which when injected into people will produce antibodies against malaria. " transforming this discovery into a product requires adherence to strict international guidelines for good manufacturing practice (gmp). "the gmp rules must be obeyed for the manufacture of vaccines, " says horii. "the rules even include requirements for the number of dust particles allowed per cubic metre in the manufacturing facilities. the executives at the kanonji institute of biken are interested in manufacturing the se malaria vaccine, and have provided their gmp-compatible facilities for producing test material for the initial trials. this is our contribution to society. " the se malaria vaccine has passed safety trial tests in japan, and clinical trials in uganda are scheduled for this year. the vaccine is expected to be available for general use within five years. like adjacent strands of a β-sheet, the study of proteins and the development of osaka university are intimately linked. the relationship dates right back to the earliest days of the university as a modern institution in , although it was not until that a laboratory dedicated to the investigation of the organic chemistry of proteins and amino acids was set up in the faculty of science. the current institute was opened two years later and has since grown into one of the premium centres for protein research in the world. although the ipr is active in all areas of protein science, it plays a particularly prominent role in the development and management of the worldwide protein database (wwpdb), of which it was a founding member in . currently the protein data bank japan (pdbj: www.pdbj.org) located at the ipr and led by haruki nakamura acts as the first point of reference for scientists from the asia-pacific. in this role, the pdbj processes - % of the total world depositions to the wwpdb, amounting to around , structures each year. as well as curation and registration of data, the pdbj develops tools that allow users to access the data in a range of different formats and to search the protein structures in the database for points of structural similarity, such as functional sites, backbone shape and molecular surface. the ipr also carries out ground-breaking research in ab initio computational chemistry relating electronic states to the three-dimensional structure of proteins and related systems. this has provided important information about questions of fundamental importance in biosystems, such as asymmetric electron transfer in the photosystem ii reaction centre -one of the key mechanisms in photosynthesis. whilst the majority of the data in the wwpdb is generated by x-ray diffraction analysis -a technique that allows the structure of a molecule to be obtained directly by analysing x-rays that have passed through a crystal of the test substance -not all proteins can be examined using this method. "some proteins just don't crystallize nicely enough for x-ray studies, " explains toshimichi fujiwara, head of the laboratory for molecular biophysics at the ipr, "but fortunately in many cases they are suitable for analysis by nmr. " nuclear magnetic resonance (nmr) is a technique that uses differences in the way radiation is absorbed by atoms within a molecule when placed in a strong magnetic field to gain information about the molecule's structure. fujiwara's group has already deployed solid-phase nmr techniques extensively in the study of membrane proteins that are either not sufficiently soluble for solution-based nmr or that undergo drastic structural change during dissolution, which renders the structural data obtained of little use. "it's all about sensitivity, " explains fujiwara. "the more sensitive the atoms in the sample are to radiation, the better the data we can obtain. " however, in many cases the sample is not sufficiently sensitive to the low-energy radio waves of conventional nmr to allow the collection of detailed structural information. to get round this problem, fujiwara's group is developing new 'terahertz' technology using high-field dynamic nuclear polarization. in this technique, electron spins, which are very much more sensitive to irradiation, are energized by terahertz-frequency radiation and used indirectly to excite nuclear spins in the sample at very low temperature. this has allowed accurate measurements of microgram quantities of protein, rather than the tens of milligrams normally needed, at sensitivities up to - times that previously possible. the terahertz project is carried out in collaboration with academic and industrial partners including jeol, at which fujiwara worked for seven years before moving into academic research. currently at the experimental stage, the plan is to place the technology at the heart of an international nmr resource centre along with existing high-powered conventional nmr instruments such as the mhz machine scheduled for delivery in early . "we want to see what is really possible with nmr, " says fujiwara. the electrostatic molecular surface of the rat liver vault. an example of the complex computational chemistry performed at the ipr by haruki nakamura's group. - yamadaoka, suita, osaka - , japan the oral cavity is responsible for many crucial activities that most of us take for granted: breathing, eating, defence against pathogens, language and facial expressions. however for many people -those suffering severe periodontal disease -these basic functions become a challenge that dramatically affects their quality of life. the ability to apply the most effective preventive and therapeutic approaches to periodontal care should be a top priority, but far too many dentists are satisfied with conventional techniques. not enough energy goes into introducing new biomedical methods based on the latest molecular and cellular biology research. the osaka university graduate school of dentistry aims to correct this situation. "many people still see dentists as practicing the art of pulling teeth, filling cavities or putting in dentures, but that image is completely outdated, " says toshiyuki yoneda, dean of the graduate school of dentistry. yoneda argues that discoveries concerning the function of genes, the immunological system and the nervous systems have opened the door to an era of new treatments. "molecular biology has revolutionized dentistry, " he says. one obstacle to this advance had been the divide between the clinic and the scientists' workbench. "dentists don't really understand research and researchers don't really understand dentistry, " says yoneda. "many dentists are clinically oriented and they don't care about science. " the graduate school of dentistry is an attempt to resolve this problem. of the school's staff, are researcher/practitioners who hold both doctor of dental surgery (dds) and doctor of philosophy (phd) degrees, showing their ability to bridge the two cultures. "there is no doubt that this is important for the development of high-level healthcare, " says yoneda. the school was given a vote of confidence for its approach in from the japanese government, which bestowed on the school a center of excellence grant worth up to million yen annually. the grant allowed yoneda to establish the frontier dentistry/oral biology (fdob) program. for the past seven years, the program has been at the forefront of integrating molecular and cellular biology into dentistry practice. the program has focused on three topics: management of oral infection, support of oral development and biological stimulation of the oral cavity. the results have been impressive. publications originating from the school have had a scientific impact comparable with the world's strongest programs, including harvard. an important outcome of this research from the patients' perspective is progress on a treatment for regenerating bone based on fibroblast growth factor (fgf- ). this treatment, pioneered at the osaka university school of dentistry hospital and now in phase-three clinical trials, has already helped many patients in the late stages of periodontal decay to regrow the bone that holds their teeth in place. "some of these people can't eat, they can't bite and the gums get infected with bacteria, " says yoneda. he hopes such examples will help convince the general public that 'advanced dentistry' can play a vital role in improving their quality of life. seminars and open forums held by the fdob program show people what the latest research can do. "what we' d like them to see is that our basic science supports the clinic. people are very interested and come with a lot of questions, " he says. another key role of the fdob program and the graduate school of dentistry is education, with a mission to train dentists capable of deepening our understanding of the biological phenomena of the mouth at the molecular level. the educational scope is widened through the exchange of graduate students with foreign countries, and by hosting international symposia and establishing joint research programs with foreign institutions. these international activities, along with english educational programs at the school, will enable japanese dental researchers to build names for themselves. efforts by japanese dental researchers to interact with their peers from other countries, present scientific findings at international conferences and publish their findings are often hampered by inadequate english language ability. yoneda's program aims to train the next generation to undertake research, be active in the clinic and be involved in the international research community. by doing so, the school will help to give japanese dentists the respect they deserve and their patients the high-quality treatments they need. "people underestimate japanese dentistry at the moment, but we are world leaders in the field, " he says. regeneration of tooth-supporting bone weeks after topical application of fgf- . this type of pioneering research, directed by shinya murakami, has direct applications in dental care. akemichi baba has his own perspective on finding treatments for psychiatric disorders. such disorders are complex, and those researching treatments have a hard time knowing where to start. baba has provided a solid foundation. he has spent nearly two decades characterizing pituitary adenylate cyclase activating peptide (pacap), a polypeptide first isolated in and known to play an important role in the camp pathway. baba and his team of researchers soon took the lead in examining pacap by cloning it, carrying out genomic analyses, identifying its receptors, tracing its involvement in molecular pathways and localizing its distribution. it turns out that the peptide is mainly expressed in the brain -an exciting finding that suggested a potentially important role in brain function. that interpretation turned out to be correct. using modern gene-targeting methods, baba's team found that when the pacap gene was knocked out, mice suffered the tell-tale signs of schizophrenia: hyperactivity, reduced ability to adjust to disturbances (lower pre-pulse inhibition), a tendency to jump around explosively, cognitive dysfunction and depression-like behaviour. it was a triumph of 'reverse pharmacology' , an approach in which scientists study phenotypic effects of a disturbance to a complex system. baba knows that the phenotypic expression of genes also depends on certain environments, so he has also been studying relevant environmental factors. baba's pacap knockout mouse is on its way to becoming a new model for human schizophrenia. the mice show improvement when given anti-psychotics. and analogies with humans are supported by the fact that a certain mutation in the pacap gene, found to be significant in schizophrenic patients, is associated with smaller hippocampal volume and poor memory. what causes schizophrenia and how should it best be treated? with his mice, baba is on his way to finding out. hiroyuki mizuguchi is perfecting methods for introducing genes into cells at will -a technology that offers the unprecedented ability to manipulate cells for biomedical research -to produce vectors for gene therapy and potentially to carry out life-saving clinical therapies. adenovirus vectors accomplish this feat, with the added advantage that they express the gene of choice without disturbing the host's dna. but conventional adenovirus vectors also have problems, such as being unable to penetrate cells that lack a certain type of receptor. mizuguchi has experience in solving such tough problems. "we want to find a way to take advantage of the merits, limit the problems and find a new generation of adenovirus vectors that can serve even more functions, " says mizuguchi. mizuguchi pioneered an efficient way to make adenovirus vectors through a patented approach that is now commonly used worldwide. and with several publications and patents on a new kind of adenovirus vector, he is well positioned to make even more breakthroughs. satoshi obika found a recipe that works. twelve years ago, he pioneered a technology that stabilizes nucleic acids, whether dna or rna, by adding an extra bridge in the molecule's sugar. the 'bridged' nucleic acid (bna) outdid his expectations, binding to complementary rna with an affinity more than , times that of normal dna. "i was very surprised, " says obika. bna can be used to increase the specificity of detection in common dna microarrays or polymerase chain reactions. the specificity will also allow 'antisense' blocking of dna transcription or rna translation, which could make dna-based clinical therapies possible. obika designs a successful bna roughly once per year, each with its own rna or dna targets, paving the way to a future full of discoveries. yasuo tsutsumi, a toxicologist, thinks people should be worrying more about the smaller things, and his animal studies back up that position. he studies silica materials composed of molecules smaller than nanometres in diameter, which are increasingly being used in cosmetics, food and drugs. his first question: do they get into the body? the answer he found in his mouse and pig studies is 'yes' -even when applied to the skin, which is supposed to be the toughest barrier to pass. the second: where do these nanoparticles go in the body? the answer: into many vital organs, including the brain. the last question: are they safe? "we don't know yet, " says tsutsumi, "which is exactly why we have to extend our studies on nanotoxicology as quickly as possible. " tsutsumi wants to bring these facts to our attention. "we believe these products are safe, without any evidence, " he says. "i want to grasp the risk involved. " "basic science is essential to advances in human understanding, " emphasizes kiyoshi higashijima, dean of the graduate school of science. "and it is our chief focus in research and in education. " higashijima is quick to note that the emphasis on basic science is just as productive in spawning real-world applications as in illuminating scientific fundamentals. "our faculty and students tackle issues in bioscience from diverse perspectives, including macromolecular science, physics and even mathematics, as well as biology and chemistry. we expect our people to do world-class research, and they have responded impressively. multidisciplinary initiatives leverage our strengths in individual disciplines, and multilateral international exchange broadens our horizons further still. " epitomizing the world-class work in bioscience at osaka university is innovative research by three of the graduate school's professors of biology: hiroki nishida, tatsuo kakimoto and hisao masukata. all three are leaders in their fields, and each has captured worldwide attention with their discoveries. nishida's research on ascidians (sea squirts) has yielded insights into embryonic development in chordates. "ascidians are among the simplest of chordates, " notes nishida, "and scientists have elucidated the fates of nearly all of their embryonic cells. so those humble marine creatures are a superb laboratory for studying embryonic development, including patterns of gene expression, determinants of cell fate and cellular interaction. we have learned how to manipulate the fates of embryonic cells in ascidians artificially. our findings fortify the foundation for work in developmental medicine, including stem cell therapies. " kakimoto, meanwhile, has contributed greatly to parsing the mechanism of intercellular communication. he discovered a biosynthesizing enzyme and receptor for cytokinin, which figures prominently in intercellular communication and in regulating cellular differentiation in plants. and he has gone on to identify regulators of cell multiplication and cellular positioning. "i'm interested, " kakimoto explains, "in the basic principles that determine form in plants. so i am studying the communication and the signalling networks that fulfil those principles. " masukata describes his pioneering work on chromosomal replication as the result of a long-standing fascination. "the replication, " he observes, "is subject to different structural control mechanisms, depending on the stage of the cell cycle and even on the position of the chromosomes. that mystified me early on, and i was determined to solve the mystery. " masukata's quest has illuminated important aspects of chromosomal replication. for example, he has helped identify the action of heterochromatin protein , the chief determinant of chromosomal structure in fission yeast. and masukata is broadening his research with an eye to achieving a more comprehensive grasp of chromosomal replication. active in cutting-edge chemistry at osaka university is akira harada, a professor of macromolecular science. he has received plaudits for his work on artificial molecular machines. "organisms, " harada comments, "exhibit different kinds of motor movement, including the linear motion of muscles and the rotary motion of atp synthesis and of flagella. scientists have devoted immense time and effort to analysing biological motor functions with an eye to harnessing them for various applications. i have built self-assembling structures by combining the ring-shaped molecule cyclodextrin with linear macromolecules, and i have succeeded in controlling the movement of the molecules relative to each other. the next step in my research is to develop applications for my molecular machines in sensors, catalysts and other applications by combining them with bioactive molecules, such as antibodies. " koichi fukase, a professor of chemistry at osaka university, regards organisms as communities of molecules. "my work unfolds, " he quips, "in the realm of biomolecular society. " fukase works to verify the action of biologically active molecules in observable functions. his approach has led him into a diversity of projects, including research on the synthesis and functioning of molecules that regulate innate immunity; analysis of the synthesis and functioning of sugar chains on cell surfaces and in glycoproteins; and the imaging of sugar chains, macrobiomolecules and cells. he pioneered the application of positron emission tomography (pet) to analysing glycoproteins, and his 'glycopet' imaging technique has provided visual verification of the decisive effect of sugar chains on protein dynamics. an ascidian tadpole larva, just before hatching. hiroki nishida's group uses ascidians as a laboratory for embryonic research. advertiser retains sole responsibility for content |naturejobs| september "sugar chains and proteins are just the beginning, " declares fukase. "we could assemble those and other biomolecules into structures for performing useful functions, such as recognizing patterns in the communities on cell surfaces and regulating the molecular communities inside cells. that approach could even provide an effective means of correcting autoimmune disorders and targeting anticancer drugs at tumours. " michio murata, a colleague of fukase in osaka university's department of chemistry, is also doing important work in the realm of biomolecular society. murata works to elucidate biological phenomena by reconstituting the biomolecular communities required for various functions. for example, he analyses the interaction of biomolecules and pharmaceutical compounds inside a liposome membrane as a model for the cell membrane. using this approach, murata has demonstrated that membrane lipids figure prominently in the pharmaceutical action of antibiotics. "the cell membrane is a classic biomolecular society, " remarks murata. "i look forward to developing methodologies for grasping the exact roles that lipids, proteins and other biomolecules play in that society. " these achievements are but a small sample of the cutting-edge work under way in bioscience at osaka university. some of the other bioscience researchers at the university who have earned international acclaim include hitoshi watarai in analytical chemistry, yasuhiro kajihara in organic biochemistry and yasuhisa mizutani in biophysical chemistry. the mei centre is a major osaka university initiative that brings together researchers from various fields to collaborate on projects in medical sciences, bioengineering and bioinformatics. the centre is funded, mostly externally, to the tune of around one billion yen per year and houses principal investigators who also maintain primary affiliations with other divisions of the university. "in one sense it's a 'virtual' department, " says yoshihisa kurachi, director of the mei centre. at the heart of this virtual department is the global center of excellence for an in silico medicine-oriented platform that forms the basis for collaboration with leading institutions from new zealand, the us and the eu. the aim of the platform is to compile physiome databases of dynamic mathematical models describing biological systems -from the molecular and cellular level right through to organs and individuals -based on experimental data on the fundamental chemical and physical properties of physiological entities. these models, whether a liver, a heart or even an entire human skeleton, are made available for download from a morphology database (www.physiome.jp) for integration into biological modelling software, allowing the effects of given stimuli on the structure-based physiological function to be modelled and investigated. current applications include a study on the risk of drug-induced cardiac arrhythmia for pharmaceutical drug candidates, and the use of morphology data to predict optimal parameters for joint replacement surgery and rehabilitation aftercare packages. the mei centre offers tuition in medical engineering and informatics to postgraduate students from osaka university and other institutes in the region. although the classesunique in japan -are currently only an elective course module, there are plans to develop a fully accredited postgraduate degree program in the near future. and what of the future? the dream is to construct a complete in silico human from mathematical data, but it's a dream -admits taishin nomura, leader of the in silico medicine project -that may never be realized. "building a model of a human being is probably impossible, " he says candidly, "but at least we can continue to improve human understanding. " an in silico human. the mei centre's in silico medicine project aims to build an open platform for multi-scale mathematical models of the human body and its functions. in , fbs established an integrated five-year graduate school course to nurture students with multifaceted skills in the biosciences. "this course is extremely popular, " says fujio murakami, dean of the fbs school. "we usually receive around applicants for only places. " the success of research and education at fbs has been underscored by its selection for one of the japanese government's global center of excellence (g-coe) programs. the program supports doctoral students by arranging summer schools, international exchanges and courses on improving their english language skills. in addition, the g-coe invites leading researchers from overseas to give lectures and seminars to doctoral students affiliated with the program. fbs is also collaborating with the national institute of information and communications technology (nict). "in , we will launch a project with the nict on imaging the flow of information in the brain, " says murakami. the new , m interdisciplinary research center will house members of this project working on such areas as bio-communications and dynamics, imaging technology and computation. key tools for this project include magnetic resonance imaging, magnetoencephalography and two-photon microscope imaging. "fbs is responsible for cell imaging at the immunology frontier research center, a world premier international research center, " says murakami. "we have a lot of expertise to offer the biosciences. " murakami is using neural imaging to study the movement of cells in the brain. "cell movement in the brain is extremely important, " says murakami. "lack of cell movement can lead to schizophrenia, for example. we use a combination of in vitro electroporation, green fluorescent proteins and confocal fluorescence microscopy to image labelled cells. " murakami encourages students to "enjoy the discovery" of science. "we recently started in vivo experiments for a firsthand look at cell movement and brain functions, " he says. yasushi hiraoka pioneered fluorescence microscopy for imaging the structure of living cells. he holds concurrent posts at fbs and the kobe advanced ict research center of the nict. "i succeeded in the fluorescence imaging of living cellular structures because of the strong involvement of nict researchers. in particular, i have worked with tokuko haraguchi in kobe for over two decades, where i covered engineering and she, the biological aspects of this research. " hiraoka's microscopy allows information to be procured from individual living cells, a major advance compared with conventional methods that yield only 'averaged' information. in , hiraoka published a high-profile article on high-resolution, stereoscopic, three-dimensional time-lapse fluorescence microscopy to monitor chromosomes. his work established fluorescence microscopy as a tool for the observation of single living cells. hiraoka emphasizes that the research environment and human resources were critical for success. his group of researchers at the nict consisted of % male and % female members. over two decades, the female staff continued their research after maternity leave. "this is an exceptional research environment in this country, " says hiraoka. "our success was based on the accomplishments of a minority group. " "tokuko haraguchi and i have organized courses on live cell imaging biannually for the last seven years, " says hiraoka. "we usually have two to three times more students applying than we can handle. " "we had many challenges over the last years, " says hiraoka. "irradiating cells with strong light killed them. we resolved this by developing a high-sensitivity automated imaging system. synthesis of low-toxicity dyes was another milestone, with one immediate application of monitoring the effects of anti-cancer drugs on individual cells and related drug screening. " "i want to exploit the wealth of specialties at fbs for in vivo, functional imaging of cell movement in the whole body, " says hiraoka. live images of dividing human cells. triple staining shows chromosomes (blue), centromeres (magenta) and microtubules (green). drug discovery is getting costly and time consuming: it can now take a decade or so from the discovery of candidate compounds to the marketing of a new drug, with total investments of us$ million to us$ billion. the introduction of highperformance computers, however, can drastically shorten the time for research and development in experimental biology, which remains one of the most important fields for drug development. from to , the japanese government sponsored the biogrid project with the objective of making it possible for users anywhere to access cutting-edge data processing power by networking and coordinating the vast databases, software and other information technology resources hosted on supercomputers at geographically distributed research institutions. the main purpose of the initiative was to speed up the research and development of drug design by introducing high-performance computing into the steps of cell modelling, drug screening and investigations of drug efficacy. to take full advantage of the project, biological and computational researchers established a non-profit organization called the biogrid center kansai in osaka in . the centre is presided over by shinji shimojo, an executive researcher of the national institute of information and communications technology (nict). members of the biogrid center -biotechnology venture firms, osaka university, research institutes and pharmaceutical companies -have access to powerful computing tools such as 'mypresto' for modelling of proteins and drug docking, and ' amoss' for quantum mechanics calculations. members also have access to spring- in kobethe world's most powerful synchrotron radiation facility -and supercomputers at osaka university. the centre aims to accelerate technology transfer, strengthen collaborations between academia and industry, and ultimately enhance the opportunities for drug innovation and the creation of start-ups. the picture looks bright, but collaborations rarely work well without strong commitments from each of the participants. "that's why we decided to design attractive projects on our own, " says tsuneaki sakata, a director of the biogrid center and visiting professor at the cybermedia center of osaka university. the flagship scheme is called the pharmaceutical innovation value chain, an open platform that allows members and non-members around the world to contribute their expertise in any stage of drug development towards the common goal of generating innovative drugs. currently, a venture company called kringle pharma is taking advantage of technologies provided by participants in the value chain for the development of cancer treatments. "it is important to prompt researchers -particularly younger ones -to support the value chain and develop their skills without expecting immediate returns. " says sakata. last year, the value chain produced kyoto constella technologies, a spin-off from kyoto university. initiated in , the value chain is funded by the knowledge cluster initiative ( nd stage) of the japanese ministry of education, culture, sports, science and technology, and the 'consortium r&d projects for regional revitalization' program of the ministry of economy, trade and industry. as part of the biogrid center's expansion of the value chain beyond national borders, the center has created an opportunity to form a memorandum of understanding between the biomedical cluster kansai, which includes contributors in kyoto and kobe, and a biotech cluster in alsace, france. in three years, the biogrid center is expected to benefit from the next-generation supercomputer being built near kobe with funding from the japanese government. the new supercomputer will be able to perform at a rate of petaflops, or operations per second. the government has identified simulation of the human body as one of the 'pillar' projects for this new facility. in august, the biogrid center held the first workshop discussing suitable applications for the advanced supercomputer, such as predictions of drug toxicity. yet there still remain many gaps between computational scientists and biologists, and also between industry and academics, concerning intellectual property and the best approach to research. "we must increase the opportunities and incentives for interactions for everyone, " says sakata. the pharmaceutical innovation value chain. venture companies and research institutes interested in commercializing drugs can seek collaboration partners from the biogrid center kansai's value chain project. the ncvc has pioneered medical care and research in areas including revascularization of the heart and brain during acute and superacute phases, less-invasive heart surgery, artificial heart and lung technology, and genetic diagnosis and therapy. "our research is based on close collaboration among industry, academia and government, " says hashimoto. "this translational approach to research is important for conducting clinical trials, for drug development, and for a myriad of medical instruments and devices. " highly acclaimed breakthroughs by the ncvc researchers include the discovery of the novel hormone 'ghrelin' , the rapid diagnosis of thrombosis, peptidomics/proteomics and the development of artificial hearts and lungs. new buildings and initiatives are in the pipeline for . "next year sees some major administrative changes at the ncvc, " says hashimoto. "we will reorganize into an 'independent administrative institution' from april . this will give us more autonomy from the japanese government to hire specialists -including highly motivated scientists from overseas -and to define new areas of research. " kenji kangawa is an internationally acclaimed biochemist, director general of the ncvc and a researcher who was pivotal in the discovery of the hormone ghrelin, publishing a series of high-profile papers on the topic in - . "most researchers were looking for this hormone in brain extracts, " says kangawa. "the assumption was that this hormone was produced in the hypothalamus, and hence related to the brain. but quests to find this hormone in the brain were not fruitful. " kangawa and his group decided to look in the stomachs of rats and humans. "if our competitors, especially at large industrial labs, had looked in the stomach, then they would have found it before us, " says kangawa. kangawa was the first in the world to isolate other important bioactive peptides, including brain natriuretic peptide (bnp), c-type natriuretic peptide (cnp), and atrial natriuretic peptide (anp). the discovery of anp, which is isolated from the heart, showed that the heart acts as both a pump and an endocrine organ that secretes hormones. kangawa stresses that the unique research environment at the ncvc -combining basic research within a busy hospital -has played a major role in his search for peptides. "new peptides lead to new areas of research. " translational research on hormones has advanced rapidly, with anp and bnp already being used for treatment and diagnosis by physicians. ghrelin is also undergoing clinical trials for the treatment of anorexia nervosa and related illnesses. the director of the department of pharmacology, naoto minamino, is searching for new bioactive peptides by a strategy based on peptidome analysis. "we use mass spectrometers to identify peptides in tissue extracts, " says minamino. "but there are still many issues to resolve because the peptides are easily degraded. " two approaches, 'activity-first' and 'peptide-first' strategies, are being investigated. "the key to indentifying peptides and proteins accurately is to keep cells and tissues intact. " minamino and his colleague kazuki sasaki applied peptidome analysis to the conditioned media of cultured cells -a procedure known as 'secretopeptidome analysis' . using this approach, they successfully acquired high-quality secretory peptide data by reducing the degradation fragments of cellular proteins. "it is possible to propose processing pathways for precursor proteins, and to predict the peptides generated from precursor proteins, " says minamino. these studies based on the peptide-first strategy will allow the identification of new bioactive peptides and biomarkers, and will increase the probability of computer-based prediction of bioactive peptides and biomarkers. the results of research on the functions of peptide hormones are critical for the development of new drugs for the treatment of heart failure, hypertension and metabolic syndrome. another example of the basic-to-translational research being conducted at the ncvc is toshiyuki miyata's work on thrombosis -the clogging of blood vessels. miyata is director of the department of etiology and pathogenesis, and has been investigating the diagnosis and treatment of the life-threatening systemic disease known as thrombotic thrombocytopenic purpura (ttp). effective diagnosis of ttp requires detection of adamts -an enzyme that cleaves the protein responsible for blood clotting (von willebrand factor; vwf). adamsts regulates diagnosing ttp using frets-vwf . cleavage of frets-vwf by adamts results in fluorescence of the intact peptide. normal blood will thus fluoresce increasingly over time, while adamts -deficient plasma will not produce fluorescence. advertiser retains sole responsibility for content |naturejobs| september spotlight on osaka the molecular size of vwf in the blood, and an absence of adamsts leads to the build-up of vwf, with subsequent dangers of ttp. "monitoring the activity of adamsts is critical for early diagnosis of ttp, " says miyata. "with my colleague koichi kokame, we determined that the minimum region recognized as a specific substrate by the adamts enzyme was only amino acids, from d to r , of vwf. " this discovery led to the development of the fluorescence resonance energy transfer (fret) assay, which allows the activity of adamts to be checked within one hour, instead of the - days required using conventional methods. "we worked with industrial partners to chemically synthesize a fluorogenic peptide, frets-vwf , containing the amino acids, " says miyata. "the fret design is a fluorescent molecule attached to a quenching group. if the molecule is cleaved by adamts , then fluorescence is observed, but in the absence of adamts , cleavage does not occur and fluorescence is quenched. " frets-vwf and related fluorogenic substrates are now sold commercially by vendors including osaka-based peptide institute inc. taking discoveries from the lab to the bedside is critical in translating scientific discoveries into real-world medical applications. "the ncvc was selected as a super-tokku region by the japanese government, " says yoshiyuki taenaka, director of the advanced medical engineering center. "super-tokku refers to a region designated to pursue high-level research and development based on close industrial, academic and government collaboration. " taenaka's speciality is mechanical circulatory support systems, such as artificial hearts and lungs. "our success in translational research at the ncvc paved the way for our selection by the government for the super-tokku project, " says taenaka. "the project is for five years, with people each being funded with about two million dollars per year. " industrial collaboration is imperative for developing implantable artificial organs. "our interaction with mitsubishi heavy industries, bridgestone, dic corporation, toyobo corporation and nipro corporation is important to get through the ' critical path' , " says taenaka. the director of the department of artificial organs, eisuku tatsumi, in collaboration with taenaka, is a pioneer of artificial hearts and lungs. "worldwide, there is a move towards the longterm use of artificial hearts, 'destination therapy' as it is known, as opposed to temporary support during recovery from heart disease, " says tatsumi. ventricular assist devices (vads) are examples of temporary support devices that are commonly used for short periods after heart attacks. "in japan, the toyobo vad is widely used, " says tatsumi. the ncvc is focused on developing a portable pneumatic vad system and an implantable axial flow pump vad system. "the size and weight of vad systems are important factors for improving quality of life, " says tatsumi. "our portable mobart-ncvc pneumatic vad driver, which has been widely used in clinical cases, is compact and weighs only . kg. " an even more compact vad driver that weighs less than kg and fits in a wearable shoulder bag is expected to be commercially available in two years. the implantable axial flow pump vad is being developed by the ncvc in collaboration with mitsubishi heavy industry and aist. this vad is less than the size of a d-type battery and weighs only g. "our device is durable because of its contact-free hydrodynamic bearings. importantly, the small size enables it to be used by children as well as adults, " says tatsumi. commercial products are expected within three years. there is also demand for artificial lung systems (pcps/ecmo systems) for use in emergency life support and over longer periods of time for individual patients. however, current systems are complex to set up and operate, are bulky, and require the use of anticoagulants. the biocube ncvc series of ecmo systems developed by the ncvc in collaboration with dic, toyobo and nipro solves these problems. "the use of anticoagulants can be minimized, or even made unnecessary, with the biocube ncvc series. they are compact, mobile platforms that will find applications in the out-of-hospital arena, such as at road accidents resulting in lifethreatening lung damage, " says tatsumi. the results of translational research at the ncvc will enhance the quality of life for patients, and act as seeds for new areas of medical research. "when i started at nibio, few people were interested in vaccine development. immunology progressed, but manufacturers are too small to do much r&d, " says yamanishi, a virologist with decades of experience studying herpes and other viruses. following panics caused by the sars coronavirus, the h n avian flu and most recently the h n pandemic flu, governments and industry have started paying attention. yamanishi leads a super special consortium in which universities, national research centres, pharmaceutical companies and hospitals aim to create next-generation vaccines for hiv, malaria, influenza and other infectious diseases. the consortium's goal is to improve vaccines by studying new vectors that will enable single vaccines to convey immunity to multiple infectious diseases. nibio has significant capabilities in the genetic recombination technology needed to create these vectors, which will make vaccination programs easier and cheaper. the consortium is also developing nasally, dermally and orally administrated vaccines as safer and more convenient alternatives to injections. "this type of immunization is closer to our natural immunity, " says yamanishi. the consortium will develop new adjuvants to enhance the effect of vaccines and allow the use of smaller doses. uncertainty about how adjuvants work has been an obstacle to their implementation, but japanese scientists have led the analysis of toll-like receptors and other related components of the immune system. "a new generation of effective adjuvants will come from japan, " says yamanishi with confidence. pharmaceutical companies are increasingly using embryonic stem (es) cell-derived cell lineages for in vitro testing of drugs. but es cells are in short supply. induced pluripotent stem (ips) cells, first created in japan in , offer an alternative. ips cells are made by reprogramming somatic cells to an embryo-like state. scientists can do this with anyone's cells, offering an infinite source of fresh cells with defined genetic backgrounds. research groups by the dozens have embraced ips cell technology. before they can be used in drug development, however, they need to be standardized and quality controlled in a manner that facilitates clinical studies by pharmaceutical companies. "japan is leading the examination of ips cells, " says nibio's hiroyuki mizuguchi, "but most university groups stop at the research level. " mizuguchi leads a super special consortium to establish a drug toxicity testing system based on ips cells. such a system will limit controversial a drug revolution in the making the unique role of nibio in japan. nibio combines extensive collaboration with basic research, valuable biological resources and project funding to support the pharmaceutical industry. advertiser retains sole responsibility for content |naturejobs| september spotlight on osaka animal testing, rein in ballooning drug development costs and put japan at the forefront of highly competitive global efforts to understand the genetic background of adverse drug reactions. nibio is creating a cell bank that will eventually hold ips cell lines. standardizing the protocol for banking ips cell lines is a labour-intensive process requiring analysis of cell morphology, cell surface antigens and gene expression profiles, and monitoring for unexpected changes. "we need to be able to grow the cells in a strictly defined medium so that we can differentiate them stably and with a high level of reproducibility, " says miho furue-kusuda, who is in charge of the bank. analyses using the cells will benefit from nibio's toxicogenomics database, the largest in the world, which collects data on the effects of more than drugs in rats. standardized protocols for producing ipsderived cells will be the foundation for new guidelines on their use in pharmacological testing. mizuguchi hopes to have the protocols in place within two years. "japan is the only country moving forward so quickly towards the use of ips cells to validate new drugs, " says mizuguchi. nibio is developing other resources that will give a crucial boost to pharmaceutical studies. for example, nibio' s dna bank has started to make available quality-controlled resources derived from patients suffering from rare diseases. the bank distributes cdna clones from chimpanzee and crab-eating macaques along with related genetic sequences. a unique experimental mouse has also been established at nibio. whereas conventional severe combined immunodeficiency (scid) mice can only hold transplanted human tissue for a few weeks, nibio's 'super-scid' mice maintain human tissue transplants, such as lung, thyroid and skin, for a year or more, a boon for drug development and the study of environmental hazards. in addition, nibio holds mice that develop various diseases spontaneously. "we can watch the natural development of chronic diseases rather than making artificial disturbances with mutagenesis or transgenes, " says nibio's tohru masui. the resource bank also includes a cell bank of cultures of human brain, lung, liver, kidney, gut and blood cells, as well as many samples of mesenchymal stem cells and cells derived from patients, which are important for drug development and regenerative medicine. through extensive quality control and monitoring, nibio ensures that there is no contamination by microorganisms or other cell types. this is not easy considering a recent study by nibio's arihiro kohara showing that % of cell cultures in japan contain mycoplasma. "these figures are similar to those seen all over the world, " says masui. "it shows the importance of quality control. contamination has a huge impact on the reliability and repeatability of experiments. " the vital experimental resources available through nibio also include primate and medicinal plant collections. the research center for medicinal plant resources has four bases across the japanese archipelago, representing different climatic areas. new restrictions on crude drug exports in china as well as quality-control problems prompted the cultivation of several important medicinal plants locally. the centre cultivates and preserves over , plant species to supply seeds and seedlings, and offers guidance on cultivation technology to research institutes. "it's the most comprehensive collection in japan, " says yamanishi. the tsukuba primate research center, with , cynomolgous macaques, also offers a unique resource. the monkeys are mated in specifically selected pairs so that hereditary factors can be traced. the centre carries data on the monkeys' age, laboratory test results and family histories stretching back years, enabling researchers to predict, for example, which monkeys might experience high blood pressure. "we know the entire history of these monkeys, " yamanishi says. the monkeys, used at nibio for vaccine and other research, are also available to scientists and pharmaceutical companies throughout japan for research on disorders such as hereditary diseases, diabetes and cardiovascular disease. yamanishi hopes that these resources, maintained at high levels of quality, and the research that nibio supports will give the pharmaceutical industry a push. "these kinds of research and resources are essential for drug development, but neither the universities nor the drug companies are doing it. we've only been around for four years, but we're already starting to see results. " a n international group of young, talented and highly motivated scientists at the osaka bioscience institute (obi) is at the forefront of research on systems neuroscience, olfactory mechanisms, retinal systems and sleep mechanisms. the obi is a non-profit organization conducting research in the biological and medical sciences. with around researchers in five departments, the obi has been at the forefront of research in bioscience, with breakthroughs that include elucidating the mechanisms governing cell death, sleep and consciousness. the obi has a unique history, being established in following an initiative by former osaka mayor yasushi ohshima as part of the th anniversary celebrations of the osaka municipal government. "oshima wanted to create something for the future of osaka, " says osamu hayaishi, former director of the obi and present chairman of the obi's board of trustees. "osaka is the commercial heart of japan. oshima asked a panel of experts from academia, industry and government to recommend a way of creating something for the people of osaka. bioscience was considered important because many local companies made products such as pharmaceuticals and alcoholic beverages based on bioscience and medical science. this proposal led to the birth of the obi. " research at the obi is funded by osaka city, the japanese government and private foundations. this scheme for funding research is unique. "we could say this system is 'too unique' , " says hayaishi. "almost all research in japan is funded by the national government. even in the us, the national institutes of health are supported solely by the federal government. our approach is different. many companies have donated funds to build and run this institute. our research is based on a solid industrygovernment-academia relationship. " for an international perspective, the obi advisory board asked arthur kornberg -the noble laureate in physiology or medicine who clarified mechanisms in the biological synthesis of deoxyribonucleic acid -to be a consultant to the obi. the first chairman of the board was kenzo saji, former president of suntory, who had a doctorate in chemistry. "i was appointed the first director of the obi, " says hayaishi. "i think i was chosen because of my extensive experience in international research. i had spent ten years in the us as section chief at the national institutes of health and teaching at universities. after returning to japan, i held faculty positions at the universities of kyoto, tokyo and osaka -my alma mater. i also had visiting positions at harvard, vanderbilt and the karolinska institute, amongst others. so i knew many young and active researchers all over the world. " in contrast to other research institutes in japan, which offer permanent tenured positions, the obi employs young scientists on fixed-term contracts ranging from three to ten years. "this was my idea, " says hayaishi. "it was and still is an unorthodox approach to research in japan. we offer well-paid positions, with an excellent research environment hosting state-of-the-art facilities. a turnover of scientists is important for innovative research. during my directorship i allowed our researchers to choose their own areas of research. " at the time, hayaishi's approach was thought to be too idealistic by some administrators at monbusho -the former japanese ministry of education -and some doubted whether this approach would succeed. as if to disprove their critics, obi scientists have made many major breakthroughs over the last years. examples include identification of the basic mechanisms of apoptosis by shigekazu nagata and clarification of the control mechanism of sleep by osamu hayaishi. the quality of obi research was highlighted in when the obi was ranked at the top in scientfic impact factors for publications on molecular biology and genetics from to . internationally recognized as an institute at the leading edge of research " we provide an excellent research environment for ambitious scientists to spend % of their time doing research, " says hayaishi. "i personally interviewed all applicants. i believe that people wishing to conduct research at the obi have great courage and confidence for considering working in this small, private institute. " shigetada nakanishi is the present director of the obi. himself an accomplished scientist, he is renowned for elucidating the molecular nature of the glutamate receptor combining xenopus oocyte expression systems and electrophysiology. "i studied under dr hayaishi at kyoto university, and years ago i was a member of the obi advisory committee, meeting annually with the directors of the obi, " says nakanishi. "when i was an advisor, i was impressed by the fact that the researchers at the obi were tackling problems in completely unknown fields -sleep and cell death were new fields back then. the discovery of the mechanism and inhibition of cell death by obi scientists is acknowledged as being a major advance in understanding cell cancer. " hidesaburo hanafusa, who worked at rockefeller university, was the second director of the obi between and . "unfortunately, dr hanafusa became ill, and i was appointed director four years ago, " says nakanishi. "our situation has changed recently because we now have to compete with large universities who are using the same approach towards research as us. my main mission now is to devise ways of competing with major universities such kyoto, tokyo and osaka. " given the changing research landscape, nakanishi is making changes to compete with new approaches to research in bioscience. "i have decided to define certain areas of research and find talented principal investigators for these areas, " says nakanishi. "one of the new areas of research i want to expand is brain science, or more precisely, systems neuroscience. we know that many molecules are involved in brain functions, but we still do not know how the brain actually functions. we need to clarify the dynamic mechanism governing information processing and integration in the brain. this is systems neuroscience. " nakanishi has recently appointed a young scientist who is now working with her husband at the obi on olfactory systems -the sense of smell. "these scientists found that olfactory memory is divided into areas, " says nakanishi. "one area is the genetically controlled olfactory mechanism. for example, certain animals show a fear response towards the smell of a fox; this is a hereditary response. but animals also learn smells by training. our scientists have found that these two fear responses are divided, and they have elucidated the neural information paths responsible for each individual type of memory recognition. " "i studied molecular biology before coming to the obi, " says nakanishi. "now i have shifted towards systems biology, and in particular, systems neuroscience. " research in the future will focus on mechanisms of memory learning in the brain using systems biology, as well as the olfactory mechanism, visual retinal systems and information processing. sleep and consciousness are also major topics, such as how caffeine acts on the brain. the obi's unique approach to basic research is reflected in the construction of its spacious and eye-catching main building, designed by the famous architect, kenzo tange. the institute is equipped with state-of-the-art facilities including recombinant dna laboratories, genetic analysers and laser confocal microscopes. the well-stocked library and comfortable meeting spaces provide an ideal research environment. a mouse lacking dorsal olfactory sensory neurons. by targeted expression of the diphtheria toxin gene, researchers at the obi successfully bred a mouse without odour-evoked innate fear responses. - - furuedai, suita, osaka - , japan tel: + - - - web: www.obi.or.jp/index .html advertiser retains sole responsibility for content |naturejobs| despite pioneering research by many japanese biologists, the application of this expertise has proceeded slowly. but change is coming quickly.nibio was established in april by the japanese government to fill crucial gaps in the path from discovery to drug development. a unique institute, nibio carries out basic science aimed at developing shared technologies at its laboratories in the saito life science park, standardizes and shares biological resources throughout japan, and funds external research in a similar fashion to the national institutes of health in the us. "it's the only place like it in japan, " says koichi yamanishi, director general of nibio.the japanese government has recognized nibio's special mission in several ways. in , nibio was awarded two coveted 'super special consortium' designations for the development of cutting-edge medical care -two of only that were approved nationwide. nibio has also taken a leadership position in the japanese government's knowledge cluster initiative -a five-year project to stimulate cooperation between research institutes, universities and industry in biomedical sciences. nibio maintains a host of joint research projects with pharmaceutical manufacturers in fields ranging from toxicogenomics and biomarker exploration to animal models and vaccine development. "we meet regularly with companies to ask what they need from us, " says yamanishi. t he national institute of advanced industrial science and technology (aist), with a network of research centres across japan, promotes the creation of innovative ideas for society through 'full research' -a multidisciplinary approach to research from basic studies through to product realization. aist kansai is no exception.the aist institute is japan's largest public research organization, employing about , researchers at nine locations throughout the country and hosting almost , visiting scientists from universities and industry in japan and overseas."i was appointed president of aist in april after serving as chairman of the board of mitsubishi electric corporation, " says tamotsu nomakuchi. "needless to say, sales and profits are important in industry. but aist's role is to define and solve problems, anticipate future industrial needs and develop innovative technology for a sustainable society. " 'medicine' and 'engineering' are keywords that define research conducted at aist kansai on maintaining and promoting good health in an ageing society. "life sciences, and in particular, medical engineering, form the basis of multidisciplinary research at aist kansai, " says nomakuchi. this research is carried out at the research institute for cell engineering (rice), the health technology center and the institute for human science and biomedical engineering (hsbe)."at aist, i want to use my experience of working in industry, and i hope to act as a bridge connecting industry and academia in osaka as well as other aist centres in the rest of japan, " says nomakuchi.masayuki kamimoto, director of aist kansai, is proud of his centre's multidisciplinary capacity. "in addition to life sciences, we also have research projects in areas including the environment, energy, information technology and materials science, " he says. recent internationally recognized breakthroughs by researchers at aist kansai include cell cultivation protocols, carbonnanotube molecular actuators and biomarkers for parkinson's disease. self-repair of damaged bone and cartilage is extremely difficult in the elderly. hajime ohgushi, director of the research institute for cell engineering, is addressing this problem using stem cells for 'regenerative medicine' ."i use mesenchymal stem cells, which exist in bone marrow, to repair damaged bone tissue, " says ohgushi. "this is my contribution to regenerative medicine. "in , ohgushi and his group reported pioneering work on the regeneration of bone tissue using tissue-engineered bone derived from the patient's own mesenchymal stem cells (mscs).however, in spite of the tremendous advances as a result of this work, clinical applications of this approach were limited because the ability of the cells to proliferate and differentiate declined dramatically over time."we succeeded in activating the mscs by introducing nanog or sox -genes expressed by embryonic stem cells -into the mscs, " says ohgushi. introduction of only the sox gene was ineffective, and a combination of sox and a protein called basic fibroblast growth factor (b-fgf) was required."in , shinya yamanaka reported that introducing - transcription factors into the skin cells of an adult led to these skin cells gaining pluripotency like embryonic stem cells, " says ohgushi. "this report was the birth of induced pluripotent stems cells, or ips cells. we also succeeded in generating ips cells from mscs, therefore the technology may be available for regenerative medicine based on mscs. " kinji asaka and his group at the research institute for cell engineering is developing low-voltage electroactive polymer (eap) actuators to produce light-weight, directly fitting artificial muscles for rehabilitation. eap actuators are operated by applying a voltage to move and bend sheets of polymer actuators. "conventional polymer actuators rely on chemical reactions within liquid electrolytes to convert the applied voltage into mechanical motion, " says asaka. "they have short lifetimes and slow response times. "in asaka's approach, movement is produced by changing the internal volume of a polymer composite. "we mix millimetre-sized, single-walled carbon nanotubes with an ionic liquid to produce a gel that is dried to form highly conductive, free-standing sheets, " says asaka.the actuators were produced by cutting fifteen mm strips from the carbon nanotubes, sandwiching a soft polymer film between the strips, and finally applying an alternating voltage across the carbon-nanotube electrodes. low-voltage electroactive polymer (eap) actuators. these devices form the basis for light-weight, directly fitting artificial muscles. the actuator works at frequencies of - hz without notable deterioration of the actuator structure or range of motion over more than , continuous operations.these ' dry' actuators are extremely robust and flexible, and show rapid response to alternating voltages of as low as . v. "we could use roll-to-roll manufacturing for mass production of arbitrarily shaped actuators, " says asaka. the development of diagnostics technology for chronic diseases is a priority at aist kansai. parkinson's disease is a neurodegenerative disorder in which dopamine-producing brain cells are lost. the disease affects millions of people worldwide, and comes with symptoms that include trembling hands, stiff limbs and inability to balance."we are developing protocols for early diagnosis and treatment of parkinson's disease based on the detection of biomarkers, " says yasukazu yoshida at the health technology research center.yoshida's team found the gene park (dj- ) to be critical for early diagnosis of parkinson's disease. the next step for the researchers was to develop a means of detecting this biomarker in blood samples. "we successfully prepared a monoclonal antibody and developed a competitive enzyme-linked immunosorbent assay system for an oxidized form of dj- , known as cys- -oxidized dj- , " says yoshida.the concentration of oxidized dj- in blood samples of unmedicated parkinson's disease patients was about ten times greater than that detected in control and medicated patients. "future plans include improving the performance of the measurement system and introducing this technology into hospitals for actual clinical applications, " says yoshida. "we also want to establish our approach as an international standard for diagnosing parkinson's disease. " sunao iwaki is a group leader at the institute for human science and biomedical engineering. his group is at the forefront of developing technology for diagnosis and sensory prosthesis support for people with higher brain dysfunctions, such as dementia and language difficulties. "we are trying to unlock the mysteries of the brain using our integrated non-invasive brain imaging technique, " says iwaki. "it is extremely important to elucidate not only 'where and when' but also 'how' various parts of the brain interact. "iwaki's group has combined magnetoencephalography (meg), electroencephalography (eeg) and functional magnetic resonance imaging (fmri) with innovative information algorithms to allow non-invasive imaging of the brain with high spatiotemporal resolution. meg detects magnetic signals from neural networks in the brain, eeg maps changes in electric potential over the scalp due to neuron activity, and fmri detects brain activity by the degree of oxygenation of the blood. a system integrating these three analyses has been used to identify the neurophysiological mechanism of bone-conducted ultrasound (bcu) -the detection of sounds above khz not directly discernable by humans. "the bcu experiments led to the development of a bcu hearing aid, " says seiji nakagawa, senior research scientist at the hsbe institute. "our trials showed that % of profoundly deaf people could hear simple words, and more than half perceived some form of sound. in the future, we hope to commercialize this technology. "the bcu hearing aid could replace cochlear implants, which require time-consuming and physiologically taxing surgical procedures.non-invasive brain imaging system. a combination of meg, eeg and fmri analyses allows visualization of spatiotemporal neural dynamics (top) and neural interactions (bottom). key: cord- - tpef n authors: komiya, kosaku; yamasue, mari; takahashi, osamu; hiramatsu, kazufumi; kadota, jun-ichi; kato, seiya title: the covid- pandemic and the true incidence of tuberculosis in japan date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: tpef n nan a recent report in the present journal focused on the decreased incidence of tuberculosis during the covid- pandemic in taiwan. a declining trend in influenza following the covid- outbreak has already been indicated in brazil, singapore and japan. [ ] [ ] [ ] according to the infectious diseases weekly report japan, besides influenza, the incidences of mycoplasma pneumoniae pneumonia, respiratory syncytial virus infection, infectious gastroenteritis, epidemic keratoconjunctivitis and other droplet or contact transmission diseases are also markedly lower than those in the same periods in previous years. the covid- pandemic has brought about lifestyle changes, including the encouragement of wearing masks, handwashing, maintaining social distance and suspension of mass gatherings. these prevention measures for severe acute respiratory syndrome coronavirus (sars-cov- ) transmission may have contributed to a decline in many types of infectious diseases. lai et al. suggested that these interventions for infection control may also positively influence pulmonary tuberculosis. however, whether or not the decline in tuberculosis incidence is actually due to these prevention measures, as with other respiratory infectious diseases, is unclear. tuberculosis can be pathologically divided into primary and secondary (reactivation) tuberculosis. an increase in the numbers of elderly patients with newly diagnosed tuberculosis has been observed worldwide, and this disease typically develops as a result of reactivation of a latent tuberculosis infection a long time, usually several dozen years, after the initial tuberculosis infection. as such, the infection control measures enacted to prevent sars-cov- transmission a short time after the covid- outbreak are not expected to influence the trend in tuberculosis incidence. in japan, as seen in taiwan none. the covid- pandemic and tuberculosis in taiwan. the journal of infection different patterns of influenza a and b detected during early stages of covid- in a university hospital in são paulo, brazil. the journal of infection unintended consequence: influenza plunges with public health response to covid- in singapore. the journal of infection seasonal influenza activity during the sars-cov- outbreak in japan ministry of health law, diseases nioi tuberculosis in the global aging population the authors thank mr. shinya takano (srl, tokyo, japan) for providing data. key: cord- -mvlib ol authors: hatachi, takeshi; michihata, nobuaki; takeuchi, muneyuki; matsui, hiroki; fushimi, kiyohide; yasunaga, hideo title: early steroid pulse therapy among children with influenza virus-associated encephalopathy date: - - journal: j intensive care doi: . /s - - - sha: doc_id: cord_uid: mvlib ol background: influenza virus-associated encephalopathy (iae) can lead to neurological sequela and mortality among children. therefore, instant recognition and therapeutic intervention for iae are crucial. in some clinical subtypes of iae, steroid pulse therapy might be beneficial, especially when it is administered in the early phase. however, early identification of patients who may benefit from steroid pulse therapy is sometimes difficult. we aimed to assess the effectiveness of early steroid pulse therapy among children with iae. methods: in this retrospective observational study, we used a national database that covers half of the acute care inpatients across japan to identify inpatients aged ≤ years with a diagnosis of iae between july and march . unfavorable outcome was defined as a composite outcome of sequela including japan coma scale ≥ at discharge, requiring tracheostomy, mechanical ventilation, enteral tube feeding, rehabilitation at discharge, or in-hospital death. propensity score matching was performed to compare unfavorable outcome and in-hospital mortality between patients with and without steroid pulse therapy within days of admission. results: among patients included in the study, the mean age was . years, and . % were male. the overall in-hospital mortality was . %, and the proportion of the unfavorable outcome was . %. we observed no significant difference in the unfavorable outcome between matched patients ( patients in each group) with and without early steroid pulse therapy ( . % vs . %; p = . ) or in-hospital mortality ( . % vs . %; p = . ). conclusions: we did not observe the effectiveness of early steroid pulse therapy on patient outcomes among children with iae in our study population including all clinical subtypes of iae. further studies considering severity of illness are warranted to determine whether steroid pulse therapy is beneficial, especially for specific clinical subtypes of iae. acute encephalopathy, one of the most serious complications among children with influenza virus infection, can lead to neurological sequela and mortality [ ] . neurological symptoms and death occur very early from the onset of influenza symptoms [ ] . instant recognition and intervention for influenza virus-associated encephalopathy (iae) are therefore crucial. iae has been reported worldwide [ ] [ ] [ ] [ ] [ ] , but most frequently in japan [ ] [ ] [ ] . this implies difference in genetic background of patients between japan and other countries [ ] . iae is generally classified according to clinical subtypes including clinically mild encephalitis/ encephalopathy with a reversible splenial lesion (mers), acute encephalopathy with biphasic seizures and late reduced diffusion (aesd), and acute necrotizing encephalopathy (ane) [ ] . in some iae cases, increased levels of cytokines in the serum or cerebral fluid were observed [ ] [ ] [ ] , and immunomodulatory therapy such as steroid pulse therapy was therefore proposed and is widely used in addition to systemic support and anti-influenza agents [ ] [ ] [ ] . some previous studies reported that only "early" steroid pulse therapy could be beneficial among children with iae [ , ] , while most frequent clinical iae subtypes such as mers had favorable outcomes without aggressive therapeutic interventions including steroid pulse therapy [ ] . however, identification of clinical subtype of iae or prediction of patient prognosis are sometimes difficult in the early phase when steroid pulse therapy is thought to be beneficial [ , ] . therefore, it is necessary to assess whether early steroid pulse therapy is beneficial among children with iae as a whole. in this study, we aimed to assess the effectiveness of early steroid pulse therapy on patient outcomes among children with all subtypes of iae using a nationwide database in japan. this was a retrospective observational study using a nationwide database. all data were retrospectively abstracted from the diagnosis procedure combination database in japan. this database is a national inpatient database that includes administrative claims and discharge abstract data from more than participating acute care hospitals across japan. all academic hospitals are obliged to contribute to the database, and community hospitals participate voluntarily. approximately % of all acute-care inpatient data of all ages in japan are included in the database. the details of this database were described in a previous study [ ] . patients aged ≤ years who were admitted to hospitals with a diagnosis of iae and discharged between july and march were included in the study. diagnosis of iae was identified according to documented diagnosis of influenza virus infection and encephalopathy during the same hospitalization. diagnosis of influenza virus infection was determined according to the international classification of diseases th revision (icd- ) codes of j (influenza due to identified zoonotic or pandemic influenza virus), j (influenza due to identified seasonal influenza virus), and j (influenza, virus not identified), as well as a manually documented diagnosis of influenza virus infection. diagnosis of encephalopathy was determined according to diagnostic codes including virus-associated encephalopathy (icd- code: g ), acute encephalopathy (g ), and delirium due to other medical condition (f ). when a patient was admitted twice or more to the same hospital with a diagnosis of iae during the study period, only the first admission with a diagnosis of iae was included. we excluded the following patients: ( ) those transferred from other hospitals because their therapeutic interventions at the initial hospital were unknown, ( ) those transferred to other hospitals within days of admission because their outcomes were difficult to determine, ( ) those who died within days of admission to avoid immortal time bias, and ( ) those aged < month at admission to avoid including unproved congenital diseases such as metabolic disorders. unfavorable outcome was defined as a composite outcome of neurologic sequela and death during the hospital stay including ( ) the japan coma scale (jcs) ≥ at discharge, ( ) requiring tracheostomy during hospital stay, ( ) requiring mechanical ventilation at discharge, ( ) requiring enteral tube feeding at discharge, ( ) requiring rehabilitation at discharge, and ( ) in-hospital death. these unfavorable outcomes did not include those that already existed before admission with iae. jcs comprises four categories: alert, -digit (awake without stimulation), -digit (awake with stimulation), and -digit codes (non-awake with stimulation) [ ] . each category has subcategories ( , , in the -digit code; , , in the -digit code; and , , in the -digit code). jcs indicates that a patient did not open his/her eyes spontaneously; however, he/she opened them in response to a normal volume of voice. anti-influenza agents included peramivir hydrate, oseltamivir phosphate, laninamivir octanoate hydrate, and zanamivir hydrate. intravenous antiepileptic drugs included thiopental, thiamylal, propofol, midazolam, diazepam, fosphenytoin, phenytoin, phenobarbital, and levetiracetam. vasoactive agents included adrenaline, noradrenaline, dopamine, and dobutamine. steroid pulse therapy was generally administered for consecutive days and comprised mg/kg bodyweight of intravenous methylprednisolone per day [ ] . we defined steroid pulse therapy as intravenous administration of any steroid equivalent to methylprednisolone ≥ mg/kg body weight during the hospital stay that was initiated within days of admission. we collected patient demographic data such as age, sex, body weight at admission, and data on the hospitals they were admitted to. congenital anomaly was detected according to the icd- codes q - , and epilepsy was detected according to the icd- codes g and g . epilepsy did not include patients diagnosed after the incidence of iae. we collected the jcs at admission and at discharge, tracheostomy, mechanical ventilation, enteral tube feeding, and rehabilitation during the hospital stay. patients with missing data were excluded. we also collected the in-hospital mortality rate of patients, date of admission and discharge, and therapeutic interventions described above. categorical variables were evaluated using fisher's exact test. continuous variables were evaluated using the student's t test. propensity score matching was performed with : nearest-neighbor matching without replacement using a caliper width of . of the pooled standard deviation of the logit of the propensity scores between patients with and without steroid pulse therapy [ ] . the following factors were included to estimate propensity scores: age category (< year, - years, - years, - years), sex, congenital anomaly, epilepsy at admission, jcs category at admission ( , - , - , and - ), and the following factors that were implemented within days of admission: anti-influenza agents, intravenous antiepileptic drugs, acetaminophen, mechanical ventilation, vasoactive agents, computerized tomography or magnetic resonance imaging, cerebrospinal fluid examination, admission to intensive care unit, and immunoglobulin. we calculated absolute standardized differences to check the balance in the distributions of these factors in the unmatched and propensity scorematched groups with and without steroid pulse therapy. absolute standardized differences of < % are generally considered negligible imbalances [ ] . we then compared the patient outcomes between the propensity score-matched groups with and without steroid pulse therapy using fisher's exact test. a post hoc sensitivity analysis was performed using propensity score matching for patients with an icd- code of g (virus-associated encephalopathy) or g (acute encephalopathy) and excluding those with code f only (delirium due to other medical condition). statistical significance was defined when two-sided p values were < . . all analyses were conducted using stata version . (stata corp llc, college station, texas). this study followed the strengthening the reporting of observational studies in epidemiology (strobe) reporting guideline [ ] (supplementary data). during the study period, patients aged ≤ years were admitted to hospital with a diagnosis of iae and discharged. among these, patients were transferred from other hospitals, patients were transferred to another hospital within days of admission, and patients died within days of admission. two patients were transferred from other hospitals and were transferred to other hospitals. there was no neonatal patient aged < month. consequently, patients were included in the analysis (fig. ) . among those included in the analysis, the mean age was . years ( % confidence interval (ci), . - . ), ( . %) patients were male, ( . %) patients had a congenital anomaly, and ( . %) patients had preexisting epilepsy at admission. these patients were cared for in hospitals across japan. the mean length of stay was . days ( % ci, . - . ). the overall inhospital mortality was out of ( . %) among all children with iae identified in the database and out of ( . %) among patients included in the analysis. table shows the patient characteristics and therapeutic interventions among unmatched patients with and without early steroid pulse therapy. among patients with steroid pulse therapy (≥ mg/kg) initiated within days of admission, ( . %) received ≥ mg/kg of steroids. the median initiation day of steroid pulse therapy was . days ( % ci, . - . ). table also shows the characteristics and therapeutic interventions administered within days of admission between propensity score-matched patients with and without early steroid pulse therapy. absolute standardized differences were ≤ % for most of the characteristics, except for epilepsy ( . %). there were no missing data in the database regarding the variables included in table . among patients included in the analysis, the number of patients with each unfavorable outcome is shown in table . there were ( . %) patients with the composite unfavorable outcome. the mean length of stay was . days ( % ci, . - . ) and . days ( % ci, . - . ) among patients with favorable and unfavorable outcomes, respectively (p < . ). there were no missing data in the database regarding the outcome variables included in table except for jcs scores for deceased patients; therefore, none of the patients were excluded from the analyses. table shows patient outcomes between propensity score-matched patients with and without early steroid pulse therapy. there was no significant difference in the proportions of the unfavorable outcomes between matched patients with and without early steroid pulse therapy ( . % vs . %; p = . ) or in-hospital mortality ( . % vs . %; p = . ). when performing a post hoc sensitivity analysis by propensity score matching for patients with an icd- code of g (virus-associated encephalopathy) or g (acute encephalopathy) (n = ), the proportions of the composite unfavorable outcomes were out of ( . %) and out of ( . %) in patients with and without early steroid pulse therapy, respectively (p = . ). in this sensitivity analysis, in-hospital mortalities were out of ( %) and out of ( . %) in patients with and without early steroid pulse therapy, respectively (p = . ). in this retrospective observational study, we identified pediatric patients admitted to hospital with a diagnosis of iae during seven influenza seasons between and . of these, we included eligible patients in the analysis and identified that the mortality rate was . %, and the unfavorable rate was . %. we observed no significant difference in patient outcomes between the propensity score-matched patients with and without early steroid pulse therapy in our study population, including all clinical subtypes of iae. iae is generally diagnosed when a patient with influenza virus infection develops neurological symptoms such as delirious behavior, seizure, vomiting, consciousness disorder, and coma [ , ] . these neurological symptoms usually occur within a few days of influenza virus infection symptoms such as fever, rhinitis, and cough [ ] . although, there are no established diagnostic criteria, we included patients who were clinically diagnosed with iae by physicians at each hospital and identified those patients according to icd- codes and documented diagnosis in the database. in our study population, the mean and median ages were . years and . years, respectively (the most frequent age was year), and . % were male, similar to previous studies [ , ] . additionally, a previous study fig. flowchart of patients in the study. dpc database diagnosis procedure combination database, iae influenza virus-associated encephalopathy. on the basis of the international classification of diseases th revision, ( . %) patients out of patients included in the study had the g code (virus-associated encephalopathy), ( . %) had the g code (acute encephalopathy), and ( . %) had the f code (delirium due to other medical condition) including duplications. of these, patients had g and g codes, had g and f codes, and had g and f codes reported that . % of children with iae had no underlying disease before the onset of iae [ ] , which was similar to our study ( . % without congenital anomaly or epilepsy). therefore, most of the patients with iae were previously healthy children. among iae as a whole, in-hospital mortality was as high as . % in a questionnaire study from to [ ] ; thereafter, it improved to . % in another questionnaire study between and [ ] , was . % in a national surveillance database study between and [ ] , and was . % in our study between and . similarly, the proportions of patients with neurologic sequela or mortality were reported as . % in - [ ] , which improved to . % in - [ ] and . % in - in our study population. the differences in mortality and proportions of unfavorable outcomes may be explained by recent improvements in patient outcome among children with iae [ ] , differences in case definition and severity of iae, reporting bias according to the study design, and differences in the definition of unfavorable outcome. however, although patient outcomes including sequela and mortality have improved recently, they remain unacceptable. epilepsy patients did not include those diagnosed after the incidence of influenza virus-associated encephalopathy iae includes variety of clinical manifestations such as aesd, ane, and mers [ , ] . in patients with iae, brain dysfunctions are thought to be caused by cerebral edema [ ] . although the pathogenesis of iae is poorly understood, it may involve viral invasion to the central nervous system, inflammatory cytokines, metabolic disorders, and genetic susceptibility [ ] . influenza virus is rarely detected in blood or cerebrospinal fluid samples, which suggests direct invasion of the virus into the central nervous system, although this is uncommon [ ] . however, increased cytokine levels in serum or cerebral fluid were observed among children with iae [ ] [ ] [ ] . although chemokines and proinflammatory cytokines play essential defense roles against influenza virus [ ] , exuberant immune responses with excessive levels of them described as a "cytokine storm" may lead to extra pulmonary complications including encephalopathy [ ] . therefore, in some clinical subtypes of iae, immunomodulatory therapies to mitigate the systemic cytokine storm have been proposed. these therapies include steroid therapy [ ] [ ] [ ] , immunoglobulin [ , ] , cyclosporine [ ] , and plasma exchange [ ] . among these therapies, steroid therapy in the form of methylprednisolone steroid pulse therapy is documented in the japanese clinical guidelines for iae [ ] and widely used in japan [ ] . however, such an immunosuppression therapy in patient with infections might lead to more virulent illness, and evidence for these treatments are not sufficient [ ] . therefore, we aimed to assess the effectiveness of steroid pulse therapy among children with iae using a nationwide database in japan. in our study population including all clinical subtypes of iae, after adjusting for the factors in table , we did not observe a significant difference in the proportions of unfavorable outcomes or in-hospital mortality between the patients with and without early steroid pulse therapy within days. the effectiveness of steroid pulse therapy among children with iae has not been assessed previously in randomized controlled studies. a previous descriptive study identified the wide use of steroid pulse therapy ( . %) for iae in japan [ ] . they observed lower incidences of severe disability or death among children who received "earlier" steroid pulse therapy: out of patients who received steroid pulse therapy within day of the onset of iae had severe disability or death, out of patients who received therapy on the second day had severe disability or death, and out of patients who received therapy on or after the third day had severe disability or death. however, as described by the authors, the severity of illness at admission was not adjusted for in their study. another previous study investigated patients with ane without brain stem lesions (n = ; influenza related, n = ), and observed a lower incidence of unfavorable outcomes among patients who received early steroids: out of patients who received early steroids (within h) had unfavorable outcomes, and out of patients who received steroids after h or who did not receive steroids had unfavorable outcomes (p = . ) [ ] . however, that study did not adjust for the severity of illness at admission, and no difference was observed between patients who received steroids and patients who did not receive any steroids. in our study population including all clinical subtypes of iae, after adjusting for several factors such as patient characteristics, status at presentation, and therapeutic interventions, we did not observe effectiveness of early steroid pulse therapy. however, patients with some clinical subtypes of iae may have benefit from the steroid pulse therapy [ , ] . therefore, further studies are needed in this regard. the present study had some limitations. first, this was a retrospective study, and all the data relied on past records in the database. additionally, we only assessed the japanese database; therefore, the generalizability of the results is limited. second, we excluded patients who were transferred from and to other hospitals. third, data on laboratory results such as higher creatinine [ , ] , [ , ] , serum aspartate aminotransferase, alanine aminotransferase [ , ] , and lower platelet counts [ ] were not available, although they may have been associated with patient outcomes. similarly, we did not include results for electroencephalography or brain imaging such as computerized tomography and magnetic resonance imaging, which may have influenced the initiation for steroid pulse therapy. fourth, iae has several subtypes, which have different prognoses including aesd, ane, and mers [ ] for which information was not available in the database. nonetheless, we believe our results are important because identification of specific clinical subtype of iae is sometimes difficult, especially in the early phase when steroid pulse therapy is thought to be beneficial [ , ] . furthermore, although the icd- code of f (delirium due to other medical condition) may have included mild iae cases who did not benefit from the early steroid pulse therapy, the sensitivity analysis that excluded patients with the f code showed the same results. fifth, although we adjusted for potential risk factors such as requiring mechanical ventilation, vasoactive agents, or admission to an intensive care unit, we did not include validated risk adjustment tools in our analyses. sixth, our composite outcome was not validated. although validated outcome measures such as pediatric cerebral performance category (pcpc) scale [ ] may be warranted, they were not available in the database. nonetheless, because the pcpc scale includes patient consciousness or coma status, we included jcs scores, which are thought to be correlated with the glasgow coma scale scores, in our composite outcome [ ] . seventh, we did not identify long-term patient outcomes. a previous study reported that moderate or severe disability was maintained; however, mild disability may have improved thereafter [ ] . in our retrospective observational study, we did not observe a significant difference in the proportions of unfavorable outcomes or in-hospital mortality between the patients with and without early steroid pulse therapy within days of admission among children with iae as a whole. however, further studies considering severity of illness are warranted to determine whether steroid pulse therapy is beneficial especially for specific clinical subtypes of iae. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . abbreviations iae: influenza virus-associated encephalopathy; mers: clinically mild encephalitis/encephalopathy with a reversible splenial lesion; aesd: acute encephalopathy with biphasic seizures and late reduced diffusion; ane: acute necrotizing encephalopathy; icd- : international classification of diseases th revision; jcs: japan coma scale; ci: confidence interval acute encephalopathy associated with influenza and other viral infections encephalopathy associated with influenza epidemics pediatric neurological complications of pandemic influenza a (h n ) acute childhood encephalitis and encephalopathy associated with influenza: a prospective -year review neurological manifestations of influenza infection in children and adults: results of a national british surveillance study influenza-associated encephalitis/encephalopathy identified by the australian childhood encephalitis study fatal viral infectionassociated encephalopathy in two chinese boys: a genetically determined risk factor of thermolabile carnitine palmitoyltransferase ii variants epidemiology of acute encephalopathy in japan, with emphasis on the association of viruses and syndromes national surveillance of influenza-associated encephalopathy in japan over six years, before and during the - influenza pandemic characteristics and outcomes of influenza-associated encephalopathy cases among children and adults in japan the puzzling picture of acute necrotizing encephalopathy after influenza a and b virus infection in young children cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus-associated encephalopathy elevated serum levels of neutrophil elastase in patients with influenza virusassociated encephalopathy predictive value of serum interleukin- level in influenza virus-associated encephalopathy the specific therapy for influenza encephalopathy in japan guideline for the management of influenza associated encephalopathy outcome of acute necrotizing encephalopathy in relation to treatment with corticosteroids and gammaglobulin prognostic factors for acute encephalopathy with bright tree appearance prediction of acute encephalopathy with biphasic seizures and late reduced diffusion in patients with febrile status epilepticus validity of diagnoses, procedures, and laboratory data in japanese administrative data the eye response test alone is sufficient to predict stroke outcome-reintroduction of japan coma scale: a cohort study constructing a control group using multivariate matched sampling methods that incorporate the propensity score balance diagnostics for comparing the distribution of baseline covariates between treatment groups in propensity-score matched samples the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies encephalitis and encephalopathy associated with an influenza epidemic in japan acute encephalopathy and encephalitis caused by influenza virus infection detection of influenza virus rna by reverse transcription-pcr and proinflammatory cytokines in influenza-virus-associated encephalopathy prostaglandin e : the villain in the host response to influenza virus the hidden burden of influenza: a review of the extra-pulmonary complications of influenza infection. influenza other respir viruses diagnostic and prognostic factors for acute encephalopathy assessing the outcome of pediatric intensive care treatment of severe intraventricular hemorrhage by intraventricular infusion of urokinase short and long-term outcomes in children with suspected acute encephalopathy publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions th, nm, mt, and hy contributed to the conception and design of this study; th, nm, mt, hm., kf, and hy collected and analyzed data; th drafted the manuscript; nm, mt, hm, kf, and hy critically reviewed the manuscript and supervised the whole study process. the author(s) read and approved the final manuscript. this work was supported by grants from the ministry of health, labour and welfare, japan ( aa to h.y. and h -policy-designated- to h.y. and k.f.) and the ministry of education, culture, sports, science and technology, japan ( h to h.y.). the datasets generated and analyzed during the current study are not publicly available complying with data-sharing policies of the database.ethics approval and consent to participate this study was approved by the institutional review board at the university of tokyo (number: -( )). given the anonymous nature of the database, informed consent was waived for this study. the authors declare that they have no competing interests. key: cord- -yfaji cv authors: kim, yong-kyun; sohn, hong-gyoo title: disaster theory date: - - journal: disaster risk management in the republic of korea doi: . / - - - - _ sha: doc_id: cord_uid: yfaji cv to find a conclusive definition for contemporary purposes and uses, we look at many of the various definitions of disasters through cataclysmic events, historical records, public policies, laws, and organizational usage. our natural progression leads us to modern theories of disaster and disaster risk management (drm) that have had to tackle new types of disasters that are being brought about by the interconnectivity of societies, people, diseases, technology, etc., increasing in magnitude and complexity like what was seen in fukushima, japan, in and on-setting disasters like climate change. after looking at all the historical evidence, we come to a definition for the term disaster for modern usage and what it means for policy implications. risk as "the probability of harmful consequences, or expected loss (of lives, people injured, property, livelihoods, economic activity disrupted or environmental damaged) resulting from interactions between natural or human induced hazards and vulnerable conditions" (undp ) . in the national infrastructure protection plan (nipp ), department of homeland security (dhs) considered risk elements as "threat nature and magnitude, vulnerability to a threat, and consequence that could result." haddow et al. ( ) noted that risk is composed of ( ) the probability and frequency of a hazard occurring, ( ) the level of exposure of people and property to the hazard, and ( ) the effects or costs, both direct and indirect, of this exposure. the korean word for risk is "wiheom" or "wiheomdo." finally, the origin of the word "crisis" is the greek word "krinein (separate; critical moment);" and it has been used as the medical term of "decisive moment of life and death." quarantelli ( ) viewed that a crisis of a certain organization appears in the three following interrelated conditions: ( ) a type of threat including organizational value, ( ) sudden occurrence of an unexpected event, and ( ) need to respond collectively as the outcome may seem more negative otherwise. the term "crisis" is defined as "a difficult or dangerous situation that needs serious attention" in the merriam-webster's learner's dictionary (webster dictionary ) . the korean word for crisis is "wigi." the term indicates a dangerous crucial moment or time and the state to make the final decision on a critical matter. the term "wigi" is a combination of the two characters: "wi" meaning danger and "gi" meaning chance. in the same context, lee et al. ( ) defined crisis as an "incomplete state that desperately needs a significant change or the turning point to determine whether to proceed with, modify, or end an event or a behavioral process." disaster means a condition where the damage cannot be overcome without national or external assistance due to lack of capacity or resources of the damaged community or local/state governments uniqueness korea a presidential decree regulates the magnitude of disaster, which can be financially supported by the national government usa disaster is classified into emergency and major disaster declaration depending on the magnitude of the damage japan disaster caused by natural hazard was historically emphasized in act unisdr a comprehensive approach considering human, physical, economic, or environmental losses and impacts is emphasized fritz considers disaster as the disruption of the essential functions of the social system quarantelli considers disaster as what is caused by natural or technological hazard that is beyond the knowledge of modern society jeong ( ) saw the word "crisis" as natural disasters such as typhoons, heavy snowfalls, and floods; human and technical disasters such as explosion, traffic accidents, and collapse; comprehensive risk situations in various contexts such as terrorist attacks, status of diplomatic relations with north korea, failure of various policies, financial difficulties of firms, stores, and state, crimes, spread of diseases, and various scandals. the terms related to disaster can be summarized as table . . summarizing the discussions above, "disaster" can be conceptualized as follows: • a state that cannot be recovered to the original state with the ability of the community due to great damages in human life and property • inclusive of disasters triggered by natural, technological, or social hazard the term "disaster" will be used embracing natural, technological, and social disasters that the whole community need to cope together reflecting the characteristics of contemporary societies. therefore, in this book, we will define "disaster" as "a status of community or nation's being seriously damaged by natural, technological or social cause and difficult to recover from the damage with its own resources, requiring the whole community to cope together." this definition is similar to the definition of disaster by unisdr and consistent with the definition by laws in korea, japan, and the usa. originates from the old french word "sauf" meaning "not damaged or not harmed," or the latin word "salvus" meaning "not damaged or healthy" the condition of being safe from undergoing or causing hurt, injury anbo security originates from se (¼without, liberation) + cura (¼care, concern, or distress) the state of being protected or safe from harm wiheom (do) originates from the spanish word "risque," known to be used from the mid-seventeenth century a probability of harm that encompasses threat/hazard, vulnerability, and consequence wigi crisis originates from the greek word "krinein" (separate) dangerous crucial moment or time in this context, referring to "jaehae" as natural disaster and "jaenan" as human-caused disaster is not appropriate, which had been commonly used in korea from to ; it is still partially used. both "jaehae" and "jaenan" should be used as terms pointing out natural, technological, and social disasters. to be more precise, "jaehae" means the damage caused by hazards, and "jaenan" means the situation that creates or is likely to create damage. like the concept of disaster, its categorization is also very diverse. the typical one is a categorization by disaster cause. the other ways include categorization by the characteristics of the event, phase-related categorization, and categorization by surgical and medical disaster. in korea, the current framework act on the management of disasters and safety classifies disasters into natural and social disaster. its original version enacted in classified disasters into natural, human-caused, and social disasters, but the same act when it was amended in integrated "human-caused disaster" and "social disaster" into "social disaster." thus, disaster in the current korean law is divided into "natural disaster" and "social disaster." the categorization by disaster cause is a typical way about disaster type. most of renowned organizations dealing with disaster data, such as center for research on the epidemiology of disasters (cred) and munich re, and many scholars have used disaster cause as the principle of disaster categorization. the official categorization of disasters in korea is "natural disaster" and "social disaster" regulated by disaster-related laws, which are differentiated by causing phenomenon. the countermeasures against natural disasters act, formerly the disaster control act and the disasters and safety act, defined disaster-related terms as below: • countermeasures against natural disasters act (act no. , amended on march , ) the purpose of this act is to prescribe necessary matters concerning natural disaster prevention or recovery and other countermeasures against natural disasters, in an effort to preserve national land and to protect lives, bodies, and properties of nationals as well as key infrastructures from disasters caused by natural phenomena, such as typhoons, floods, etc. this act defined "jaehae" as any damage caused by "jaenan" and encapsulated "jayeon-jaehae," meaning natural disaster and "pungsu-hae," meaning disaster triggered by hydrological hazard as specific examples of "jaehae." article (definitions) the definitions of the terms used in this act shall be as follows: . the term "jaehae" means any damage caused by "jaenan" under subparagraph of article of the disasters and safety act (hereinafter referred to as the "framework act"); . the term "jayeon-jaehae" means any disaster caused by typhoon, flood, heavy rain, strong wind, wind wave, sea wave, tidal water, heavy snowfall, lightning, drought, earthquake (including any earthquake-caused tsunami), yellow dust or other natural phenomena corresponding thereto, from among disasters falling under subparagraph ; . the term "pungsu-hae" means any disaster caused by typhoon, flood, heavy rainfall, strong wind, wind wave, sea wave, tidal water, heavy snowfall or other natural phenomena corresponding thereto. • disaster control act (enacted on july , , act no. and abolished on june , ) this act, abolished when the disasters and safety act was established on march , , states that the purpose of the act is to establish a disaster and safety control system of the state and local governments, establish a system for prevention of and countermeasures against disasters as well as emergency rescue, declare a special disaster area, and define matters such as emergency measures necessary for disaster management in order to protect the lives and property of citizens from disasters due to artificial causes. this act was enacted to prepare for human-caused disasters, such as arson and building collapse, rather than natural disasters, such as drought and floods. thus, it targets technological disasters, such as explosions, collapse of bridges, traffic accidents, and chemical, biological, and radioactive accidents, like the leakage of hazardous material and environmental pollution incidents. • framework act on the management of disasters and safety (enacted on march , , act no. ) the framework act on the management of disasters and safety that took effect in categorized disaster into natural, human, and social disaster and defined them as follows: article (definitions) the terms used in this act shall be defined as follows: . the term "disaster" means any of the followings, which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state: (a) disasters caused by a typhoon, flood, downpour, strong wind, wind and waves, tidal wave, heavy snowfall, lightning, drought, earthquake, sandy dust, red tide, ebb and flow and other natural phenomena equivalent thereto; (b) damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto; (c) damage caused by the paralyzation of the state's backbone systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases. . the term "overseas disaster" means those which actually cause or are likely to cause any harm to the lives, physical safety and property of citizens of the republic of korea outside the territory of the republic of korea, and which require the government to take measures thereon. • framework act on the management of disasters and safety (partially amended on august , , act no. ) types of disasters were categorized into natural and social disasters in the disasters and safety act partially amended on august , , defining them as follows. the korean government classifies standardized disaster types on the standard crisis management manual as shown in table . according to the statutory classification of disasters summarized above. article (definitions) the terms used in this act shall be defined as follows. . the term "disaster" means any of the followings which actually causes or is likely to cause any harm to the lives, physical safety and property of citizens and the state. (a) natural disasters: disasters caused by a typhoon, flood, downpour (the korean word "howoo"), strong wind, wind and waves, tidal wave (the korean word "hae-il"), heavy snowfall, lightning, drought, earthquake, sandy dust (the korean word "hwangsa"), red tide (the korean word "jeokjo") outbreak, ebb and flow (the korean word "josu"), and other natural phenomena equivalent thereto (b) social disasters: damage beyond the scale prescribed by presidential decree, such as a fire, collapse, explosion, traffic accidents, chemical, biological and radioactive accidents, and environmental pollution incidents and other accidents similar thereto, and damage caused by the paralyzation of the state's critical systems, such as energy, communications, transportation, finance, medical treatment and water supply, and by a spread, etc. of infectious diseases under the infectious disease control and prevention act, and contagious animal diseases under the act on the prevention of contagious animal diseases enforcement decree article (scope of disasters) "damage beyond the scale prescribed by presidential decree" in subparagraph (b) of article of the disasters and safety act (hereinafter referred to as the "act") means the following harm: . harm to human life or property for which measures at the level of state or local government are required; . other harm deemed by the administrator of the national emergency management agency to be necessary for disaster control, and which is equivalent to the harm referred to in subparagraph damage on cultured marine products and fishery production facilities due to a discoloration of seawater to red or light brown caused by an explosive growth in phytoplankton density ※ article of the act on the prevention of and countermeasures against agricultural and fishery disasters 「methods regarding the monitoring, prediction of red tide, and damage prevention」(directive of ministry of maritime affairs and fisheries) drought a prolonged shortage of water supply in a certain region due to a period of below-average precipitation or insufficient available water resources: it can be categorized as a meteorological drought, a hydrological drought, an agricultural drought, or a socioeconomic drought depending on its usage tidal wave damage by level rise due to the effects of tides, weather tidal waves, swells, and abnormal waves in costal or estuarine waters social disaster forest fire trees, weeds, fallen leaves, etc. in a forest or in an area adjoining to a forest are burned by a fire artificially or naturally ignited ※ article of the forest protection act chemical accident all situations that occur because a chemical flows out or leaks out to humans or the environment due to the fault of a worker as at the time he/she works, such as replacement of facilities, defects in facilities or deterioration of facilities, a natural disaster, a transport accident, etc. ※ article of the chemicals control act (continued) disaster type definition water pollution accident that oil, toxic substance, or sewage or wastewater is discharged to river, causing continuous large-scale suspension of water intake or perish of fishes and significant effects on the people's living and natural ecosystem large-scale marine pollution accident accident caused by the crack of oil tank due to sinking, stranding, or collision of a ship resulting in a significant and extensive damage to the people's living and natural ecosystem and requiring comprehensive measures at the government level utility-pipe conduit accident occurred on the facility installed underground for smooth maintenance, aesthetic improvement, preservation of road structure, and smooth traffic flow by accommodating underground facilities (electricity, gas, waterworks supply facilities, communication facilities, drainage system, etc.) commonly collapse of dam accident of leak or overflow occurred due to natural disaster such as large-scale flood or earthquake or structural defect of dam, concerning the collapse of dam large-scale subway accident accident occurred due to the collision, derailment, fire, explosion or flooding of electric train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale high-speed railroad accident accident occurred due to the collision, derailment, fire, or explosion of high-speed train in operation, causing casualty or expecting the suspension of train operation for a long period of time large-scale fire at multiuse facility accident at a facility used by the many and unspecified general public concerning which has a high concern of large casualty and property damage in case of large-scale fire radiation exposure from a neighboring country situation requiring national protection measures due to a large-scale radiation exposure or radiation contamination exposure accident from a nuclear facility of neighboring country marine vessel accident accident occurred due to the collision, fire, explosion, stranding, sinking of vessel, loss of loaded cargo, or other hull damage large-scale casualty accident at workplace accident of large-scale casualty such as physical explosion related to tasks at the workplace, largescale collapse accident at construction site, choking accidents due to oxygen deficiency, acute poisoning accident due to exposure to chemicals, or other equivalent accidents multiuse facility large building collapse accident accident at a facility used by the many and unspecified general public concerning large casualty and property damage in case of collapse of building (continued) paralysis of essential national financial information function due to natural disaster, strike, terrorism, and electronic infringement on the financial information system which is the key national infrastructure safety of nuclear facility disaster caused by radiation leak or radioactive contamination from a nuclear power plant and research reactor facility electric utility serious impediment to national life and paralysis of national functions due to anxiety about the electricity demand and supply caused by increased demand, defected facilities, and social conflicts demand and supply of oil serious hazard to the national safety and the stability of national economy due to imbalance between demand and supply of oil caused by foreign oil producing countries health and medical services serious hazard to national health due to the paralysis of healthcare system drinking water national crisis situation such as suspension of drinking and industrial water supply due to water pollution, destruction of facility due to natural and artificial disasters, and strike and cyber terror cargo transport situation which may cause or lead a significant crisis in the national logistics system due to refusal of (continued) by cred and munich re the cred is a research institute founded in by lechat, a professor in belgium and has been studying international disasters and disputes for over years. it has been a world health organization (who) collaborating center since and is operating an international disaster database called emergency events database (em-dat). the disaster classification system of em-dat is in four levels: generic group, subgroup, main type, and subtype. generic group, the highest level of the system, classifies disaster into natural and technological disasters. the natural disaster category is divided into six subgroups: biological, geophysical, climatological, hydrological, meteorological, and extraterrestrial disasters. each subgroup in turn covers disaster types and more than subtypes. more detailed information can be found in the em-dat report (cred ) . natcatservice provided by munich re group in germany is another database related to disasters that wins global recognition, using a natural disaster classification system similar to em-dat. natcatservice enters all natural disasters that cause human and physical damages regardless of scale, but it does not mention artificial/technological disasters (munich re ) . in , cred and munich re developed and presented a joint standard classification system for natural disasters (cred and munich re ). this can be considered the international standard classification system for natural disasters. this system classifies disasters into five levels, dividing disaster into two generic disaster groups of natural disasters and technological disasters. natural disasters are then divided into six disaster groups, geophysical, meteorological, hydrological, climatological, biological, and extraterrestrial, each of which is then classified into main type, subtype, and sub-subtype. however, this system is limited in that it cannot present a detailed classification system for artificial/technological disasters, as munich re does not have the classification for such disasters. threat and hazard identification and risk assessment the threat and hazard identification and risk assessment (thira) is a four-step common risk assessment process that has been designed by the dhs in the usa. this process helps the whole community identify risks, and enables the community to build local resilience to the risks. according to thira, threats and hazards are divided into three disaster type definition people in cargo truck transport business to cargo transport as a group without justifiable grounds resulting in suspension or setbacks of cargo transport gps signal interference service interruption of national infrastructure due to intentional or unintentional interruption of gps signal reception for national core infrastructure and service using gps cosmic radio wave disaster related to radio wave occurred due to change in electromagnetic energy existing outside of the earth's atmosphere types as listed in table . . the natural hazards are the types that result from acts of nature, such as flood, earthquakes, drought, pandemics, epidemics, or more. the technological hazards are the types that result from the accidents or the failures of systems and structures, such as transportation accidents, collapse, hazardous materials spills, or more. the human-caused incidents are the types that result from the intentional actions of an adversary, such as terrorism, sabotage, cyber incident, chemical attack, biological attack, or more. classification of disasters by their cause has been useful for deciding governmental actions toward disaster recovery because it clearly indicates the responsibility of who will pay for what, e.g., supporting disaster victims and/or providing disaster relief. however, the categorization is not useful for disaster response because the response way is not different depending on its cause. therefore, a new prism is needed. kim et al. ( ) have analyzed the occurrence of large-scale disasters in korea since and the government's response to them and have proposed new optics for classifying disaster types for advancing disaster responses. to this end, two important elements were considered: the evolutionary or devolutionary pattern of the magnitude of disaster damage and the roles and responsibilities of responding agencies. regarding the first element of the pattern of disaster damage, three types can be derived: ( ) events with measurable prediction of large-scale damage, ( ) events with unpredicted high social shocks with quick evolution and devolution independent of its magnitude, and ( ) events slowly evolving to large scale. large-scale damage caused by typhoons, torrential rain, and heavy snowfall can be predicted according to weather forecasts and precedence. train crashes and building collapses are types of disasters that suddenly evolve and devolve, leaving not only physical damage but also a shock to the social fabric. finally, foot-andmouth disease, infectious disease, and red tide are disasters that commence on a small scale, or with little notice, but over time become considerable disasters that require large-scale disaster response tactics. the reason why this classification is important is that the response system, such as the timing of the operation of the central disaster and safety countermeasure headquarters (cdschq) and the central disaster management headquarters (cdmhq), is different depending on the progress or evolution of the damage. secondly, the scope of the overall coordination that cdschq should undertake varies depending on whether there are one or more disaster management authorities. the role of the coordinating authority becomes important for typhoons, torrential rain, and heavy snowfall, since they are types of disasters that simultaneously cause damage to various facilities, which will result in various agencies, such as the ministry of construction, the ministry of agriculture, the ministry of environment, etc., engaging the disaster together. for effective response to these types of disasters, it is important for the coordinating authority to support the human and material resources necessary for the various disaster responses by the disaster management agencies. on the other hand, for the disasters such as aircraft accidents, infectious diseases, and dust storms, expertise and experience are concentrated in specialized disaster management authorities, meaning it is efficient for the agencies responsible for those disasters to respond. the category by kim et al. ( ) is very useful in developing an optimum disaster response system fitting the disaster cause and evolutionary path. in this book, we will propose the disaster category for effective disaster response based on the category by kim et al. ( ) as outlined in table . . dombrowsky ( ) suggested event-related concept and phase-related concept as disaster categorization. the event-related concept is composed of time, space, and severity, while the phase-related concept is composed of pre-emergency phase, emergency phase, warning, and post-emergency phase. methods of classifying disasters include one in terms of emergency and medicine. in the field of emergency and medicine, disaster is classified into surgical and medical disasters. the purpose of this categorization is to treat efficiently patients during disasters by securing effectiveness of first aid on-site and hospital treatment. surgical disasters are mostly those in which victims are injured, and they refer to disasters in which damage from physical disasters appears in the form of injury. medical disasters or disease disasters refer to chemical disasters that cause hindrance to respiratory organs and metabolic systems due to the leakage of chemicals, radioactive matters, or toxic agents. this classification method has significance in that it is possible to predict the conditions of the patients in disasters and effectively manage medical resources accordingly (kim and lim ) . in case of instant disaster with single primary response agency, such as dam failures, major rail accidents, and maritime accidents, the cdschq and the cdmhq are established simultaneously; the cdmhq takes charge of response, and the cdschq coordinates the pan-governmental support primary response agency takes charge of its own responsibility in the early stage of the event. as the damage evolves, the charge is transferred to the cdschq. this type of event includes infection, animal disease, red tide, and green tide contemporary society is becoming vulnerable to new types of disasters, such as new epidemics including ebola virus and middle east respiratory syndrome coronavirus (mers-cov), new animal diseases including bovine spongiform encephalopathy (bse) and hpai, and complex disasters such as the tohoku earthquake and tsunami in along with typical types of disasters like typhoon, fire, and building collapse. in addition, the characteristics of complexity in modern times require new and innovative approaches that are different from current response ways. there has recently been much research in various fields, such as sociology and science of public administration, in order to understand what increases disaster complexity in modern times. this section will review representative disaster theories and find how these theories explain major disasters that occurred in korea and around the world. based on this understanding, we will recommend the desirable policy change for effective disaster response in korea. representative theories, such as heinrich's law, normal accident, risk society, and complexity theory, will be reviewed in the following section. herbert william heinrich, who was an assistant superintendent at an american insurance company, presented an important study in based on his analyses of a wide range of accidents. he coined the notion of heinrich's law, which states that, for every major accident, there have been preceding minor accidents and signs of anomaly (heinrich ). heinrich's law is therefore also known as the law of : : . in other words, an accident that is large enough for people to notice is only a small tip of an iceberg and is always antedated by countless other accidents and happenings that warn of the upcoming disaster. through a scientific statistical approach on industrial disasters that were once believed to take place unexpectedly, heinrich found that massive disasters occurred due to negligence on minor defections. originally, the heinrich's law was applied to industrial disasters, but it is now extended to rules related to accidents, disasters, and failures throughout all kinds of areas in contemporary society. the : : rule is meaningful in that it sets up a rule to explain quantitatively the process of an evolutionary incident, starting from a small accident and resulting in a catastrophic event. he also applied the domino theory to disaster areas and indicated that in order for disasters to take place, an inappropriate "direction of flow" occurs sequentially: many causes interact with complexity, and those causes generate interwoven influences. as a result, phenomenon called disaster or accident takes place, and ultimately it causes human and physical disasters, which is a major argument of the domino theory. the theory emphasizes fundamental elements before accidents to take place and explains there are three potential elements as follows: the first condition -human genetic component or socially undesirable phenomena the second condition -flaws caused by the first condition the third condition -unsafe actions, mechanical and physical, according to the second condition among the three conditions that cause a disaster, the first condition of inherited elements or social environment and the second condition of inherited or acquired human defects are hard to be rectified; however, the risk by the third condition can be significantly reduced through safety education and strengthened safety devices. in other words, heinrich insisted that if the third condition is effectively eliminated, disaster can be prevented before it occurs (heinrich ) . the sampoong department store collapse accident in korea is a typical example of the heinrich's law. the accident happened because no countermeasures had been taken even though there were many signals before the accident. the department store opened its business with inborn structural problems during the construction process: about tons of installation equipment was installed on the rooftop, which was four times more than its original design load, and reinforcing bars were out of place. along with problematic construction, poor management was another cause of the accident: minor symptoms such as cracks in the ceilings and damage to the floor of the rooftop (potential elements of ) were overlooked by staff and maintenance crews. in addition, although customers and employees raised several concerns about the general health of the building, such as vibrating sound from an air conditioner and many cracks in the wall, no specific actions were taken, even after receiving evaluations from experts ( small accidents). neglecting potential elements eventually led to one huge accident with casualties (one massive accident) (lee et al. ) . we found that most of the major social disasters, such as the seongsu bridge collapse, the sampoong department store collapse, the daegu subway fire, and the sewol ferry sinking accident, have common things to cause the disasters: improper alteration of use, insufficient safety culture, insufficient safety inspection, and insufficient safety infrastructure. heinrich's law indicates the direction of disaster response that korea should follow: institutional reforms for strengthening disaster and safety management, improvement of safety inspection, expansion of safety education, and enhancement of disaster and safety infrastructure. in the book normal accidents, charles perrow, a professor of sociology at yale university, insisted that complex systems, such as nuclear power plants, chemical factories, aircrafts, ships, dams, and gene manipulation, hold a risk of tragedy. thus, there are some unavoidable accidents called normal accidents that have a high probability of occurring regardless of safety measures and devices (perrow ) . charles perrow proposed the normal accident theory based on the empirical evidence of the three mile island (tmi) nuclear power plant accident that took place in pennsylvania in , the first nuclear power plant crisis to have occurred. perrow ( ) defines a normal accident, which he also refers to as a system accident, as "an event that involves the unanticipated interaction of multiple failures, reflecting the characteristics of high-risk system in which multiple and unexpected interactions of failures are inevitable." perrow's theory insists that accidents in today's cutting-edge technological societies are closely related with complex technological and mechanical structures with built-in risks and those risks are therefore a normal part of our lives. an extremely complex system, in which individual technologies interact with one another closely and inseparably through an endless loop, is prone to catastrophe caused by a series of breakdowns whenever one of the interwoven elements begins to dysfunction. it is nearly impossible for humans to predict such technical failures (park ) . perrow noted that conflicts of interests might exist between a given organization and its members and that organizations are also subject to external political and social environments. technical solutions are therefore not enough, particularly as it is impossible to ensure the total control and containment of risks in such highly complex private facilities as nuclear power plants and petrochemical factories, at which minor errors can lead to complete failures (perrow ). the three mile island accident is considered as the typical example of normal accident. this brought widespread panic to the usa as large portions of one of the reactor's cores partially melted, releasing radioactive gases and hazardous iodine into the surrounding environment. the direct cause of the accident was officially recorded as a mistake by operational staff; however, the complexity of the disaster is a hidden root cause of the catastrophic event. therefore, as perrow indicated, complex systems with built-in intensive risk, such as nuclear power plants, require that centralized and decentralized management should be considered simultaneously. there have been a lot of industrial accidents around the world. accidents, such as the india bhopal chemical factory accident, the chernobyl nuclear power plant radiation leak, and the bp gulf oil spill, indicate that industrial safety measures need constant improvement through comprehensive understanding about high-risk technology and systems. some scholars criticized that the normal accident theory overemphasizes the vulnerability of high-risk facilities; the high reliability theory is a typical example of the critics. professors from uc berkeley and weick, an organizational theorist, proposed the high reliability theory based on research of organizations fraught with disaster risk, such as national aeronautics and space administration (nasa), the nuclear industry, aviation, nuclear carriers, swat, and massive petrochemical facilities in the usa. the high reliability theorists insist that sophisticated quality controls, a settled safety culture, built-in safety by cross-check, and continuous education and training can prevent disasters at high-risk facilities. irrespective of these critics, perrow's normal accident theory provides a beneficial lens through which we may view and analyze risks in contemporary society. because the theory explains risks as a matter of organizational characteristics of the ecosystem, it overcomes the shortcomings of the science and technology-centered approach. it provides sociological explanations for organizational risks that are likely to cause massive collateral damage, such as in petrochemical factories and nuclear power plants (jeong ). after the earthquake that occurred on september , in korea, social interest in the safety of high-risk facilities such as nuclear power plants and old industrial complexes has increased. in accordance with etkin ( ) , who noted that careful thought should be given to the construction of complex tightly coupled systems, the korean government should entirely overhaul the safety of risky facilities such as nuclear power plants, industrial complexes, and chemical plants. moreover, there is a need for society-wide attention and efforts to provide a more sophisticated safeguard system. around the s, an in-depth exploration on new risks that occurred in modern society was conducted by european scholars, such as luhmann, giddens, and beck (lee ) . ulrich beck, the german sociologist, suggested the concept of risk society as a solution to structural and deep-rooted problems of industrial societies, including science and technical safety issues, which started in the mid- s (lee et al. ) . in his book world risk society ( ), ulrich beck argued that the modern society is a "risk society" replete with risks all throughout and emphasized the multiplication of cross border risks and international dangers that single nationstates cannot tackle on their own (beck ) . during his lecture at seoul national university in , beck pointed out the similarities between the asian financial crisis in the s and the chernobyl nuclear plant accident in , stressing the importance that all nations must work together to reduce the global risk on the basis of a common understanding that they are facing the same global risk. beck also considers this emerging risk as a result of the de-bounding of traditional national boundaries in the spatial, temporal, and social dimensions. beck defined risk society as a society where socially produced risk is inherently accompanied by socially produced wealth (oh ) . he also insisted that industrial society should be addressed through a comprehensive perspective, which includes social, historical, and technological views (beck ). as the world entered into a contemporary society, new types of risks, combined with secondary, supernatural, and artificial uncertainty, had arisen, and those risks were beyond a dimension of traditional response methods (lee ) . the background with such phenomena includes a complex structural change that is understood as the term of postmodernity. generally, risk, unlike danger that indicates direct and physical loss, is based on the probability of prediction or control: a new concept of "uncontrollable risk" needs urgent attention because this type of risk denies the applicability of currently available risk theories and risk control mechanisms, arising new threats (beck ). the concept of risk society by beck has a critical meaning to korea's drm. the increased risk can be also found in urbanization in korea. lee et al. ( ) emphasized the increased risk due to urbanization by showing the increased disaster risk following urban development with the change of green space to paved road. korea has also suffered from newly emerging disaster risks, such as severe acute respiratory syndrome (sars), foot-and-mouth disease, and mers-cov. therefore, an innovative and cooperative approach to disaster risk suggested by beck needs to be reflected in designing resilient future. complexity theory began from researching complex natural phenomena such as meteorology. just as lorenz proved with the butterfly effect, the contemporary society embedded with complex network needs to take different countermeasures against disaster. the fukushima nuclear power plant accident of japan, a great flood in thailand, and new types of epidemics such as mers-cov are representative examples of disasters that the contemporary society is newly facing. the complexity theory is useful for understanding the characteristics of contemporary disasters and finding appropriate countermeasures. on march , , the fourth strongest earthquake struck japan, since japan started to observe earthquakes, with a mega tsunami and aftershock that caused more than , deaths. also, the earthquake destroyed the nearby fukushima nuclear power plants and caused radiation leakage. this disaster showed signs of complex disaster. due to the fukushima accident, air, soil, ocean, and underground water were exposed to radioactivity, and damages from contamination influenced largely not only japan but also the whole world continuously. the severe flood in thailand is a typical example that a disaster that happened in one country affected the regional economy. due to a heavy rain, combined with low topographic characteristics of thailand and high tide of seawater, two thirds of the land in thailand, including parts of bangkok, was flooded, causing significant damage to the entire manufacturing industry. the flood did not affect just thailand. for example, many japanese companies' production facilities located in the central region, having developed the area for the manufacturing of automotive and electronic goods, were inundated during the flood. the intensity of the flooding caused all of the facilities to be shut down, which caused tremendous economic loss for both countries. additionally, the shutdown of the automotive plants caused a reduction of japanese auto and parts deliveries to other major markets like japan, the usa, and europe. in case of the electric and electronic industry, hard disks became in short supply due to flooding of hard disk manufacturing factories. due to this, the production of semiconductors by intel became inadequate, resulting in a short supply of semiconductors to the world's semiconductor market. moreover, it caused a price increase of computers worldwide. also, since the severe flood occurred right before the tourist season, the number of tourists in the fourth quarter decreased by approximately %, and additionally induced damages occurred in other industries, such as transportation and food services. due to the influence of the heavy flood, the estimated economic growth rate of thailand in was lowered from . % to . % (korea institute for international economic policy ). table . shows the summary of damage that occurred to the japanese economy due to the heavy flood in thailand. this table shows that the flood was not just problematic for thailand but indicates that a disaster occurring in one country does influence other countries. the contemporary society is facing new types of epidemics and animal diseases, such as sars ( ), h n virus ( ), foot-and-mouth disease ( ), and mers-cov ( ), which did not exist in the past, and such symptoms have a high possibility of becoming more frequent due to increase in trade and traveling with foreign countries. pelling ( ) argued that complexity theory possesses a very important lesson to understanding ways to cope with disasters. for example, one of the characteristics of complexity theory is "emergence," which shows a trait of disasters that take place with unexpected causes at an unexpected place. according to drabek and mcentire ( ) , "emergence" appears while people change organizations in the process of making temporary organizations and responding to disaster situations. beck's risk society and perrow's normal accident theory share the similar understanding with complexity theory in that the emerging risk in modern society is closely related with its increased complexity. therefore, the core elements of complexity theory, such as nonlinearity, self-similarity, fractal, self-organization, and emergence, are essential in understanding disaster characteristic and innovating disaster response methods in modern society. as disasters started to become complexed and intensified, scholars and stakeholders began to look for new theories and methods to improve disaster risk knowledge and organizational capabilities. starting in the s, several theories, such as petak's four-phase model, mcloughlin's comprehensive disaster management procedures, and the new public administration theory, were developed to deal with these new complex and intensified disasters and to help modernize and reform institutions that were becoming too antiquated to deal with modern disasters. organizations such as the usa's fema, created in the s, used the progressing knowledge and concepts to help it mature in the s, and the un and world stakeholders, who came together in the early s to produce the hyogo framework for action (hfa), used the foundations' insights to increase disaster resilience around the world. investigating the historical progress of such knowledge and the evolution of response organization is important in figuring out what logical steps should be made for a resilient future as well as in strengthening organizational capabilities to better deal with complex and intensified disasters. in , petak proposed a four-phase model to identify the role of governments and stakeholders in each disaster management phase. he divided pre-disaster management and post-disaster management according to the progress of disasters and countermeasures and explained disaster management procedures in a timesequential manner: . disaster mitigation . disaster preparedness . disaster response . disaster recovery petak emphasized that the clear delineation of roles and responsibilities of all levels of governments and stakeholders is essential for effective disaster management four main areas in japan reduced production by % nippon steel corporation adjusted release of the crude steel in japan due to effect of reduced automobile production honda suspended the operation of four-wheeled vehicle plant in malaysia toshiba had trouble in hard disk drive (hdd) production pioneer had trouble in the production of car navigation system, relocated from thailand to malaysia deteriorated business results expects billion yen decrease in sales automobile possibility of sales decrease of billion yen for five automobile companies suspended distribution suspended the transport based on flooded areas paid insurance tokio marine and fire insurance co., ltd. began investigation to provide insurance to japanese client companies (petak ) . petak's model influenced the basic structure of the framework act on the management of disasters and safety (hereafter "disasters and safety act") in korea, which is shown in fig. . . the new public administration theory emphasizes that a government can provide better public service to citizens by adopting business management principles to public management (rosenbloom and goldman ) . the theory aims to overcome the problems of a typical bureaucracy and to improve the efficiency of the public sector by adopting business management skills and by emphasizing output and outcome of public policy rather than the input to implement the policy (rosenbloom and goldman ) . the theory was accepted through the national performance review project during the clinton administration, and laid the foundation for fema's reformation (waugh and streib ) . the demand for the reformation of fema started from hurricane hugo, which hit north carolina and the virgin islands in , causing $ billion in damages. in the aftermath of hurricane hugo, the loma prieta earthquake struck california, a fire broke out in oakland, and hurricane andrew struck florida and louisiana, dealing a massive blow to the two states. disappointed by fema's ineffective response to these massive natural disasters, political groups and citizens in the usa raised the need to improve fema's performance, which was endorsed by the clinton administration in (anna et al. ). james lee witt, appointed as director of fema by president clinton, emphasized disaster mitigation and shifted from recovery-oriented policy to preventionoriented policy. additionally, he insisted that disaster mitigation investment should be based on the assessment of the effectiveness. consequently, government policy has continued to embrace this line of thinking. to quantify the future savings of hazard mitigation activities, the multihazard mitigation council (mmc) of the national institute of building sciences (nibs) analyzed three major hazard mitigation grant programs: the hazard mitigation grant program, project impact, and the flood mitigation assistance program. the independent study proposed the following significant findings: ( ) for every dollar spent on mitigation, four dollars was saved from future spending; and ( ) fema mitigation grants beget nonfederally funded mitigation activities (nibs ) . the new public administration theory affected the development of disaster management in korea: the increased investment to structural and nonstructural measures for disaster mitigation and the evaluation of the effectiveness of disaster mitigation and recovery projects in the s are typical examples of the effect of the theory. although the theory receives criticism in that it does not consider the characteristics that disaster management has as a public service, it can provide a useful prism to improve the effectiveness of disaster management policy by focusing on the outcome or performance of the policy, rather than the input. public governance has been acknowledged as a way to improve public-private partnership in disaster management. in the public governance theory, rules govern the behavior of actors not as the result of official authority or market equilibriums, but according to the consensual process among participants, based upon networks and cooperation. the concept of collaborative networks in disaster management entails the assumption that parties involved in disaster management have diverse cultural backgrounds and are bound to experience conflicts. effective cooperation in this setting requires cultural sensitivity and mutual understanding from all participants. collaborative networks, moreover, are becoming important because, in modern society with emerging and complex risks, it is not possible to ensure perfect preparations and resources for all possible disasters and because a specific organization or one single agency cannot perfectly control all response agencies involved in managing disasters (waugh and streib ) . the advantage of public governance theory is that it strengthens shared responsibility by enabling diverse social groups to participate in the decisionmaking process so as to tackle uncertainty with social intellect and make policy decisions based on the social consensus. the theory emphasizes cooperation, public participation, problem-solving, and openness as key principles of disaster management. it aims to form a collaborative risk governance system consisting of diverse actors, including governments, businesses, and civil society organizations, in which national government serves as facilitator in promoting the development of a decentralized collaborative network among local governments, nonprofit organizations, and various public services. however, there is an opinion that the public governance theory is hard to be applied to emergency response. in other words, in an urgent disaster response process where there is not enough time to make a consultation, the process of negotiation through mutual discussion and consultation is not appropriate (waugh and streib ) . the argument seems reasonable, but recent researches have shown that interagency cooperation is becoming more important in an emergency situation. moynihan pointed out that establishing network governance in advance are an essential element in achieving the two objectives of "interagency cooperation" and "coherent response" in a crisis situation for effective disaster response (moynihan ). public governance theory is also important in the development of the disaster response system in korea. the recent major disasters in korea, such as the mauna ocean resort gymnasium collapse accident, the sewol ferry sinking accident, and the middle east respiratory syndrome, taught korea important lessons that cooperation among all relevant organizations, such as on-site response agencies, local disaster and safety countermeasure headquarters (ldschq), various line ministries involved in the cdmhq, and the cdschq, is essential for effective disaster response and relief. therefore, the korean government should develop an interagency cooperation plan and execute a joint field training program; demanding the participation of all relevant organizations specified above. in late , more than federal organizations related to civil engineering and defense had fragmented responsibilities for hazard mitigation and disaster response, resulting in no coordinating organization taking the full responsibility for the entire phase of disaster management. president jimmy carter created fema in for this reason. the establishment of fema made it possible to unify the fragmented responsibilities of emergency preparedness and response resources (anna et al. ) . mcloughlin ( ) proposed an integrated emergency management model. considering disaster as an incident or condition that threatens the survival of organizations, he was concerned with the fact that troubles in cooperation among related agencies repeatedly occurred during disaster response since various public and private groups had been engaged in disaster response without comprehensive coordination. he argued that a comprehensive and integrated emergency management system could sustain administrative capabilities during emergencies and protect property and life through a series of circulation processes under the cooperation of the federal, state, and local governments. this model emphasizes that each local government and the federal government should cooperate to protect life, property, and government functions through a program of mitigation, preparedness, response, and recovery (cho ) . quarantelli ( ) also emphasized that comprehensive disaster management is essential in modern times in order to build a comprehensive and unified organization that manages all types of disasters. he argued that a dispersed or separated approach, by which each line ministry is responsible for its own disaster; devoid of a coordinating agency, cannot deal with complex and intensified disasters in modern times. additionally, he asserted that a comprehensive and integrated method be used when managing disasters because: first, disasters have become more complex and capable of destroying the functionality of typical community operations, meaning that dividing disasters into natural or human-caused ones is inadequate for effective response; second, there is an underlying commonality among governmental departments to respond to disasters; therefore, comprehensive measures are required for a unified effort; third, the planning process and its contents for disaster response by each department has commonality, thus, it is ineffective that each department develops its own response plan; finally, the shared response resources among the governmental departments reveals that a move to a comprehensive management format is feasible since vital resources are similar in each department (quarantelli ). global cooperation for effective disaster relief had been a pivotal issue among the international society. to improve the international cooperation for disaster relief to affected nations, the united nations established a responsible agency, labelled as "the united nations disaster relief organization (undro)," in . since then, the international community has been working for developing a systematic disaster management framework; disaster response plan, disaster prevention measures, and scientific and technical solutions to disaster risk have been discussed and adopted. in particular, technical approaches, such as vulnerability analysis for disasters and early warning measures, have been tried. additionally, the scientific approach played a key role in the evolution of disaster management through a variety of research on how to identify hazard and assess vulnerability (unisdr ). in , the international society began to prepare the international decade for natural disaster reduction framework to promote more systematic disaster management (unisdr ). the international community established the un humanitarian emergency assistance and the international search and rescue advisory group (insarag) mechanism in . also in , systematic mechanisms for disaster response were introduced by building the united nations disaster assessment and coordination (undac) mechanism. the international conference held in in japan adopted the yokohama strategy that emphasized the paradigm shift from recovery-oriented policies to prevention-oriented policies. in , the name of the international strategy for disaster reduction was adopted with the title "safer world in the st century: disaster and risk reduction." since then, a comprehensive policy framework to cope with increasing global risk has been developed in earnest (unisdr ). in december , a tsunami hit countries in the eastern-western-southern asian regions, such as indonesia, sri lanka, and india. in the wake of the catastrophic event, national governments, international organizations, un agencies, and other stakeholders agreed to adopt "the hyogo framework for action (hfa): building the resilience of nations and communities to disasters," to mitigate global disaster risk for - . with the hfa, disaster management moved toward building resilience based on social consensus and strengthening shared responsibilities among all stakeholders. in march , all national governments also agreed to the sendai framework for disaster risk reduction - (sfdrr), which is the first major agreement on disaster management in line with post- development agenda. the sfdrr aims for the significant reduction of disaster risk and losses with the following four priority actions: understanding disaster risk, strengthening disaster risk governance to manage disaster risk, increasing investment in disaster risk reduction for resilience, and enhancing disaster preparedness for effective response and to "build back better" in recovery, rehabilitation, and reconstruction. the drr-related global agenda in various fields will be further reviewed and analyzed in chap. in order to link them with disaster resilience in the future. in the s, massive disasters continued to occur throughout the world. national governments have reformed their disaster response system to deal with massive disasters. the usa reformed its drm system after / in , shifting from a typical natural and human-caused disaster management paradigm to comprehensive security (park and cho ) . within this context, since the s, civil defense and civil protection became important concepts in disaster management. civil defense was originally designed to protect citizens from calamities, such as war, but it was gradually expanded to encompass drm, reducing civilian casualties during disasters. alexander ( ) suggested that the role of the state and the civilian in disaster preparedness be shifted from civil defense to civil protection. this is because civil protection is an appropriate concept to reinforce the protection of people against external risks, meeting the need to protect people from typical disasters, such as natural and technological disasters, as well as emerging risks, such as new infectious diseases, terrorism, and cyber attacks, while civil defense was useful to cope with the invasion of an outside force which was a severe threat during the cold war era. one of the most important features of civil protection suggested by alexander is a shift from providing public safety service by the government to encouraging public participation. when a national government provides public safety services, it usually prefers to use a top-down approach: command and control, a hierarchical decision-making system excluding citizen's participation, strengthening law and order, and rules through the principle of confidentiality. however, the civil protection concept by alexander put stress on a bottom-up approach: emergency preparedness and response ensuring public participation and cooperation, problemsolving approach, and openness principles. as a result, risk governance is the most critical issue in civil protection, ensuring the cooperative interaction among the government, the private sector, and civil organizations to replace the existing hierarchical bureaucratic system. alexander ( ) addressed hurricane katrina as a typical failure case of civil defense with a top-down approach, requesting a shift from a top-down approach to a bottom-up approach, along with improved risk governance. this section will describe disaster response institutions in korea, the usa, japan, and other nations. the analysis of one nation's disaster response institution requires huge efforts and a large amount of work. due to limited time and space, this section will focus on the basic structure of disaster management organizations for normal times and emergency situations at national and local levels. the disaster response institutions in korea, the usa and japan will be analyzed first, and then those in australia, germany, switzerland, and the united kingdom (uk) will be analyzed. in korea, the ministry of public safety and security (mpss) is responsible for the overall coordination of the nationwide disaster response based on the disasters and safety act. after the presidential election on may , , the ministry of interior (moi) has been preparing for the revision of the government organization act, which includes the establishment of ministry of public administration and safety (mopas) by integrating mpss with moi and the establishment of korea fire service and korea coast guard as ind ependent agencies. the act is expected to be reviewed and decided by the national assembly in late . in addition, the crisis management center under the national security council in the blue house (presidnet's office) is expected to work as a control tower for national crisis, such as the sewol tragedy. the korean government has developed its disaster response system suitable for normal times and emergency situations at the national and the local levels. organizations for normal times are composed of disaster management agencies, disaster-management supervision agencies, emergency rescue agencies, emergency rescue, and relief support organizations. in addition, central, city/do, si/gun/gu committees are being operated to deliberate matters on disaster and safety management under their responsibilities. disaster management agencies take charge of all phases of disaster management activities that are related to their responsibilities. the agencies include national administrative agencies, local governments, local administrative agencies, public institutions and organizations, and other organizations prescribed by presidential decree. disaster-management supervision agencies are responsible for disaster response when a disaster or an accident occurs in accordance with the responsibilities prescribed by presidential decree. for example, the ministry of education is responsible for disasters in schools and school facilities, the ministry of environment is responsible for environmental pollution accidents, and the ministry of employment and labor is responsible for large-scale human accidents occurring in places of work. table . shows the disaster-management supervision agencies by disaster or accident type in korea. emergency rescue agencies are responsible for carrying out life rescue, first aid, and other necessary measures to protect the lives and property of citizens when a disaster is likely to occur or when a disaster occurs. the agencies in charge include fire hqs and coast guard hqs, under the mpss, city/do fire headquarters and si/gun/gu fire stations, and regional headquarters of the korea coast guard and coast guard stations. to support rescue activities, presidential decree prescribed emergency rescue and relief support agencies, which are equipped with human resources, installations, equipment, operation systems, etc., that are necessary for emergency rescue and relief service. to deliberate and decide crucial matters related to disaster and safety management at the national level, the central safety management committee, chaired by the prime minister, is operated. in addition, the safety policy coordination committee, chaired by the minister of public safety and security; the central disaster broadcasting consultative committee, chaired by a person appointed by the minister of the ministry of science, ict and future planning; the central private-public cooperative committee, chaired by the vice minister of public safety and security; and a civilian representative are being operated to advise, consult, deliberate, or sometimes decide disaster and safety management issues under their responsibilities. at regional and local levels, a city/do safety management committee, a si/gun/gu safety management committee, a city/do disaster broadcasting consultative committee, and a si/gun/gu disaster broadcasting consultative committee are operated. when disaster occurs or is likely to occur, emergency response organizations are established and operated to take timely and proper measures at national, regional, and local levels. the emergency response organizations at the national level include the cdschq, chaired by the mpss; the cdmhq, chaired by the head of the relevant disaster-management supervision agency; and the central emergency rescue control group (cercg), chaired by the head of the central fire headquarters for disaster occurring on land and the chief of the central rescue center for disaster at sea, respectively. the local emergency response organizations include the city/do disaster and safety countermeasure headquarters (city/do dschq), chaired by mayor or governor, and the si/gun-gu disaster and safety countermeasure headquarters (si/gun/gu dschq), chaired by the head of si/gun/gu. for rescue activities, the local emergency rescue control group (lercg), chaired by the head of the fire headquarters and a chief of a fire station, is operated; when disaster occurs at sea, the head of a si/gun/gu emergency rescue control group and the head of a city/do emergency rescue control group shall be respectively construed as the chief of a regional rescue center and the chief of a metropolitan rescue center under article of the rescue and aid at sea and in the river act. when disaster occurs or is likely to occur, the mpss will immediately hold a situational meeting to supervise the initial response, rescue, and first aid operations. in particular, in the event of a major disaster, the ministry will operate the cdschq and coordinate the intergovernmental disaster response activities. in case of an oversea disasters, the minister of foreign affairs shall exercise the authority of the head of the central countermeasure headquarters, and in cases of radioactive disasters, the chairperson of the nuclear safety and security commission shall exercise the authority of the head of the central countermeasure headquarters, respectively. in case government-wide integrated response is necessary, the prime minister may exercise the authority of the central countermeasure headquarters. in such cases, the minister of public safety and security, the minister of foreign affairs (limited to cases of overseas disasters), or the chairperson of the nuclear safety and security commission (limited to cases of radioactive disasters) shall be the vice head. when a disaster occurs in a jurisdiction, the mayor or provincial governor and the head of a si/gun/gu shall set up their own dschq and coordinate the response and recovery operations. in order to efficiently operate the cdmhq under article - ( ) of the disasters and safety act, the head of the disaster-management supervision agency shall predetermine necessary matters for organizing, operating, etc. of the cdmhq and exercise the authority of the head of the headquarters. also, local disaster management headquarters shall be established to work as an action team of cdmhq in the disaster area. the disaster response organizations during emergency situations is shown in fig. . . the disaster response plan in korea consists of three parts: the standard risk management manual, the working-level manual for risk response, and the manual for actions-at-scene. as of may , kinds of standard risk management manuals, working-level manuals for risk response, and , kinds of manuals for actions-at-scenes have been prepared and utilized. the standard risk management manual, prepared by a disaster-management supervision agency, delineates roles and responsibilities of related agencies in disasters at the national level, which shall be the guidelines for preparing the working-level manual for risk response. the working-level manual for risk response is a document stipulating the measures and procedures necessary for responding to actual disasters in accordance with the functions and roles of the disaster-management supervision agency and support agencies, which are stipulated in the standard risk management manual. the manual for actions-at-scene, prepared by implementing agencies, such as local governments, stipulates in detail the procedures for actions to be taken by an agency that directly performs its duties at a disaster scene. the standard risk management manual shall be prepared by each disaster management supervision agency that is regulated in table . . however, the standard risk management manual for disasters involving many disaster management authorities, such as typhoon and drought, can be prepared by the minister of mpss. the working-level manual for risk response shall be prepared by related support agencies that are designated by the standard risk management manual. the manual for actions-at-scene shall be prepared by an agency designated by the working-level manual for risk response. the head of the si/gun/gu may develop several disaster types of manuals for actions-at-scenes in consolidation as needed. the manuals delineate roles and responsibilities of the disaster-management supervision agency, related support agencies, and implementing agencies. on the other hand, the operational functions of disaster response of each agency shall be designated by action plans for disaster response by function under article - of the enforcement decree of the disasters and safety act. the functions for disaster response, similar to the emergency support function (esf) of national response framework (nrf) in the usa, are described below: . managing disaster situation . supporting emergency livelihood stabilization . supporting emergency communications . emergency restoration of facilities damage . restoring damaged energy supply facilities . supporting disaster management resources . traffic countermeasures . supporting medical and disinfection services . environmental arrangement at disaster scenes . supporting and managing volunteer work . maintaining social order . searching, rescuing, and emergency support at disaster areas . publicity of disaster management in summary, the two axes of the disaster response plan in korea are three levels of manual and functional action plans. the manual describes the roles and responsibilities of primary response ministries and related agencies, and the functional action plans describe how each agency performs its key response functions in line with its roles and responsibilities. the disaster response organizations during emergency situation are: cdschq, city/do dschq and si/gun/gu dschq for overall coordination; cdmhq and ldmhq for the implementation of their own responsibilities; cercg and lercg for search and rescue; and support agencies. both the establishment and evolution of fema and dhs in the usa had influenced the disaster management system in korea; in particular, the establishment of the national emergency management agency (nema) in and the establishment of the mpss in . in addition, the integrated disaster management formed by the dhs, the nrf, and the national incident management system (nims) has also influenced the evolution of the disaster response system in korea. dhs, established in , is responsible for national security and disaster management. fema under dhs is in charge of all phases of disaster management, including national preparedness, public and private capacity assessment, mobilization of resources for emergency management and disaster relief, and long-term recovery plans. at the state level, the disaster management department focuses on strengthening the linkage between the federal government, the state, and the local governments and assisting the local government in disaster prevention, preparedness, response, recovery, and relief. when a disaster occurs, the emergency operations center (eoc) commences operation and responds to disasters in accordance with a preestablished disaster response plan (eop, emergency operation plan, or cemp, comprehensive emergency management plan). local governments have a primary responsibility for the whole process of the localized disaster, prevention, preparedness, response, and recovery of disaster, and for the activation of eop followed by executing the eoc to respond to disasters. when a disaster that exceeds the capacity of a local or a state government occurs, federal government's assistance and involvement is requested and is provided through the joint field office (jfo). the usa performs disaster management through nrf and the nims. be that as it may, the catastrophic events on september , ( / ), and august , (hurricane katrina), saw the federal government's failure to provide proper support to state and local governments for effective disaster response. after that, there is an increasing demand for the federal government to respond to disasters proactively by federal emergency declaration to large-scale disasters and pre-deployment of federal resources to states. however, the basic principle of disaster management in the usa is still that local governments are primarily responsible for all disasters, with the support from state and federal governments concerning disasters that exceed the capacity of local governments. the disaster management organizations of the usa are summarized in fig. . . the dhs was established in january to integrate the prevention of terrorism and the function of disaster management under one department's coordination in the wake of the september , terrorist attacks and the mailing of anthrax spores. dhs took charge after the commencement of the national strategy for homeland security and the homeland security act. the dhs carries out tasks that include the suppression of terrorists' attack, minimization of damage, prevention, preparation, response, and recovery in the emergency plans for all domestic and international dangers that threaten the usa. the dhs consists of the office of intelligence and analysis (oia), the information analysis and infrastructure protection (iaip), the chemical and biological (cb) weapon management service, the state affair safety service, secret service (ss), and fema. fema, which is the most well-known organization among the various organizations of the dhs, is responsible for various tasks that include the disaster risk reduction at all levels, the reduction of property loss through various risk-based emergency management programs for the preparation, response, and recovery, and the protection of people's lives and main facilities. fema was established in as an organization coordinating the response and recovery of disasters during the carter administration. however, the initial phaseout of fema was not that high. it had grown into a member of the administrative cabinet and a ministerial level organization during the clinton administration through the establishment of an integrated response system and the strengthening of preventative and mitigation programs after failing to respond effectively to several large-scale disasters such as hurricane hugo. through the establishment of fema, various agencies related to disaster response were integrated into one agent under the direct control of the president and capable of comprehensively responding to various human-caused disasters and natural disasters. after the dhs was established, fema was incorporated and operated as a bureau under the dhs. in the early days, under the dhs, fema's status was weakened since antiterrorism and security were the first priority of the nation. however, after hurricane katrina, the importance of disaster management has been highlighted, and fema has also been strengthened as the independent deputy-minister level agency. fema, headquartered in washington, dc, operates local offices and provides regular disaster support personnel who can respond immediately in case of a disaster. the central organization for when an emergency occurs includes the jfo. the jfo is established by fema for coordinating between state governments and federal agencies after the president declares a state of emergency, and jfo plays a pivotal role in providing coordination between federal, state, and local governments and non-governmental organizations (ngos) and private sector accident supports. the head of a jfo is called the federal coordinating officer (fco), who is designated by the president and carries out the comprehensive coordination and the management of support activities using the resources secured by the federal government. in general, the state and local governments have an organization dedicated to disaster management: a disaster management department at the state level and an emergency office at the local level. a local emergency office (leo) normally consists of divisions handling emergency operation, information and communication, administrative task related to disasters, prior identification of risk, and the modification and supplementation of a risk management plan. in the event of a disaster, the eoc is set up to coordinate or support the disaster response at the site and responds to the disaster in accordance with the incident command system (ics). disaster response plans in the usa are developed at the federal, state, and local levels. at the federal level, the nrf and the nims constitute overall responsibilities of the dhs, fema, line ministries, and other agencies. the purpose of the nrf was to connect government agencies with ngos and the private sector and transparently assign and coordinate key roles and responsibilities nimbly. the nrf consists of the base document, the esf annexes (esf annex), the support annexes, and the incident annexes. table . shows the organization of the nrf. the local disaster response plan is carried out through the preparation of an eop by each area. the plan incorporates all aspects of disaster management in any given area, and it guides the roles and responsibilities of all related agencies for disaster response, depending on the size and complexity of a disaster. eop consists of the basic plan enclosed with the annex including the esf, the administrative and financial support plan, and the incident annex. nims provides national response doctrine for the whole community to work together based on the principle of the nrf. the nims defines standardized command and control principles to enable various response agencies to coordinate in the event of a terror and disaster. it provides a standardized response principle that allows federal, state, local governments, and nongovernmental organizations to respond consistently to a disaster regardless of the cause, size, location, or complexity of the disaster. to this end, it defines concepts, principles, organizations, rules, procedures, and terminology that provide a structured framework that is flexible, applicable, comprehensive, and geographically balanced for all types of disasters. the on-scene commander of the nims directs and controls the response at the incident site, and the head of the eoc is responsible for supporting the incident site while coordinating and managing local resources from outside the incident site. the components of the nims include preparedness, communication and information management, resource management, technical support, continuous management, and maintenance. in , fema developed "state nims integration" to facilitate states to adopt the nims. the guidelines require states to develop both an "emergency operation plan" and a "procedural document." the eop is a response plan that adapts the disaster response principles and the emergency support functions specified in the nrf tailored to the situation of the state. the procedural document consists of general principles for disaster response, standard action procedures, on-site operation guides, and job aids. each standard operating procedure (sop), the instruction for carrying out esf tasks to enable the smooth support for disaster response in the field and how to carry out those disaster response tasks, contains the guiding principles listed in the esf. it can be drawn from fema's actions that the modified us disaster management system would emphasize comprehensive, integrated, and mutual cooperation among the relevant organizations and stakeholders. to solidify this new emphasis, fema announced in seven guiding principles to cope with national emergencies: comprehensive, progressive, risk-driven, integrated, collaborative, coordinated, flexible, and professional (emergency management ). describe key roles and responsibilities around the nation and structures for implementing nationwide response policy and operational coordination for all types of domestic emergency events emergency support function annexes specify the federal resources and capabilities to provide emergency support for functional areas and identify coordinator, primary agency, and support agency for each functional area support annexes describe common and basic supports to the majority of incidents: critical infrastructure and key resources support, financial management support, international coordination support, etc. describe the response methods for seven incident categories: biological, catastrophic, cyber, food and agriculture, mass evacuation, nuclear/radiological, and terrorism. the basic act for disaster management in the usa is the stafford disaster relief and emergency assistance act. it gives the state the right to request support from the federal government and the right of the federal government to supplement resources to requesting state. additionally, it gives the president the right to declare emergency or major disaster in order to provide federal assistance. the homeland security act and the post-katrina emergency management reform act are also important disaster-related laws. japan has been exposed to various natural and human-caused disasters, such as earthquake, typhoon, and hazardous material contamination. to cope with those threats, japan has developed a comprehensive disaster management system at the national and local levels. the "basic act on disaster control measures," enacted in , functions as the backbone of the disaster management system in japan. the japanese government enacted the law in the wake of typhoon vera (isewan typhoon), which caused deaths, , injures, and property damage of approximately . trillion yen. since then, the "basic act on disaster control measures" has become the foundation for carrying out all measures related to disasters including emergency countermeasures and recovery as well as the disaster prevention, and this law handles disaster prevention measures in each field comprehensively. after the great hanshin-awaji earthquake on january , , japan revised the master plan for disaster prevention completely, and the "basic act on disaster control measures" was partially revised to reflect the lessons learned from the earthquake. the great hanshin-awaji earthquake, which led to a full modification of the master plan for disaster prevention, resulted in deaths, , houses destroyed completely, and property damage of trillion yen, which accounted for . % of gross domestic product (gdp) at that time. with the occasion of several huge disasters, the disaster management system in japan has been appropriately modified so that a more systematic disaster response can be provided through central disaster prevention meetings. a central disaster prevention meeting (chairman, prime minister) consists of the prime minister, the minister of disaster prevention, related ministers, the representatives of designated public agencies, and people with knowledge and experience designated by the prime minister. this meeting carries out the preparation and implementation of a master plan for disaster prevention and emergency measure plans and the examination of important items regarding disaster prevention according to the advice of the prime minister. the fire and disaster management agency under the ministry of internal affairs and communications, which is equivalent to the central firefighting headquarters in korea, was established based on article , paragraph of the national government organization act and article of the firefighting organization act. the fire and disaster management agency under the ministry of internal affairs and communications takes charge of planning and drawing up firefighting administration procedures and policies of various laws and standards. this agency has no direct right to command firefighting at the local level but handles tasks in an adversarial, instructional, and coordination role. the general affairs and planning department (bureau) and disaster prevention sections in the general affairs division have been established to carry out cooperation and coordination tasks since the cooperation and coordination between relevant local divisions are important for disaster management. the local disaster prevention meeting is divided into prefectures and municipalities. the local crisis meeting of prefectures consists of heads of local administrative agencies, self-defense forces, and superintendents of education with the prefecture governor as the manager. the head of the municipality takes charge of the municipality local crisis meeting. these two agencies take charge of contact and coordination between relevant agencies in case of a disaster and play a role in establishing and implementing a disaster prevention plan to handle each step effectively including disaster prevention, emergency disaster measures, and disaster recovery. the local emergency organization is the local disaster relief center. the local disaster relief center is installed according to a local disaster prevention plan in case a disaster is expected or a disaster occurs. the local disaster relief center is responsible for carrying out disaster prevention and emergency disaster measures related to the relevant prefecture or municipality according to the local disaster prevention plan of the relevant prefecture or the local disaster prevention plan of the municipality. the central/local and normal times/emergency response institutions of japan are summarized in table . . the disaster management system in japan has been established into central and local disaster management systems and regular and emergency disaster management systems to enable a smooth communication among divisions. japan, where various disasters occur frequently, has established various systematic disaster response plans just as the disaster management system. the disaster response plan consists of a "master plan for disaster prevention" for the central government and a "local disaster prevention plan" for the local level. the "master plan for disaster prevention" is a comprehensive long-term plan for disaster prevention prepared by the central crisis meeting, and it is based on the basic act on disaster control measures. it is responsible for determining the comprehensive and long-term plan for disaster prevention, the key points for the disaster prevention task plan and the local disaster prevention plan, and the preparation standard for the disaster prevention task plan and the local disaster prevention plan. based on such disaster prevention plans, japan has prepared for disaster prevention in three steps including prevention, emergency measures, and recovery and redevelopment. each step contains the basic policy for disaster prevention, mutual linkage around the country between public agencies and local governments, and measures to share disaster prevention information between disaster prevention agencies and residents. the central disaster prevention plan and the local disaster prevention plan are executed identically. disaster management in australia utilizes a comprehensive and integrated approach (ema ) . each provincial and local government, in accordance with federal guidelines, establishes a disaster management act that puts forward how local disaster entities should actively and effectively prepare against regional risks. such a decentralized legal system gives each state or local government the responsibility and the flexibility to carry out disaster countermeasures tailored to the characteristics of disasters, which have occurred or are to occur in each area of australia. one of the most influential events that moved opinion for the improvement of disaster management in australia was a large-scale fire called the tasmanian bushfire that occurred on february , . this large-scale fire resulted in deaths, displaced from families, and , ha of land damaged. this fire made the australian government recognize the importance of a disaster management system at the federal level, which resulted in the establishment of the natural disaster organization (ndo) in . this organization was strengthened in due to the needs of more systematic disaster preparedness, and its name was also changed to emergency management australia (ema). currently, ema performs its duty as the standard central organization for disaster management and, based on the commonwealth government disaster response plan (comdisplan), takes charge of planning and the coordination for disaster management at the state, district, and local governmental levels in australia. disaster management in australia is divided into four levels: federal, state, district, and local. the disaster management system is handled by ema at the federal level, whereas disaster management groups (dmg) -state/district/local -manage the disaster management system at the state, district, and local levels. at the federal level, the australian government has established separate disaster management systems suitable normal times and emergency situations, respectively. the ema division in the attorney general's department carries out normal disaster management: national disaster management, planning, coordination, -h disaster status monitoring, international support, and cooperation tasks. in case of emergency situations, the federal government carries out disaster management through the australian government crisis coordination centre (ccc), an emergency center coordinating disaster responses for all line federal departments and state, district, and local governments. the state, district, and local governments in australia also have two types of disaster management systems suitable for normal times and emergency situations, respectively. during normal times, the dmg is the primary organization to deal with disaster management, particularly focusing on prevention and preparedness, which is managed by the department of the premier and cabinet (premier of state government), consisting of the premier of each state government, all state ministers, and the army commander of each state government. the dmg develops and operates disaster management plans, strategies, and policies and supports state, district, and local management groups. in case of an emergency, the manager of the dmg should appoint a disaster coordinator in advance for the cooperation and coordination between the federal government, other state governments, and relevant organizations within state governments. on the other hand, the disaster coordination centre-state/district/local (dcc) was established to handle and operate a disaster management system during emergencies more systematically. the dcc consists of divisions representing each state government, the bureau of meteorology under the federal government, the australian defense force, the australian red cross, and the australian government ) insurance parliament at the senior officer level, and is responsible for decisionmaking and coordination regarding support of resources to local, district, and state governments according to the level of the disaster situation occurrence. the disaster response plans in australia have also been separated into a federal level and a local-level disaster response plan. the disaster response plan of the federal government is the comdisplan, which specifies competent disaster organizations in the six states and seven territories of australia, and the contents and procedures for resource support and cooperation between state governments. the disaster response plan at the state, district, and local levels is called the disaster management plan (dmp) or guideline-state/district/local and consists of four parts: prevention, preparedness, response, and recovery for strategic policies to deal with disaster by delineating roles and responsibilities for each phase in disaster management. the roles and responsibilities of each department and agency are specifically described in esfs. for more specific response activities, the response phase is subdivided into four steps: alert, lean forward, stand up, and stand down. two record-breaking disasters have occurred in australia since . the black saturday bushfires that occurred on february , , resulted in large-scale forest fire damage in the southeastern region of australia. more than forest fires occurred simultaneously resulting in a more significant damage occurrence, and these forest fires resulted in deaths and forest loss of , ha (victorian bushfire reconstruction and recovery authority ). a series of floods called the queensland floods occurred in western brisbane, queensland, on december , . these floods resulted in more than dead, more than missing, and approximately , homes and businesses flooded (queensland floods commission of inquiry, ). these floods were recorded as the largest floods within years, and the continuous rain for weeks made the damage greater. the australian government is in the process of improving its disaster management system in the wake of these two catastrophic events, which is worthwhile to be monitored. according to germany's basic law, the primary responsibility for disaster management rests on local and state governments, and the federal government provides financial, human, and physical assistance to local and state governments when a large-scale disaster occurs that exceeds the capacity of the local government or the state government. in the event of a disaster beyond the capacity of the state, the state will be supported by police departments and military forces in the neighboring states. and if necessary, the state receives the support of the police department, military forces, and technical support from the federal government. the federal ministry of the interior (bmi, bundesministerium des innern) is in charge of coping with major disaster or nonmilitary crisis that require the intervention of the federal government. in particular, the federal office of civil protection and disaster assistance (bbk, bundesamt für bev€ olkerungsschutz und katastrophenhilfe), established in may under the ministry of the interior, is responsible for responding to disasters in an integrated manner through systematic cooperation with other federal, state, and local governments in the event of largescale disaster. bbk has been expanded from the federal office of administration (bundesverwaltungsamt) as the importance of civil protection has increased in germany, in the wake of the september terrorist attacks in the united states in and the floods of the elbe river in germany in august . the main responsibilities of the bbk include the development of a comprehensive civil protection plan, dissemination of emergency information to citizens in crisis situations, critical infrastructure protection, and education and training for civil defense. the joint situation and information center (gmlz, gemeinsames melde-und lagezentrum von bund und ländern) in the bbk is responsible for monitoring disaster situations, disseminating disaster information, and international requests for help. when a large-scale disaster occurs in germany, the federal ministry of home affairs calls up the crisis task force (krisenstab). the task force works jointly with relevant departments within the federal government, agencies under the ministry of interior, and state liaison officers. in the event of a major type of catastrophic event that can be a national crisis, the ministry of interior will form the joint task force with responsible ministries. for example, if a nuclear accident or an illegal use of radioactive materials happens, the bmi and the federal ministry for the environment, nature conservation, building and nuclear safety (bmub, bundesministerium für umwelt, naturschutz, bau und reaktorsicherheit) will form the joint crisis management task force. when pandemics or biochemical terrorism occurs, the bmi and the federal ministry of health (bmg, bundesministerium für gesundheit) will form the joint task force. the german local system consists of the state (länder) government, the city or county (kreis) government, and the municipal government (gemeinde). disaster management organizations in germany's local system are different depending on the characteristics of each region; however, departments dealing with internal affairs mostly take charge of disaster management. in recent years, there have also been a growing number of states establishing disaster risk protection departments (gefahrenabwehr) by experiencing increased terrorism and massive natural disasters due to climate change. the central/local and normal times/emergency institutions of germany are summarized in table . . each state government develops and operates its own disaster response plan. for example, hessen is the state government that has the disaster response plan under the title of katastrophenschutzpläne (emergency plans). katastrophenschutzpläne (emergency plans) of hessen specifies the contents regarding necessary information in the event of disaster and means to be used. also, in case of a very severe disaster such as nuclear risk that targets a specific object, it is required to establish and operate sonderschutzpläne (special protection plans) separately. figure . shows the katastrophenschutzpläne (emergency plans) in hessen, germany, and it shows the contents regarding the sonderschutzpläne (special protection plans). the english translation of fig. . is: § disaster protection plans the disaster protection plans shall contain, in particular, the necessary information on the emergency aid, the alarm, and the means of assistance available in a disaster. they must be coordinated with the neighboring disaster control authorities. special protection plans are to be drawn up for special dangers. the uk, based on the tradition of local autonomy and accountability of citizen and local governments, utilizes a bottom-up disaster management system. in , the cabinet office, a guide to emergency response and recovery was released with eight core principles: anticipation, preparedness, subsidiarity, direction, information, integration, cooperation, and continuity (cabinet office a). the basic act for disaster management in the uk is the civil contingencies act (cca), . the uk has developed its disaster management system suitable for normal times and emergency situations at the federal and at the local levels (korean association for local government studies ) . at the federal level, the civil contingencies secretariat (ccs) was established in to take charge of the overall coordination of disaster management during normal times, whereas the cabinet office briefing room (cobr) and the civil contingencies committee (ccc) take charge of disaster response in case of emergency situations. the ccs is headed by the permanent secretary, vice-minister level official in the cabinet office, and it takes overall responsibility for disaster management in the uk. if needed, the minister of the home office (or the cabinet office) reports to the national assembly for sharing emergency information or hearings after hit by disaster. the ccs carries out the emergency response tasks including anti-terrorism and disaster restoration tasks and is responsible for identifying and making preparations for a crisis during an emergency situation. the cobr and the ccc are activated for the federal government to act when catastrophic events occur. the activation process and procedures are decided depending on the level of emergency, specifically at level and level ; and at . disaster theories and progress of disaster management in modern times level , the responsible minister takes the leading role as chairman, and at level , the prime minister serves as chairman. table . shows the three levels for disaster response, in which the roles and responsibilities of each agency at the federal and the local levels are delineated differently. the cobr and the ccc focus on cooperation between the federal and local governments and support tasks rather than command and control. also, highranking officials can participate in the cobr, receiving and processing a report of situation, for prompt disaster management. figure . shows the block diagram of cobr. the cca categorizes agencies for disaster response into two categories according to their roles and duties with different obligations. the category responders are the agencies that have priority for response. the category responders consist of police services, fire and rescue services, health bodies, maritime and coastguard agency, local authorities, and environment agency. the police normally control and coordinate the activities at and around the scene. there are, however, exceptions, for example, the fire and rescue service takes the responsibility at the scene of a major fire. category responders consist of a wide range of private sector bodies that have an important role, but not routinely involved in the core of multi-agency emergency response and recovery work. utilities, telecommunications and transport providers, highways agency, strategic health authorities, and health and safety executives are included in the category responders. a local resilience forum (lrf) is a private and public joint organization of local governments, military officials, corporations, and civic groups that analyzes risk factors and establishes the risk management plan. the main aim of the lrf is to assist multi-agency and multi-sectoral cooperation. the lrf has regular meetings at least once every months to strengthen regional resilience, and category responders should participate in the meetings. the lrf is not a legal entity, nor does the lrf have powers to direct its members. nevertheless, the cca and the regulations provide that responders, through the forum, have a collective responsibility to plan, prepare, and communicate in a multi-agency environment. the police serve as chairman for the lrf, and a manager is assigned to each of the areas classified according to the police administrative district. a regional resilience forum (rrf) is a high-level organization of the lrf to coordinate plans for large-scale emergencies that are difficult to handle locally and to coordinate with the central government. the rrf divides areas into local units, and a table . disaster step as the standard of disaster management system in the uk ((cabinet office, b)) step disaster support system by step step (significant) the competent agency provides supports and cooperation at central government level step (serious) the national crisis committee is organized with the competent minister as the chairman step (catastrophic) the national crisis committee is organized with the prime minister as the chairman local government office is located in each local unit, and the regional director of government offices serves as chairman. the strategic coordinating group (scg) and the regional civil contingencies committee (rccc) are local emergency organizations for coordinating multiagency cooperation. the scg is a type of accident response and recovery center consisting of government branches, military, police, fire, hospitals, and private local committees in the relevant area at the local level for accident response, and all government officials dispatched to the jurisdiction of the central government are under the direction and control of the local director. the rccc is established when an emergency crisis exceeds the local level, and its composition and system are similar with the rrf. the disaster management in the uk is shown in table . . the disaster response plan in the uk is also operated separately into national and local disaster response plans. the national disaster response plan in the uk is carried out through the national contingency plan (ncp). the ncp describes initial response, accident response, responsibility and compensation, role and responsibility of central government, international support, and cooperation for each type of disaster, and it becomes the foundation for the disaster plan of the central government. the uk has also prepared a disaster response plan for flood under the ncp since flood occurs frequently, which is the guidance for accessing specialist flood rescue mutual aid. the guidance for accessing specialist flood rescue mutual aid specifies the strategic approach of the government for preparing for and responding to a flood, and it is responsible for providing comprehensive emergency response guidelines to all flood rescue service providers including public and private volunteer organizations including the utilization of existing and future flood relief assets. also, the guidance for accessing specialist flood rescue mutual aid presents the role and responsibility for each agency regarding floods as well as the action procedure (sop) to enable prompt response in case of a flood. the local disaster response plan in the uk is carried out through the strategic emergency plan (sep). the purpose of the sep is to present the direction of regional response activities and methods, through which the disaster response strategy and disaster response plan of each cooperation organization is prepared. the sep is managed through the lrf organized in each area, and the lrf is responsible for developing a more detailed implementation plan on the basis of what was given by rrf. in switzerland, cantons and communes have the authority and responsibility for disaster response, and the federal government intervenes only in the event of a national crisis. particularly, switzerland has a disaster management system based on a close cooperation among the federal, state, and local governments, a proactive role of the military in disaster response, and a strong civil defense system. the basic act of disaster management in switzerland is the federal civil protection and civil defence act (bzg, bundesgesetz über den bev€ olkerungsschutz und den zivilschutz). at the federal level, the federal office for civil protection (babs, bundesamt für bev€ olkerungsschutz) under the federal department for defense, civil protection and sport (vbs, eidgen€ ossisches departement für verteidigung, bev€ olkerungsschutz und sport) is responsible for disaster management. under babs, national alarm center (naz, nationale alarmzentrale) operates h all around the year in order to monitor situations, disseminate disaster information, and respond to disasters when necessary. since its establishment in , naz has designated radioactivity, chemical accidents, and the collapse of bridges due to a natural disaster as major disasters, making preparations for such disasters, and naz has also established the linkage system between major facilities and competent authorities to judge the situation in case of a disaster. the office for civil protection, sport, and military (bsm, amt für bev€ olkerungsschutz, sport und militär) takes charge of the normal-time disaster . rccc (regional civil contingencies committee) . lrf (local resilience forum) . scg (strategic coordinating group) management system at the local level. bsm is divided into four departments, and the department related to disaster management is included in the citizen and nation protection division. the manager of bsm changes according to the conditions of the state government. the five local-level emergency services utilize an "integrated system" (verbundsystem) which allows for an autonomous or a cooperative response, depending on the severity of the situation. in an extended or severe disaster situation, any of the police, fire brigades, health and ambulance services, technical agencies, or civil defense organizations can partner up to handle the crisis; this can be done at the communal or at the cantonal level. additionally, based on federal government guidelines, which includes the basic principle of disaster management and civil defense, the physical plan for disaster response, and the technical and structural countermeasures, state and local governments develop and manage their own disaster response plan. for planning and preventative measures for natural hazards, the five civil-security agencies follow the national platform for natural hazards (planat) guidelines. the swiss civil-security agencies, basing their planning activities on "risk-based planning" and "integral risk management," have developed common prioritization procedural guidelines (riko) and an online tool (econome). the guideline and the online tool, based on "protection objectives" (schutzziele), are also designed to keep natural hazard management projects cost-effective. when it comes to implementing a project, the babs have developed a tool (kataplan) to identify and classify various risks and the planning of response measures by cantonal agencies. the babs have also developed a learning program (lernrisk) and assessment software (riskplan) (babs ; bfu and babs ) . until now, we have described the disaster management system in six countries. table . shows the national and local disaster management organizations during normal time and emergency situations. social advancements in contemporary society beget new techniques and technologies. thus, new risks due to the application of the new technologies are continually springing up, and difficulties predicting uncertainties increase in parallel with new risks. furthermore, due to the development of information technology, the rise of interdependence increases propagation or chain properties to expand continuity in type, range, and scope (oh ) . the contemporary society can be featured with a change in natural environment such as climate change, socioeconomic environmental change such as aging, urbanization, and polarization, international environmental change such as each country being networked, and an entrance of complexity which indicates characters such as emergence, self-organization, and adaptation. due to these changes in environment, the contemporary society can be represented as increase of complex general set up the strategic objective for national safety, crisis management, and disaster relief and established the comprehensive drm system. in summary, most developed countries are in the process of shifting from a top-down, fragmented, and hazard-oriented disaster risk management approach into a comprehensive, integrated, and human-centered approach. while this is happening, special attention is being paid to "low probability and high impact" focusing events that are located in the long tail of the power-law distribution. symbolic and practical interpretations of the hurricane katrina disaster hazard mitigation and preparedness babs ( ) leitfaden kataplan: kantonale gefährdungsanalyse und vorsorge bfu and babs ( ) pragmatisches risikomanagement mit riskplan online risikogesellschaft: auf dem weg in eine andere moderne. frankfurt am main, suhrkamp beck u ( ) world risk society cabinet office ( b) responding to emergencies the uk central government response concept of operations disaster data: a balanced perspective national incident management system dhs ( ) threat and hazard identification and risk assessment guide: comprehensive preparedness guide again and again: is a disaster what we call a "disaster? emergent phenomena and the sociology of disaster: lessons, trends, and opportunities from the research literature principles of emergency management supplement emergency management australia (ema) ( ) emergency management in australia: concepts and principles disaster theory: an interdisciplinary approach to concepts and causes disaster relief and emergency assistance act, as amended, and related authorities fema ( ) national response framework: third edition fritz ce ( ) disaster and community theory introduction to emergency management, th edn hessisches gesetz über den brandschutz, die allgemeine hilfe und den katastrophenschutz national comprehensive crisis management: theory and reality research on effective disaster relief planning disaster classification for optimal disaster response in korea economic loss due to thailand flood in and policy implications to korea business korean association for local government studies ( ) a study on the comparison of disaster and safety management system in major advanced countries study on the change of risk structure in korea and its social and cultural effect a terminological and etymological study about safety damage situation in thailand big flood and implications for korean companies building an integrated emergency management system in korea introduction of disaster prevention. donghwa tech, hwaseong-si mcloughlin d ( ) a framework for integrated emergency management the network governance of crisis response: case studies of incident command system standard crisis management manual munich re ( ) natcatservice, global distribution of insurance premiums per capita crisis management system in korea: focusing on nuclear power plant and oil transportation the role of u. s. military forces and its implication for the korean military during disaster management natural disaster and development in a globalizing world. routledge, london perrow c ( ) normal accidents: living with high risk technologies what is a disaster? the need for clarification in definition and conceptualization in research technological and natural disasters and ecological problems: similarities and differences in planning for and managing them queensland disaster management arrangements public administration: understanding management, politics, and law in the public sector victorian bushfire reconstruction and recovery authority ( ) victorian bushfire reconstruction and recovery authority day report living with hazards, dealing with disasters: an introduction to emergency management collaboration and leadership for effective emergency management oecd studies in risk management: innovation in country risk management most developed countries have put stress on developing an effective disaster response system to cope with emerging risk interwoven with complexity and climate change. the policy implications through the comparative analysis can be summarized as follows:first, many countries aim for an integrated organization that considers both natural disasters and social disasters. the usa has endeavored to build an integrated disaster management system through the establishment of fema, and since the / terror attack, the dhs has been integrated to take charge of disaster management and terrorism. the dhs set the national preparedness goal to reflect the insights and lessons learned from hurricane katrina and to build a comprehensive organizational capacity for the entire nation that includes federal, state, and local governments and the private sector.japan and the uk have an organization that control and coordinate disaster and emergency response directly under the prime minister. the cca of uk and the cabinet office of japan are both small in size, but they are characterized by a very high level of authority for the overall coordination of disaster policies. according to the types in the reorganization of disaster management proposed by the organization for economic cooperation and development (oecd), the case of the usa corresponds to the first case, "super-ministry," and japan and the uk correspond to the second type, "a relatively small and highly influential body under direct authority of the head of government" (wyman ). disaster response plans are also being developed within national and local governments using an integrated disaster management style for all hazards approaches (waugh ) .second, all stakeholder engagement for disaster response is highly recommended. the usa successfully established the disaster risk management system by promoting the engagement and commitment of all levels of governments, citizens, and ngos. australia and japan also developed standard operating procedures indicating citizenry roles and activities in case of emergencies. in addition, australia developed checklists to enhance citizenry participation for disaster preparedness. japan has enhanced citizenry participation and preparedness by strengthening citizenry education and training. korea should improve the participation of local governments, ngos, and citizens to drm by providing guidelines and incentives and enhancing the governance-based approach.finally, many countries have stressed the clear accountability of each government and interlink between the national government and local governments. most countries, such as australia, germany, japan, and the usa, empowered local governments for disaster response and engaged in the national crisis situation. in addition, the interlink between national and local disaster response planning was also emphasized. the usa improved interlink between nrf and nims at the federal level and eop at the state and local levels. in australia, the attorney key: cord- -gehesmpu authors: toyoda, hidenori; huang, daniel q.; le, michael h.; nguyen, mindie h. title: liver care and surveillance: the global impact of the covid‐ pandemic date: - - journal: hepatol commun doi: . /hep . sha: doc_id: cord_uid: gehesmpu as social distancing and strict stay‐at‐home orders have been instituted to slow the spread of coronavirus disease (covid‐ ), unintentional outcomes among those with chronic diseases including screening for the lethal hepatocellular carcinoma (hcc) may be occurring. we aimed to describe the changes in liver care before and after covid‐ restricted access to health care. we obtained data on the number of liver clinic visits, abdominal ultrasound, computed tomography, and magnetic resonance imaging using electronic query or clinic registry at three medical centers in the united states, japan, and singapore for the following periods: february to march , , , and ; and march to may , , , and . we performed trend analysis using logistic regression. in total, , visits were made to the liver clinics at the three centers: , in , , in , and , in . overall, there were no significant changes in the distribution of males and females between february and may from to , but there was a lower proportion of seniors ages years and older (p < . ). there were significant decreasing trends in the total number of liver clinic visits overall (p‐trend = . ) and in the subanalysis for chronic hepatitis b, c, and other liver diseases. hcc/cirrhosis visits also dropped from to ( . % decrease) overall and to ( . % decrease) for the us site. in addition, there was a significant decreasing trend in the number of abdominal ultrasounds (p‐trend = . ) and computed tomography/magnetic resonance imaging (p‐trend = . ) performed overall. conclusion: liver clinic visits, hepatoma surveillance, and diagnostic abdominal imaging fell dramatically as social distancing measures were instituted. care providers must find ways to recall patients for important care monitoring, including hcc surveillance. t he coronavirus disease (covid- ) pandemic has claimed , deaths globally and , deaths in the united states as of . ( ) worldwide, countries responded to the pandemic by instituting social distancing and stay-at-home orders (e.g., united states) or stay-at-home request (e.g., japan), to slow down disease spread. however, unintended consequences of these draconian measures are potentially harming others. for example, the american heart association recently reported a %- % decrease in emergency-room visits for stroke and acute myocardial infarction, with the european union reporting similar findings, while cardiac catherization labs are reporting a decrease of up to % for lifesaving treatment for those in the throes of a myocardial infarction. ( ) ( ) ( ) public health organizations have reported a decrease in childhood immunizations, to include immunizations for the highly contagious diseases of mumps and measles. ( ) recently, the american cancer society reported a decrease in cancer screenings and diagnoses. ( , ) as has been reported, liver cancer, specifically hepatocellular carcinoma (hcc), has been on the rise globally before covid- . these findings have been quite disturbing, as hcc has a high mortality rate in which less than % of those diagnosed with hcc are alive at years. ( ) therefore, extensive efforts have been undertaken to try to improve hcc surveillance by focusing on awareness of who is at risk as well as appropriate screening methods and time intervals. ( ) ( ) ( ) ( ) this report will briefly describe the experience of three hospitals located in the united states, japan, and singapore, to illustrate the impact of covid- on liver clinic visits and surveillance for hcc. this study was conducted using data from three clinical sites: stanford university medical center (palo alto, ca), ogaki municipal hospital (ogaki, japan), and national university hospital, singapore (nuh). we included all patients seen in a liver specialty clinic for follow-up. patients were identified through several means that included using electronic search to obtain the number of patients seen at the liver specialty clinic using ultrasound cpt codes for "hepatoma surveillance," and/or using a hand search of all patients seen in the liver specialty clinics for the assigned time periods, identifying those who underwent ultrasound, computed tomography (ct), or a magnetic resonance imaging (mri) for hcc surveillance. using a data collection form, we obtained the number of liver clinic visits, number of abdominal ultrasounds, and number of abdominal ct or mri scans at each study center for the following . march , ; and march to may , . the time periods were chosen to correspond with the beginning of social distancing initiative and orders for , and data from and served as controls. we also obtained demographic characteristics (age, sex, and race/ethnicity) and liver disease etiology associated with liver clinic visits. to study patients with more advanced liver disease, we obtained data for patients with hcc and/or cirrhosis. the study was approved by the institutional review board at stanford university (stanford, ca) and was conducted according to the helsinki declaration of , as revised in . we used chi-square test to compare demographic characteristics associated with liver clinic visits among the different time periods. we performed trend analysis using logistic regression to compare the number of liver clinic visits or abdominal imaging across time periods. statistical significance was defined with a two-tailed p value less than . . we performed all analyses using stata, version (stata corp., college station, tx). during the selected time periods, , visits were made to the liver clinics at the three participating centers ( , in stanford, , in ogaki, , in singapore): , in , , in , and , in . most clinic visits at the stanford site were through telemedicine after march , , while most visits at the ogaki and singapore sites continued to be regular in-person visits. overall (table ) , there were no significant changes in the distribution of males and females between february and may from article information: to and (p = . ) or in the age group - years (p = . ). however, there was a slight increase in for the age group less than and - years, but a decrease for seniors ages years or older ( . % in , . % in , and . % in ; p < . ). table lists the number of patients seen at each study site and demographic characteristics associated with liver clinic visits by study center and by time period. the sex distribution remained consistently stable over the study periods at all three centers. however, while the age distribution remained relatively stable without significant changes at the japan and singapore centers, there was a significant and notable drop in clinic visits by seniors ages years or older in the u.s. center ( . % in , . % in , and . % in ; p < . ). race/ethnicity data were available for the u.s. sites only, and showed that the distribution did not differ significantly over time. trend analysis of a total number of liver clinic visits for the month of february, march, and april for the , , and years showed significant decreasing trends overall (p-trend = . ), for the u.s. (p-trend = . ), and singapore sites (p-trend = . ), but not the japan site (p-trend = . ) (fig. ) . fig. a -d provides liver disease etiology-specific data, in which we again found a significant decreasing trend overall and for the u.s. site, but not the japan site (p-trend = . ) for chronic hepatitis c diagnosis (number of hepatitis c virus visits at the singapore site were too few for analysis) ( fig. a) . for chronic hepatitis b visits (fig. b) , the trend also significantly decreased for the total cohort (p-trend = . ), as well as for the japan (p-trend = . ), and singapore (p-trend = . ) sites. a similar decreasing trend was also observed for visits associated with other liver disease at the u.s. site (fig. c) . however, there was no significant decrease in the number of visits for patients with more advanced disease, namely those with hcc and/or cirrhosis overall (p-trend = . ) or by individual study sites, although there was a non-signficant decreasing trend at the u.s. site (p-trend = . ) (fig. d ). nevertheless, between february to march , and the march to may , period, the total number of hcc/cirrhosis visits dropped from to ( . % decrease) overall, from to ( . % decrease) for the u.s. site, and ( . % decrease) for the japan site. regarding liver imaging, there was a significant decreasing trend in the number of abdominal ultrasounds overall (p-trend = . ) and at the u.s. site (p-trend = . ), but not the japan (p-trend = . ) or the singapore site (p-trend = . ) (fig. a) . a similar decreasing trend was also observed for ct/ mri overall (p-trend = . ) and for the u.s. and singapore sites, but not the japan site (p-trend = . ) (fig. b ). although the covid- measures instituted were meant to stop the spread of covid- and to protect the general public as a whole, the measures appear to have adversely affected the routine monitoring of chronic liver disease, to include hcc surveillance. in this brief report, not surprisingly, we found that clinic visits for patients with chronic liver disease and surveillance imaging for hcc fell dramatically as the social distancing and covid- stay-at-home measures were instituted. in fact, the decrease in clinic visits and screening tests was noted to begin before the strict implementation of stay-at-home orders but then a recent informal gallup poll found that over % of patients were afraid to go to the hospital due to fears of contracting covid- . ( ) to overcome this fear, the american college of cardiology has started a campaign, cardiosmart, in which they use social media and local news stations to let people know that it is safe to come to the hospital if one is sick. ( ) given the lethality of hcc, we must also find ways to reach out to patients to bring them in for their surveillance. in fact, a delay of just a few months in hcc surveillance may be catastrophic for patients, given that the doubling time for hcc tumor volume is . days. ( ) therefore, any delay may result in either more aggressive treatment than would have possibly been needed or the potential for no longer being eligible for curative treatment. in addition to decreased hepatoma surveillance ultrasound, there was also a decreasing trend for ct/mri, which was even more concerning, as these are usually performed for diagnostic purposes. using the telehealth platform may be one way of directly linking with patients, to assure them that they are safe coming to the hospital and that the need to for continual follow-up of their liver cancer is vitally necessary. however, given the average age of patients with hcc, the use of social media may not be as effective, so sending letters through the postal system may be a necessary means to reach the patients. as seen in our report, the age group with most pronounced decrease in the number of clinic visits was those of seniors years or older. elderly patients may have a more limited capability with technology which may impair their use of telehealth. elderly patients may also be more fearful about covid- exposure based on reports of higher mortality within the elderly as reported by the media and the center for disease control and prevention. ( ) another important observation from our study is the heterogeneity of observations among the study sites. some of these may be due to cultural differences and some may be due to differences in the local government's response to covid- and subsequent morbidity and mortality. these findings were most pronounced at the u.s. site, where when shelter-in-place was ordered by local government, all regular in-person clinic visits were discontinued almost immediately replaced by almost exclusively telemedicine clinics. in the u.s., the first covid- death was only reported on march , , but the death toll quickly rose to on march , , and over , on may , , prompting extended government lock-down orders in the area of the u.s. study site of this report. although japan reported the first covid- death earlier on february , , the death toll has risen to only as of may , . although the government of japan has closed school since early march and has requested citizens to stay home, the government has not ordered a severe shelter-in-place order, and this may explain the less severe drop in health care visits at the japan center. singapore has a small population and has reported only covid- deaths as of may , , although it has more confirmed cases than japan ( , vs. , ) . singapore shifted its public health response level to "enhanced preparedness" on february , and on april , instituted "circuit breaker" measures that included closure of nonessential services and social distancing measures. ( ) we also recognize that patients may have been seen at another clinic during our study time, and therefore would not have been counted in our study. however, due to the restrictions of the pandemic as noted, we feel it is highly unlikely that a patient would go to another clinic or would have been able to be seen at another clinic. we also used internal controls as a means of controlling for this potential phenomenon and did not see such a pattern. finally, although we were not able to provide outcome data with our study, we have highlighted an area that requires study to determine the effects of delaying hcc surveillance. when future pandemics happen, health care will be better prepared to identify areas that need to continue to function, so as not to increase morbidity and mortality as an unintended consequence. due to the stay-at-home measures that have been instituted as a means of stopping the spread of covid- , liver care for patients with chronic liver disease has been suboptimal, and hcc surveillance has incurred a dramatic drop. given the lethality of hcc, health care workers must find effective ways to reach out to patients to provide assurance that it is safe to come for their hcc testing so that we do not see an increase in mortality, in which by trying to avoid one disease, patients succumb to another potentially preventable disease. covid- ) cases and deaths covid- : are acute stroke patients avoiding emergency care? reduced rate of hospital admissions for acs during covid- outbreak in northern italy reduction in st-segment elevation cardiac catheterization laboratory activations in the united states during covid- pandemic effects of the covid- pandemic on routine pediatric vaccine ordering and administration -united states medical testing, cancer screenings plunge during coronavirus outbreak -data firm analysis fewer cancer diagnoses during the covid- epidemic in the netherlands mortality assessment of patients with hepatocellular carcinoma according to underlying disease and treatment modalities prevalence of viremic hepatitis c virus infection by age, race/ ethnicity, and birthplace and disease awareness among viremic persons in the united states therapies for bruxism: a systematic review and network meta-analysis (protocol) diagnosis rates of chronic hepatitis b in privately insured patients in the united states chronic hepatitis b prevalence among foreign-born and u.s.-born adults in the united states americans worry doctor visits raise covid- risk growth rate of early-stage hepatocellular carcinoma in patients with chronic liver disease people who are at higher risk for severe illness covid- in singapore-current experience: critical global issues that require attention and action key: cord- -w yri g authors: kubo, tomoko title: divided tokyo: housing policy, the ideology of homeownership, and the growing contrast between the city center and the suburbs date: - - journal: divided tokyo doi: . / - - - - _ sha: doc_id: cord_uid: w yri g this chapter examines the generation of tokyo’s division over time by focusing on the changes in national and municipal policies on urban planning, land use, and housing, as well as the outcomes of these long-term trajectories since the s onward. by comparing with the western urban discourse, we clarify the process of how tokyo became divided in terms of residential conditions. the national government focused on the growth of major metropolitan areas, stimulating suburban development and homeownership in the suburbs from the s to the s. however, after the late s, the target shifted from the suburbs to the city centers. alongside global competition among cities, the long-term recession of the japanese economy, and social changes, continuous investment to grow central tokyo has increased in importance. in contrast, suburban neighborhoods are facing challenges related to an aging population and an increase in housing vacancies. although government intervention is necessary, there are limited possibilities for the outer suburbs to revitalize and re-grow. the sharp contrast between the growth in city centers and the shrinking suburbs, and specifically the divided tokyo, will become more obvious in the next decade. labor market, shifting from a manufacturing orientation toward domination by financial and service industries in the city center (fainstein et al. ) . as global cities have become the sites of production and coordination (sassen ) , their spatial patterns have also been transformed and are now characterized by more division, inequality, polarization, and fragmentation (sassen ; scott ) . in addition, globalization has accelerated competition among cities, and many municipal governments have moved toward entrepreneurialism (gonzalez ) . this move has been followed by real estate developers building multi-functional condominiums in city centers, targeting single women, high-income couples, urban elites, and the creative classes (florida ; kern ; koizumi et al. ; kubo and yui a; scott ) . as a global city, tokyo has experienced spatial restructuring alongside the shift to "the third wave of capitalism," characterized by the growth of city centers consequent to labor market changes and project-oriented urban entrepreneurialism (gonzalez ; jacobs ; sassen ; scott ) . this shift followed the factory production of the nineteenth century and the mass production and emergence of metropolitan urban forms in the twentieth century (scott ) . globalization has transformed the role of city regions and urban governance, promoting spatial restructuring on many scales such as within individual metropolitan areas and city centers, as well as in national and international urban networks. the fact of inter-urban competition and urban entrepreneurialism has opened up the urban space of the advanced capitalist countries to all kinds of new patterns of development, even when the net effect has been the serial reproduction of science parks, gentrification, world trading centers, cultural and entertainment centers, large-scale interior shopping malls with postmodern accouterments, and the like. the emphasis on the production of a good local business climate has emphasised the importance of the locality as a site of regulation of infrastructural position, labour relations, environmental controls, and even tax policy vis-à-vis international capital. (harvey , p. ) to succeed in the face of global and domestic competition, the tokyo metropolitan government adopted a policy of entrepreneurialism (promoting the "new patterns of development" mentioned in the above quote) beginning in the first decade of the twenty-first century (boland ; gonzalez ; jacobs ) . this policy shift not only increased the number of urban condominium developments (kubo and yui a ) and encouraged population recovery (miyazawa and abe ; yabe ) but also intensified the growth of inequality within the tokyo metropolitan area (hirayama ; jacobs ) . urban growth in central tokyo and shrinkage in the suburbs became obvious (kubo ) , and the emergence of "hot and cold spots" divided the city, even in central tokyo, because entrepreneurial urban policies exacerbated the unevenness of urban development (smith ; hirayama ) . the neo-liberal national policies that stimulated urban renewal through the deregulation of urban planning, building standards, and the housing market (e.g., the urban renaissance special measure law of ) strongly supported the re-growth of central tokyo from the beginning of the twenty-first century onward. for instance, the deregulation of the floor-space ratio enabled super high-rise buildings in commercial and industrial areas in central tokyo, offering bonus floor space to projects providing vacant public spaces that could serve as evacuation sites in natural disasters or other emergencies (kubo ; abe et al. ) . new-build gentrification, ranging from the development of vacant commercial, industrial, or reclaimed land to super high-rise buildings, has transformed central tokyo's landscape since the beginning of the twenty-first century (lutzeler ) . additionally, many areas surrounding train stations and airports offered large tracts of vacant land that was well-suited to huge urban renewal projects. with the facilitating influence of municipal policies, these areas were targeted for new urban development in tokyo and other large japanese cities. to understand the recent urban spatial restructuring in the context of globalization, the trajectories of urban housing policies and urban governance as well as their outcomes (spatial structures) must be examined. in this chapter, we examine how urban and housing policies have affected urban spatial structures, caused a divide between the city center and the suburbs, and transformed the urban lifestyle in the tokyo metropolitan area. starting with a chronicle of urban policies in japan, we review the post-war housing policies that stimulated suburban homeownership and the horizontal expansion of the tokyo metropolitan area. then, the recent spatial restructuring accompanying the emergence of urban governance under globalization is examined. we focus on the housing market from the beginning of the twenty-first century onward and on aviation governance. through globalization, transit hubs such as airports have gradually become centers of urban development projects, known as transit-oriented development. focusing on aviation governance as a case study helps to clarify the spatial restructuring that has occurred in recent decades in the tokyo metropolitan area. the discussion is positioned within the context of the transformation of the spatial structure of the tokyo metropolitan area in modern times. most differences between housing patterns in japan and those in western cities are a result of the unique family system and household ownership trends in the late nineteenth and early twentieth centuries in japan. after the meiji restoration in the s, which opened japan to new influences and industrial progress, a civil law was passed establishing, that is, ie-seido (the family institution). japan had had a long history as a feudal society in which traditional lords and families owned most of the land and houses. however, this new law passed in the late meiji era categorized people by family unit or parentage and allowed them to select their jobs and place of residence. the law also allowed people to own real estates, such as houses and land if desired, although the family patriarch made decisions regarding the latter. therefore, the history of land and house ownership by individuals in japan is relatively short. even as recently as , the homeownership rate was only % in major japanese cities (ronald ) . importantly, family members did not share equally in the family's property. patriarchs held majority control and transferred their properties to their eldest sons, who traditionally continued to look after their parents in the family home. as a result, the other sons and the daughters left the original family and formed new families through marriage. this meant that the younger sons (i.e., the male heads of the new families) needed to find jobs in other areas, which led to residential mobility from rural areas to the growing, densely populated urban zones. thus, the housing custom based on the patriarchal family institution, which was supported by the ideology that homeownership should be dominated by men, was legally codified in japan (ronald ). because few women had paying jobs, they lived with their parents until marriage. a few exceptions were found in metropolitan centers such as tokyo and osaka. for example, in tokyo, rental apartments for working women were built by dojunkai, a government institution for housing development established for the purpose of urban reconstruction after the great kanto earthquake of (kageyama ). in general, however, there were few possibilities for women to own houses or to overcome the patrimony that was dominant during this period. although the family institution law was abolished in , soon after the second world war, the old patriarchal and multi-generational housing customs remained until recently in many rural areas, and also influenced household composition in the growing urban areas. for example, even in the s, more than % of couples who had been married for more than years lived with one of the spouses' parents (kato ) . although people tended to move to metropolitan centers in the booming postwar period in search of jobs, they hoped to eventually buy a detached house. thus, suburban nuclear families gradually became a common family form in metropolitan areas. the suburbanization of modern japanese cities started at the beginning of the twentieth century, when western urban planning methods such as the development of "garden cities" were adopted. in , volunteers from the regional division of the ministry of home affairs of japan published a book entitled "den'en toshi" which literally translates as "garden city." as the depleted condition of land in rural areas had become a serious issue, the japanese government developed energetic campaigns to improve rural infrastructure. connecting the conveniences of urban areas with rural amenities as garden city developments were intended to do was considered an attractive method for improving the rural environment. little attention was paid to the notion that these areas should be self-sufficient or to the conception that workplaces are to be located near residential areas. the main concern was how to create a good residential environment for urban and rural settlements (suzuki ) . in japan, the garden city movement was started in osaka in by kobayashi ichizo, a powerful entrepreneur who founded the hankyu toho group. the main developers were private railway companies aiming to attract potential residents along their rail lines. for example, in , kansai tochi inc. launched a plan to build ohmino den'en toshi in sakai city, osaka. the plan focused on a very large area (approximately . ha) near the kita-noda station of the nankai electric railway koya line, and it clearly displayed the typical landscape of a garden city, with radiating roads extending from a central roundabout. although the planners had visited letchworth garden city and welwyn garden city in the united kingdom to study them, they were mostly interested in these cities' landscapes and in determining how to provide better, well-planned residential environments outside large cities. these interests were prioritized over adopting the maintenance and management systems, theory, or concepts such as providing rental housing for the working classaspects that were central to the garden city idea (kikuchi ) . the movement then spread to tokyo, as manifested in the epoch-making project conceptualized in the "senzoku, tamagawa plan," which is now considered a symbol of the luxurious residential district of den'en chofu. the plan was implemented in the senzoku, o'okayama, and tamagawa areas of tokyo suburbs by den'en toshi company ltd. ("garden city company"), a direct predecessor of the tokyu railroad company and its related businesses. it was built by shibusawa hideo, the son of shibusawa eiichi, a powerful entrepreneur in the late nineteenth century who consulted for the company. the company obtained - ha of land on which to build their envisioned utopia. the senzoku area was first to be developed in , followed by tamagawa-dai and o'okayama beginning in . particularly in tamagawa-dai (now called den'en chofu), the design of the landscape was strongly influenced by sir ebenezer howard's garden city concept, comprising a concentric structure with radial roads extending from a main station. similar developments emerged in the nagoya metropolitan area, where municipal planners adopted the idea of "san-rin toshi" (literally meaning "forest city"), constructing hillside developments in the yagoto area (hotta ). these planners paid great attention to retaining the natural landscape and the inclination of the hillside when building roads and in other types of land use. the great kanto earthquake of destroyed tokyo, stimulating modern urban redevelopment projects led by goto shinpei (edgington ) and fueling the relocation of major urban industries and housing developments from central tokyo to the west, where the stable musashino terrace had sustained minimal damage. in contrast, a large part of central tokyo had been destroyed in the earthquake and the fire that followed because its reclaimed lowland was filled with high-density wooden apartments. in the s and s, undeveloped open land on the musashino terrace welcomed large housing developments, defense industry construction (e.g., nakajima aircraft industry ltd.'s musashino factory), and company housing for employees. today, these modern suburban development areas are considered a part of the center of the tokyo metropolitan area. post-war suburban residential developments have expanded beyond prefectural boundaries alongside the popularization of a caroriented lifestyle. these changes have been supported by national policies aimed at stimulating suburban/urban development, for example, by constructing highways and reclaiming land for large-scale developments. this is discussed in depth in the following sections. many post-war spatial, urban, and housing regulations established in the s developed the fundamental urban systems and institutional structures of japan (abe et al. ) . for instance, a spatial plan based on the comprehensive national land development act of determined development measures in japan. this act was characterized by a hierarchical framework comprising national, regional, and prefectural levels of administration. in the s, the most urgent issue that housing policies had to address was the housing shortage after the war. the national government established major housing policies for this purpose. for example, the japanese housing finance agency was set up in to finance homeownership by high-income families. following this, the japan housing corporation began developing housing complexes in and later developed huge housing estates in the suburbs to supply affordable housing for middle-income people. the government also built social housing for less affluent families (japan federation of housing organizations ; kageyama ). these post-war policies were regularly modified, eventually shifting from welfare state policies aiming for equal growth in industry, urban infrastructure, and quality of life throughout japan to neo-liberal policies that stimulated inequality between core metropolitan centers and the surrounding regions (forrest and hirayama ). the result was that tokyo emerged as the clear winner, followed by osaka and nagoya. this led to the creation of the tokaido megalopolis and turned other areas into shrinking regions (abe et al. ) . one example is the change in procedures regarding social housing, which from the to the s was preferred by young married couples who wanted to save money to buy their own homes in the near future. they tended to spend the years when their income was relatively low dreaming of climbing the housing ladder leading to homeownership. later, because the income restriction became stricter over time, social housing came to accommodate mainly low-income households, single-parent families, and single older adults (hayakawa ; yui yui , . the comprehensive national development plan of stimulated industrialization throughout japan and encouraged the formation of new industries in middle-and small-sized cities. to overcome the problems in cities related to rapid urbanization in major metropolitan areas, many city governments developed their own urban zoning plans, delineating their urban areas into two categories: ( ) urbanization promotion areas that stimulated urban and economic development with strict zoning plans; and ( ) urbanization control zones that strictly controlled urban development to maintain a pleasant rural and agricultural environment (the classification was based on the city planning act of ). the comprehensive national development plan of stimulated economic and urban growth in seven major metropolitan areas (tokyo, osaka, nagoya, sapporo, sendai, hiroshima, and fukuoka) by expanding high-speed transportation systems nationwide. these major cities strengthened their management function, concentrating on large enterprises, manufacturing, and transportation systems. along with the economic growth of major metropolitan areas, suburban neighborhoods were developed by the public and private sectors in the and s, accommodating the growing young middle-income people and providing them with a pleasant residential environment. planning concepts such as the garden city or the neighborhood unit (perry ) were adopted from the west. after the excessive horizontal expansion of major metropolitan areas, the comprehensive national development plan aimed at decentralization, and new business districts in the suburbs were developed through collaboration between municipal and national governments in major metropolitan areas. yokohama city's minato mirai , chiba city's makuhari new city (see chap. ), and saitama city's omiya sonic city represent public suburban development in the tokyo metropolitan area (sato (sato , sato and arai a, b) . however, the collapse of the japanese bubble economy rapidly drained energy from middle-and small-sized cities after the s. consequently, the closure of central shopping streets and the aging of shop owners accelerated the process of decline. gradually, an empty space expanded in many japanese cities (shinohara ) . in major metropolitan areas, suburbanization led to population loss in the city centers. to deal with this problem, the national government passed the urban renaissance special measure law in , a neo-liberal spatial policy promoting deregulation in urban planning, land use, and housing supply. through city center redevelopment, governments expected to overcome the long-term economic recession and stimulate rebuilding projects to increase the number of disaster-proof buildings (abe et al. ). this policy strengthened the power of central tokyo and facilitated recovery in terms of both population and economic activity in the first decade of the twentyfirst century (koizumi et al. ; miyazawa and abe ; yabe ). by the next decade, the spatial divide between the growth in the city centers and the lower levels of investment and greater aging in the suburbs became inevitable (hirayama ; kubo ; kubo et al. ; yui et al. yui et al. , ; see also chaps. , , and ). since the s, the national and municipal governments in japan have focused on urban development and on enlivening major metropolitan areas. during the long-term economic recession beginning in the s, central tokyo was targeted for economic development to stimulate the japanese economy. today, city centers can accommodate their workers in newly built condominiums, resulting in the out-migration of the younger generations from the outer suburbs. the complementary relationship between city centers offering job opportunities and the suburbs providing accommodation for these workers has gradually dissolved. in the next section, we focus on changes in the residential preferences of the younger generations. in japan, the ideology of homeownership became increasingly valued during the period of rapid economic growth beginning after the s (ronald ) . however, the high costs of land and housing meant that there was a lack of affordable housing options in metropolitan centers, which led to a major suburban boom from the to the s (tani ) , mostly concerning detached houses (hasegawa ; matsubara ) . the boom was fueled by migrants from japan's rural areas who relocated to major growing cities such as tokyo in search of jobs (tani ) . consequently, the rate of homeownership had reached % at the beginning of the s (hirayama and ronald ) . soaring central city land prices in the and land-oriented property ownership, the so-called "myth of real property," strengthened the new preference for detached houses in the suburbs (van vliet and hirayama ). many people climbed the housing ladder, seeking to attain social presence, stability, and security (hirayama b) . thus, suburban housing developments expanded horizontally beyond prefectural boundaries from the to the s. for instance, tokyo's suburbs expanded as far as km from central tokyo, requiring a two-hour daily commute. rental houses were built mainly for single people and students by private developers and for low-income families by the public sector. however, the inferior building materials and facilities in these housing units compared with owner-occupied detached houses highlighted the clear inequality between housing types. this resulted in a range of housing classes, spearheaded by owner-occupied units, followed by rental housing and public housing units (hayakawa ) . this residential segregation on the basis of socio-economic status and housing class has strengthened over time. the long-term economic stagnation beginning in the s stimulated neo-liberalism in japanese society, where the housing supply and mortgages depended on private companies and self-responsibility was emphasized (hirayama a) . the relationship between family, welfare, and housing in society has transformed dramatically over time (ronald and lennarts ) . major changes have occurred in housing patterns in large japanese cities. the preference for suburban detached houses has rapidly faded, resulting in more condensed city center living because of greater interest in central housing (see also chaps. and ). this can be attributed to the following factors (kubo and yui b) . first, japan has experienced relative economic stagnation since the collapse of the bubble economy, and the labor market has been restructured, including the outsourcing of many industrial jobs to lower-wage countries and growth in the service, information, highly specialized financial, and creative sectors in the domestic market. this is similar to the experience in many global cities since the s (sassen ) . this stagnation has led to the neglect of old industrial sites, the selling of company housing to allow companies to survive the recession, and the expanding unused reclaimed land along the river and bay areas in cities, which increased the availability of land for redevelopment. furthermore, an increase in white-collar and service/information jobs in central areas has led to an increased new housing demand nearby. second, excessive horizontal expansion of the suburbs has led to increased concerns regarding long commutes. during the long-term recession, many companies reduced their staff sizes, and the employees they retained have had to work longer hours to complete the same volume of work. reducing commuting time thus became an important strategy for dual-income couples to create sufficient time for housekeeping and childcare along with their full-time jobs (koizumi et al. ) . city center living was thus seen as an attractive option because it reduced the time necessary to commute to work. third, the rapid aging of the japanese population and the declining fertility rate have both affected residential preferences. young women have gained more lifestyle independence and occupational freedom, especially in the service sectors. in , only . % of women were unmarried by age - years, but this had increased to . % by . the number of single women with significant purchasing power has increased in large cities (kubo and yui b) . in addition, more women keep their jobs after marriage, and living in the city center rather than in the suburbs provides them with more opportunities for white-collar, service, and information jobs (koizumi et al. ) . since the second decade of the twenty-first century, the japanese population has been declining, as have functional urban areas in the country (ezaki ) . furthermore, the suburbs have not been attracting new investment (hirayama ) , rendering them less popular as residential areas. suburban areas began to face challenges caused by the aging of their residents and an increase in vacant housing (kubo and yui ; naganuma et al. ; yui et al. yui et al. , . both the aging of the remaining residents and the out-migration of the younger generations increased population aging in many neighborhoods (kubo et al. ; nordvik and gulbrandsen ; yui ) . according to the statistics bureau of japan's housing and land survey ( ), more than % of owner-occupied houses were either newly built or newly purchased, and a strong preference for newly built houses dominated the owner-occupied market. in contrast, only . % of owner-occupied houses were previously owned by others. the fragile existing housing market and dramatic decline in mobility after homeownership in japan have increased the percentage of older adults in the population of homeowners. population aging and an increase in vacant housing have led to shrinkage of the suburbs, which were developed in the s and s yui et al. yui et al. , . a declining level of public investment and limited access to the financial resources necessary to revitalize suburban neighborhoods have fueled this shrinking process. the question is whether suburban stagnation is merely a stage of the neighborhood lifecycle or a longer-term shift. private developers generally tend to avoid new developments in stagnant areas because of the high level of risk involved (ley ) . here, government mortgages, subsidies, and other forms of support such as public-private cooperation and tax incentives play an important role in minimizing the perceived risk (ley ; smith ) . however, the japanese government has thus far not intervened in suburban development (see also chaps. and ). in contrast, the growth of central tokyo is becoming increasingly important in the context of competition among global cities. tokyo will host the olympic and paralympic games in (tokyo will be postponed until due to the expansion of covid- ), encouraging more public investment in the economic development of the city's central area (gonzalez ) . this investment will exacerbate the urban divide between growing central tokyo and the shrinking suburbs. city center renewal is explained in more detail in the next section. from to , the number of condominium housing units sold increased from , to , in central tokyo (fig. . ) . figure . shows the rapid growth of condominium developments and the decline in the average price of condominiums in tokyo during this period. in these years, many potential homeowners were the children of post-war baby boomers and had grown up in metropolitan suburbs. because their parents had owned real estate in major metropolitan areas, this group of potential homeowners tended to have preferences regarding lifestyle and the family-housing the neighborhood lifecycle is one of the classical urban ecology models of american cities. neighborhoods are transformed over time through the following stages (hoover and vernon ): ( ) development; ( ) expansion through intervention and the transition of specific neighborhood characteristics in terms of housing and residents into surrounding areas; ( ) stagnation and shrinkage of neighborhoods as a result of the deterioration of buildings and filtering-down effects, inadequate maintenance, and insufficient investment caused by a lack of access to financial resources; and ( ) renewal involving demolition and/or redevelopment via either public or private investments. the ability of residents or communities to access financial resources at various points in this cycle has led to the decline and (re)growth of neighborhoods in the united states, which has also been strongly affected by federal and state policies (metzger ) . relationship that differed from those of their parents. they preferred living in condominiums in the city center over moving to the suburbs to purchase a home. the growth in the number of condominiums has facilitated this new lifestyle trend. condominiums attract single women in their s and s with significant purchasing power who previously lacked alternatives that would satisfy their need for high-quality housing (see also chap. ). the upsurge in the number of condominiums in central tokyo was strongly affected by neo-liberal housing policies introduced in the s, such as deregulation of urban developments, the mortgage market, and building standards, which encouraged urban development projects. kubo ( ) has noted that this deregulation of the mortgage market strengthened the diversification of long-term housing loans by private banks and public institutions, on which most first-time homeowners depend. for example, before , the japan housing finance agency (formerly the government housing loan corporation) provided long-term, low-interest housing loans called "flat ," which had an average annual interest rate of less than . %. other private finance agencies providing long-term housing loans for middle-class homeowners followed the interest rates set by the japan housing finance agency. beginning in , the japan housing finance agency's interest rate gradually rose, but other private finance agencies continued to provide long-term housing loans at lower rates. thus, potential homeowners were easily able to purchase a home by selecting one of these long-term, low-interest loans (kubo ) . another change was that the deregulation of building standards and urban development stimulated the construction of super high-rise buildings. since the s, the japanese government has promoted rebuilding and providing parks and wide roads for disaster prevention, and deregulating the floor-space ratio was the simplest way to achieve this. the total design system of , for example, specified that a floor-space ratio bonus would be provided for development plans involving a certain amount of vacant land. after , the japanese government accelerated these deregulation processes, including the relaxation of floor-space ratios, enabling the construction of super high-rise buildings in central tokyo. currently, developers can apply for floor-space ratio bonuses if their projects include vacant public spaces that can serve as evacuation sites during emergencies. as mentioned above, the population in central tokyo declined through the early s. however, the deregulation of the floor-space ratio facilitated the construction of super high-rise residential buildings, enabling potential homeowners to purchase condominiums and increasing the popularity of living in the city center. as a result, population recovery was seen in central tokyo from the beginning of the twenty-first century. according to japan's population census (statistics bureau of japan ), . % of the country's inhabitants lived in central tokyo in . from to , japan's population dropped by , people (a decline of . %). conversely, central tokyo gained , people (an increase of . %) during the same period. this clearly contrasts with population trends in the largest metropolitan centers in other parts of japan. more importantly, population growth in central tokyo is supported by the new and growing "going solo" lifestyle (klinenberg ), which is not generally accepted in other parts of japan. as self-responsibility and the privatization of property began to highlight the advantage of city living, single women emerged as a new class of urban homeowners in tokyo, and the situation in central tokyo came to mirror the increasing popularity of women living alone in metropolitan areas in many other parts of the world. in canada, for example, kern ( ) found that women's condominium ownership grew under neo-liberal policies in central toronto. in the following sections, we examine neo-liberal urban policies and transitoriented development, which emerged in the first decade of the twenty-first century. the transformation of aviation governance in the tokyo metropolitan area illustrates the increasing importance of urban governance (harvey ) in ensuring tokyo's competitive advantage. a strategy promoting transit-oriented development in the first decade of the twenty-first century changed the role of airports in the tokyo metropolitan area. the tokyo metropolitan government ( ) has cited improving the transportation system and urban infrastructure as one of eight strategies aimed at increasing tokyo's competitiveness. the challenge of hosting the olympic and paralympic games in and the rise of east asian cities in the global economy pushed the national and municipal governments in a neo-liberal and entrepreneurial direction after the first decade of the twenty-first century (gonzalez ) . the municipal government initiated transit-oriented development to enable better access within tokyo and to increase passenger traffic. reflecting municipal policies, private transportation companies, such as the east japan railway company, tokyo metro, and japan airport terminal co. ltd., launched several renewal, improvement, and new development projects on their properties (see, e.g., east japan railway company ). regarding the domestic urban political context, here, we focus on the processes of deregulation and privatization of airport logistics and related businesses in japan. traditionally, after the s, the national government owned and operated japanese airports, the public sector dominated the logistics of the airport business, and the private sector managed related facilities at airports. this unique system resulted in higher landing fees, especially at haneda airport, and inefficiency in airport operations (the council of the growth strategy of mlit ). along with the long-term economic recession in japan since the s, neo-liberal policy deregulating and privatizing public-owned services has been the key in maintaining tokyo's competitiveness. as the population in most japanese cities started to shrink and economic conditions worsened after the first decade of the twenty-first century (kubo and mashita ) , maintaining tokyo's strength was expected to contribute to the recovery of the japanese economy. against this backdrop, the council of the growth strategy of the ministry of land, infrastructure, transport and tourism (mlit) launched "the growth strategy of mlit ( )," which included six strategies designed to improve japanese airport management. these were: ( ) internationalization of japanese airports by offering better access to other asian countries; ( ) updating the infrastructure of haneda and narita airports to increase tokyo's competitiveness; ( ) introducing private skills and financial resources to ensure more efficient airport management and operation; ( ) achieving a better debt-income ratio at kansai airport; ( ) improving the essential domestic airport network; and ( ) accepting low-cost carriers to maximize benefits for passengers (the council of the growth strategy of mlit ). following the publication of this report, the management rights for airport logistics and airport-related facilities were awarded to private companies. as the land ownership of the major airports remained with the national or municipal governments, this took the form of management contracts rather than full privatization. this transformation of aviation governance after the first decade of the twenty-first century was also affected by the socio-economic context stemming from chronographic and chorographic interactions. the following section examines the case of the tokyo metropolitan area's efforts to deal with the complicated interactions underlying the recent transformation of aviation governance. in this section, we examine the transition in terms of location, governance, and functions of the three airports in the tokyo metropolitan area-namely, haneda, narita, and ibaraki (fig. . ) . first, in relation to japanese aviation governance since the s, we examine the transition and development of the airports in the tokyo metropolitan area. takamatsu ( ) has identified three phases in the transition of japanese aviation governance from the s to the present. first, during the post-war reconstruction period, the japanese government established airport systems through public funding and government leadership to stimulate the development of the airport industry in japan (from the to the s). second, along with the establishment of the domestic airport system through public initiatives, the government promoted aviation liberalization in the s and s. finally, drastic changes occurred in aviation governance beginning at the start of the twenty-first century, compared with the situation when the airport industry was established. in the first period of aviation governance in japan, the japanese government dominated the aviation industry and its operation. details regarding operating systems in domestic airport companies and related businesses were determined by the national government, which was also responsible for developing the domestic airport system. refurbishment and new construction at haneda airport commenced after the united states occupation forces partially returned control over haneda air base to japan from to (mlit kanto region office ). government-controlled aviation systems were officially launched in the fiscal year , and the government started to formulate long-term plans for aviation systems in (takamatsu ) . the rapid increase in air transportation exceeded haneda airport's capacity, even if the airport were to be extended, meaning that a new airport was required. in , the ministry of transport (formerly the mlit) was determined to establish the public corporation naa to manage the operation of a new airport. in , the national government selected narita city in chiba prefecture as a possible site for this airport (narita international airport corporation ), but this was strongly protested by local farmers. later, radical activist groups joined the protests, resulting in a series of terrorist attacks, multiple deaths (including suicides in the aftermath of these attacks), and numerous injuries during the s through the early s. meanwhile, the demand for air transport continued to exceed the capacity of haneda airport, which was problematic. opening a new airport became an urgent priority for the government, which took political action to obtain land through eminent domain for the construction of a new airport in . this fueled even more vigorous protests. after several postponements, narita airport was finally opened in (naa ). the abovementioned protests and concerns about residents' health because of noise pollution resulted in restrictions being imposed regarding the time of day when aircraft could depart from narita airport. moreover, many local landowners continued to protest against nearby airport-related development, and the national and municipal governments introduced special measure laws and organized official meetings between airport representatives and area residents to discuss the allocation of public funds to the improvement of urban infrastructure, the residential environment, and public and cultural facilities. as a result of these efforts, tax income from narita airport and related companies, and cooperation between the public sector and residents' associations, narita city is now often listed among the most livable cities in japan (kubo et al. ; see also chap. ). after the opening of narita airport, haneda airport focused on domestic flights and narita concentrated on international flights. during this first period in the development of aviation governance, strict government control over the operation of airports, the limited capacity of haneda airport, and protests related to the establishment of narita airport affected urban development around the airports. people expected airports to be built away from settlements, and the "not in my backyard" mentality was widespread at this time. however, the situation changed with the progress of globalization and the consequent demand for new airports as urban development hubs in major metropolitan areas. the second period of aviation governance in japan was characterized by a dual system consisting of the continuation of the ministry of transport's strong leadership in managing airport systems and the rise of capitalist competition among private entities involved in the aviation industry (takamatsu ) . the deregulation of the aviation industry began during this phase. international flights, which had traditionally been dominated by japan airlines, the national public airline, were opened to other airline companies such as all nippon airways. furthermore, competition among domestic carriers was stimulated, and japan airlines was privatized. in addition, the ministry of transport allocated significant public funding to airports in major metropolitan areas. from the s to the s, suburban areas horizontally expanded beyond prefectural boundaries. in conjunction with the development of narita airport, narita new town was built for employees of the airport and related industries. a variety of housing types and prices in the new town created a welcoming environment both for these employees and for workers commuting to central cities such as chiba city and tokyo (kubo et al. ) . the special measure law on urban development near the airport ensured that the new town offered a pleasant and walkable residential environment. the law also stimulated urban development in the rural areas surrounding the airport and the old center of narita city, which had prospered since the tenth century as a temple town of the mount narita shinsho-ji temple (suzuki et al. ) . the horizontal expansion of the suburbs and an increasingly car-oriented lifestyle required new development and investment. national policies and public interventions accelerated the building of highways, high-speed railways, and other urban infrastructure in the suburbs (abe et al. ) . the government relocated some urban functions from central tokyo to the surrounding suburbs, and public interventions promoted suburban development from the s to the s (abe et al. ; kubo ) . for example, major research institutions and the tokyo university of education (now the university of tsukuba) were relocated to tsukuba science city in a project led by the national government. in addition, industrial parks were developed in areas with a high level of transportation convenience such as kashima port; the cities of chiba, yokohama, and saitama were selected as sites for new office-center development in the suburbs; and the national and municipal governments promoted multi-functional urban development (see fig. . ). in relation to the airport specifically, large parking areas, shuttle-bus service between the airport and nearby parks and hotels, and railways connecting narita and central tokyo were developed in response to rising demand from international travelers as japan's economy grew during the s. the bursting of the economic bubble in the s affected development around the airport, and the neo-liberal policies that were adopted in the following decades fueled the transformation of aviation governance and urban development in the tokyo metropolitan area. the mlit's aviation subcommittee reports in and and the amendment of the airport development law of have strongly affected the environments surrounding airports and the airport-related industries (takamatsu ) . now, in the face of economic globalization and neo-liberalization, two primary goals have been announced: increasing the competitiveness of major japanese metropolitan areas in the global market and improving the economic efficiency of airport management. the government has transferred its dominant role in the management of air transport and airport operations to private companies. at the same time, major japanese metropolitan areas have undergone a dramatic transition from suburbanization to the recovery of the city center. in tokyo, this has largely been the result of neo-liberal policies such as the urban renaissance special measure law of and related policies launched by the national and municipal governments (kubo ) . these initiatives have led to the concentration of public and private investment in central tokyo since the beginning of the twenty-first century (hirayama ) , and proximity to transportation systems connecting key facilities with central tokyo has become increasingly important. the strong and widespread desire for better access to transport systems also applies to urban retail, business, and entertainment developments. more urban facilities have thus been built near transportation cores such as train stations, bus terminals, and airports. along with the deregulation and new urban development trends, haneda airport's terminals were rebuilt to include facilities such as event halls, wedding venues, and shopping malls (haneda tokyo-international airport ). in addition, national (the mlit), municipal (metropolitan tokyo), and lower-level (ota and shinagawa wards) governments implemented a town plan involving haneda airport and its surrounding areas in (the council of haneda airport relocation problem ). the zepp haneda entertainment hall, which is the main facility of the townplanning project, will be completed in . this project aims to utilize airport areas for the development of retail, business, and entertainment facilities, while also maintaining parks and recreation facilities for residents. the re-internationalization of haneda airport and the opening of ibaraki airport both occurred in , enabling better access between central tokyo and major transportation hubs (tanaka ). at narita airport, in addition to similar renewal projects to that undertaken at haneda airport, development aimed at addressing the increasing needs of inbound tourists began at the beginning of the twenty-first century. this was partially because narita airport's popularity rapidly declined following the re-internationalization of haneda airport because of narita's distance from central tokyo (see fig. . ). the mlit launched the "visit japan" campaign in the first decade of the twenty-first century to stimulate inbound tourism to japan, and international airports were expected to play dual roles of both nodes and destinations for inbound tourism. in the narita region, the increase in asian tourists in the first decade of the twenty-first century fueled the development of urban tourism in narita city. hotels offered free shuttlebus services connecting the airport, hotels, the narita train station, shopping malls, and the old temple town. meanwhile, narita city published tourist guides in several languages and developed an english-language website providing information for tourists, and many restaurants and local shops in the old temple town and shopping malls prepared multilingual menus or brochures to welcome international tourists (suzuki et al. ) . the third airport in the tokyo metropolitan area, ibaraki airport, which is run by a governmental corporation, commenced operation in . it offers both domestic and international flights, the latter mainly to east asian countries, using low-cost carriers and private charter flights (ibaraki airport ). this airport is connected to central tokyo by a highway system and bus service. with the most affordable bus service and the lowest landing fees of the three airports in the tokyo metropolitan area but with limited facilities in the airport building, ibaraki airport was intended to serve as the secondary airport in the tokyo metropolitan area (ibaraki airport ). the three airports serving the tokyo metropolitan area play complementary roles in improving tokyo's global competitiveness, resulting in different urban development strategies. haneda, as the most important hub with the best access to central tokyo, offers diverse urban functions both in the airport buildings and in the surrounding zones. with a similar level of urban development, narita serves as a node connecting several tourist attractions in the region, and ibaraki supplements the other airports' operation by addressing the need for low-cost carriers and private charter flights. figure . shows a comparison of the functions of these three airports, each of which plays a different role and fulfills different urban development functions. haneda represents vertical, transit-oriented development, along with the central city revival trend in tokyo. narita is characterized by horizontal and network-based urban development, and ibaraki exemplifies the traditional location of airport developments, isolated from residential areas and having less effect on nearby regions. in the age of globalization and neo-liberalization, the three airports in the tokyo metropolitan area play complementary roles in forming a unique spatial structure in terms of urban development. the chronology and chorology of aviation governance at each airport, evident in changes in policies, urban development trends, the socioeconomic context, and human behavior, have led to these differences among the three airports. in this section, we summarize the findings highlighted in this chapter. figure . provides an overview of the tokyo's transformations in spatial structure and their driving forces over the centuries. we focus on the economy, urbanism, development, and transportation as driving forces. from the s to today, tokyo's city regions have expanded west of the musashino terrace. the city regions formed a conurbation along with the development of the factory system, which scott ( ) has termed "the first wave of capitalism." the government and businesspersons such as shibusawa eiichi established the modern railroad system, and entrepreneurs applied western urban planning methods in japan. factories and housing for employees supported the expansion of city regions west of tokyo. suburban development then gradually expanded to the north and the east. the ecological model of the urbanization process, which focuses the ecological model of the urbanization process was established by the chicago school and its followers. the most well-known variations of this approach are the concentric zone model (burgess ), the sector model (hoyt ) , and the multiple nuclei model (harris and ullman ) . abe ( ) examined early studies in the field of japanese urban geography by kiuchi ( kiuchi ( , and tanabe ( ) , which attempted to apply these models to japanese cities. yui ( ) reviewed late twentieth century western studies on housing and urban residential patterns (johnston ; knox and pinch ; robson ) and concluded that each of these western models assumed urban residential segregation in terms of income, race, and class. therefore, we cannot directly apply these models to japanese cities where these forms of residential segregation do not exist, there may be less ethnic diversity, and housing market conditions and family structures differ from those that spatial structure urbanism / transportation / development the s to the s the first two decades of the st century developments along rail lines by private companies (the first wave of capitalism) th century urbanism from the s to the s, the mass production economy ("the second wave of capitalism", scott ) required the horizontal expansion of the metropolitan area. government intervention in urban development, transportation, and the housing market led to suburban homeownership becoming more popular. to supply this type of housing to the growing middle class, suburban development expanded beyond prefectural boundaries, reaching a km commuter belt at the peak of suburbanization and integrating prefectural centers such as mito city into the tokyo metropolitan area (see also chap. ). in addition, there has been a suburban relocation of some previously urban functions such as research institutions (e.g., tsukuba science city) and industrial production facilities requiring proximity to transportation systems (e.g., ports, highway interchanges, or airports). a new airport was established, and the complementary roles played by haneda and narita airports supported tokyo's growth. suburban mixed-use developments such as makuhari new city were built by the national and municipal governments within a km commuter belt around central tokyo. the suburbs within a - km commuter belt became the focus of housing estate developments targeting the middle classes in the tokyo metropolitan area. since the beginning of the twenty-first century, tokyo has experienced spatial restructuring alongside globalization, neo-liberalization, and a shift toward a cognitive-cultural economy under "the third wave of capitalism" (scott ) . the national and municipal governments have promoted the renewal of the city center through the deregulation of urban planning and of the housing and mortgage markets. they have also encouraged transit-oriented development, and major train stations, highway interchanges, and airports became hubs for urban development. with global competition among cities, the long-term economic stagnation in japan, and ongoing social changes, the continuous investment in and growth of central tokyo are becoming increasingly important. to create a "strong tokyo," entrepreneurial urbanism emerged at the beginning of the twenty-first century, positioning central tokyo as the clear winner. a focus on urban redevelopment and the consequent population growth in the city center have fueled this trend over the past two decades. in this context, suburban neighborhoods face challenges related to aging populations and increases in vacant housing. although government intervention is necessary, there are limited possibilities for the outer suburbs in terms of revitalization and growth. the sharp contrast between the growth in the city center and the decline in 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geographical strategies to make better solution key: cord- - agazyre authors: dewit, andrew; shaw, rajib; djalante, riyanti title: an integrated approach to sustainable development, national resilience, and covid- responses: the case of japan date: - - journal: international journal of disaster risk reduction doi: . /j.ijdrr. . sha: doc_id: cord_uid: agazyre abstract the covid- pandemic has led to historic economic fallout. to protect public health and stabilize incomes, governments have implemented massive fiscal stimulus packages. these fiscal supports are crucial, though there is concern that sustainable and resilient development will be sacrificed in the rush to preserve incomes and industries. the aim of the paper is to review whether the japanese governments’ responses in terms of financial stimulus considers longer term resilience and sustainability. this paper reviews pertinent academic literature and publicly available data from governments and organisations. the research is a rapid analysis of emerging information provided by the government of japan and other international organisations. using the case of japan, this paper suggests that it is possible both to protect public health and essential services, while also promoting resilience and sustainability. japan's integrated solutions show that pandemic response can include accelerated decarbonization and resilient, sustainable development. the paper also warns also that failure to act on long-term sustainability risks increased inequality, higher opportunity costs, cascading hazards, and further retreat from planetary thinking and globalism. trillion (approximately usd trillion), subsequently increased to jpy . trillion on april . on may , that amount was doubled, with the total reaching jpy . . most observers expect several more stimulus packages in japan and elsewhere as the public-health crisis worsens. measures to recover from the present crisis are virtually certain to include long-term investments in health, energy, water, transport, and other critical infrastructure (lu, ) . climate-aware individuals and institutions recognize the present crisis as an opportunity -indeed an imperative -for resilient, decarbonizing and equitable structural change. certainly covid- has dramatically reshaped the policymaking debate by: ) bolstering public authority over private agency, ) elevating collaborative planning over reliance on price signals, ) expanding the scope of all-j o u r n a l p r e -p r o o f hazard resilience to emphasize pandemic risks, and ) vastly increasing public awareness of the need for holistic resilience. thus, back on march , un secretary general guterres was able to insist that " [t] he recovery from the covid- crisis must lead to a different economy" without attracting a flurry of criticism ). yet many questions whether this ideational change will become institutionalized and lead to inclusive resilience. there is no guarantee that national-level countermeasures will come to embody the agenda, let alone serve as a basis for productive global cooperation. there is also no consensus on precisely what constitutes best practice in decarbonizing and disaster-resilient infrastructure, and hence no simple guide for selecting and implementing such urban systems. many voices equate sustainability with green energy -notable solar and windwithin a context wherein "green recovery" has become the byword for transcending fossil fuels. but simply investing in more solar and wind does not seem sufficient. data on global renewable investment between - show that china is the leader, followed by the us and japan (unep/bnef, ). but those prodigious investments alone did not reduce emissions, increase equity, and otherwise achieve the agenda goals. equally, if not more important, is the resource-efficiency of urban and semi-urban communities, the inclusivity of governance, and the myriad other factors that are key to the agenda. the scope of the challenge is underscored by international energy agency estimates that the covid- shock -though unprecedented -will only lead to an % reduction in co emissions in , relative to (iea, ). to be sure, that decline in emissions accords with the call for annual co reductions of . % to limit warming to . degrees (evans, j o u r n a l p r e -p r o o f table shows that there were three key pillars in the december stimulus: national resilience plans (nrp) and disaster reconstruction; economic risk countermeasures; and "post olympic games" legacy investment in society . , sdgs-inclusive society. of course, the olympic games are postponed to , and in the end may not even be held. but that does not mean the investment in critical infrastructures is, ipso facto, wasteful. the table also separates the jpy . trillion public spending from the jpy trillion total of public and private spending. the latter figure is achieved via national government support for local government spending (through direct and indirect subsidies), low-interest loans to foster business investment, and similar mechanisms. in terms of precise content among the three categories, the nrp share is jpy . japan's integrated paradigm: society . , sdgs and national resilience far more important for our purposes here is that japan's actions are evolving in a holistic paradigm. for one thing, they are part of an integrated, resilience-oriented "society . " industrial policy regime that predated covid- (mavrodieva and shaw, ; cao ) . we saw earlier that this policy regime was already heavily funded in japan's pre-covid, december stimulus. and that pre-covid stimulus comprises a core element of the larger april stimulus noted earlier. society . 's policy arms include such critical cyber-physical linkages as "post g" next-generation communications, remote-sensing for disaster risk reduction, as is shown in a key test of any such ostensibly collaborative initiative is how well it diffuses and how purposefully engaged the actors are. by may of , all of japan's prefectures had adopted their own regional versions of the nrp. moreover, as a having analysed japan's financial stimulus in responding to covid- response from section to , we turn to examine how integrated financial and economic stimulus can foster a rights-based, low carbon, resilient and sustainable recovery. the year was slated to be the "super year" of global action toward sustainability. the sdgs -also known as the global goals -were adopted by all united nations member states in as a universal call to action to end poverty, protect the planet, and ensure that all people enjoy peace and prosperity by . the sdgs are integrated-that is, they recognize that action in one area will affect outcomes in others, and that development must balance social, economic and environmental sustainability. through the pledge to leave no one behind, countries have j o u r n a l p r e -p r o o f committed to fast-track and prioritize progress for the most disadvantaged. that is why the sdgs are designed to achieve several 'zeros', including zero poverty, hunger, aids, and discrimination against women and girls. broad stakeholder participation is needed to reach these ambitious targets. the creativity, knowhow, technology and financial resources from all elements of society are necessary to achieve the sdgs in every context. moreover, through nationally determined contribution (ndcs), countries show how they plan to meet the paris agreement on climate change (unfccc, ). in addition, the sendai framework for drr calls for a comprehensive, multi-hazard and coherent approach (sfdrr, ) . objectively speaking, integrating the agenda is even more important in the midst of covid- . as irena ( ) puts it, "the goals set out in the united nations agenda and the paris agreement can serve as a compass to stay on course during this disorienting period. they can help to ensure that the short-term solutions adopted in the face of covid- are in line with medium-and long-term development and climate objectives." the un has also emphasized that the recovery from the covid- crisis "must lead to a different economy" (un, ). the international resource panel (irp) has also pointed out that resource-efficient green stimulus packages can lead to cost savings, new industries, and equitable economic growth (irp, ). evn so, public policy is clearly distracted by political polarization, geopolitical tensions, and other factors. in this section, we outline key implications if the fiscal response does not conform with the agenda, both at the national level in japan, and also globally. emergency spending only to perpetuate the patently vulnerable status quo risks forfeiting opportunity to reduce a range of negative externalities that erode human health and welfare and have been implicated in the retreat of democracy (eiu, ). concerning the us, philip alston, the un special rapporteur on extreme poverty and human rights, pointed out that "[l]ow-income and poor people face far higher risks from the covid- due to chronic neglect and discrimination, and a muddled, corporate-driven, federal response [that] has failed them" (ohchr, ). the un j o u r n a l p r e -p r o o f has also warned that coronavirus-driven debt crises threaten poor countries already at risk. it recommends accelerating investment in resilient infrastructure, strengthening social protections, enhancing regulatory frameworks, and strengthening the international financial safety net and framework for debt sustainability. and it strongly advises that this action be coordinated lest the larger opportunity be lost in the gaps among governance and other silos (undp, ). opportunity costs are the pecuniary and other potential benefits forgone when one alternative is chosen over another. specifically concerning fiscal countermeasures to covid- and its economic fallout, failure to include climate action risks accelerated global warming. we have seen that the scale of covid- counter-measures is already enormous, unprecedented in peacetime. most countries lack the fiscal capacity and political will to continue such spending beyond one or two years, let alone for decades. focused investment in public health systems -particularly in least developed countries -could reduce economic costs from future outbreaks as well as contribute to containing the present one (mckibbin and fernando, ) . for example, analyses suggest that in the us alone social distancing measures lead to net benefits of $ . trillion (thunstrom et al, ) . these kinds of benefits could be multiplied by making the health investments climate-smart as well. global connectedness has led to increased complexity. that complexity carries with it benefits for some but at the same time a hidden cost for all: fragility (jensen, april ). in finance, for example, interconnected institutions benefit from complex networks and reduced transaction costs, but have produced a fragile structure the ongoing fiscal stimulus packages have raised deep concerns among fiscal conservatives, market liberals and others concerning over-reach of tax/debt states. the nearly inevitable increases in taxation and outrage over the contents of ongoing fiscal relief (eg, for airlines) could further weaken political coalitions supporting climate action and global engagement. in order to achieve a virtuous cycle of j o u r n a l p r e -p r o o f mitigation and adaptation, stakeholder inclusion and co-benefits must be maximized in least-cost solutions. fiscal and other resources are not infinite, and agenda solutions have to be robust against emergent political hazards. in this regard, we have shown that japan's national resilience program has built a strong foundation for equipping japan to respond to large-scale crises from covid- . its financial stimulus is characterized by long-term consideration for national and local resilience, along with a vision of society . . japan has a powerful track record of managing large scale crises and complex recovery from natural and man-made disasters. it offers a pertinent example of how to do integrated planning, implementation, and revision through multi-stakeholder and increasingly all-hazard platforms. japan integration of covid- countermeasures and resilience merits closer study for lessons on least-cost and synergistic mitigation and adaptation. we would also urge that japan use it stimulus packages even more productively, as a window of opportunity for implementing long-term sustainability and resilience. in conclusion, it is a tall order to draft and implement a coherent strategy of covid- countermeasures coupled with the agenda. global partnerships are critical to formulating and disseminating a global response. that is why goal of the sdgs centres on strengthening global partnerships among national governments, the international community, civil society, the private sector and other stakeholders. the agenda thus embodies the shared responsibility and global solidarity essential to making covid- recovery a major step in the long journey back from planetary boundaries and towards sustainable and resilient communities. asian development outlook (ado) : what drives innovation in building resilience against biological hazards and pandemics: covid- and its implications for the sendai framework analysis: coronavirus set to cause largest ever annual fall in co emissions world bank, planning for the economic recovery from covid- : a sustainability checklist for policymakers japan cabinet secretariat comprehensive economic measures to create a future with security and growth cabinet office of japan, comprehensive economic measures to create a future with security and growth covid- dashboard policy responses to covid the great lockdown imf, b world economic outlook update building resilient societies after the covid- pandemic a simple way to close the multi-trillion-dollar infrastructure financing gap, the world bank disaster and climate change issues in japan's society . -a discussion mapping the covid- recession by economic countermeasures securing critical raw materials supply is key to the response to global trends in renewable energy investment this is, above all, a human crisis that calls for solidarity" | united nations un sol, 'solidarity, hope' and coordinated global response needed to tackle covid- pandemic, says un chief the recovery from the covid- crisis must lead to a different economy covid- situation reports funding the fight against covid- loss and damage and limits to adaptation: recent ipcc insights and implications for climate science and policy impacts of . c global warming on natural and human systems an ipcc special report on the impacts of global warming of . °c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty paris agreement climate proposals need a boost to keep warming well below c the benefits and costs of flattening the curve for covid- key: cord- -foodz c authors: ueno, tomoaki; kurita, junko; sugawara, tamie; sugishita, yoshiyuki; ohkusa, yasushi; kawanohara, hirokazu; kamei, miwako title: surveillance by age-class and prefecture for emerging infectious febrile diseases with respiratory symptoms, including covid− date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: foodz c object the covid- outbreak emerged in late in china, expanding rapidly thereafter. even in japan, epidemiological linkage of transmission was probably lost already by february , . from that time, it has been necessary to detect clusters using syndromic surveillance. method we identified common symptoms of covid- as fever and respiratory symptoms. therefore, we constructed a model to predict the number of patients with antipyretic analgesics (ap) and multi-ingredient cold medications (mic) controlling well-known pediatric infectious diseases including influenza or rs virus infection. to do so, we used the national official sentinel surveillance for infectious diseases (nossid), even though nossid data are weekly data with day delays, on average. the probability of a cluster with unknown febrile disease with respiratory symptoms is a product of the probabilities of aberrations in ap and mic, which is defined as one minus the probability of the number of patients prescribed a certain type of drug in ps compared to the number predicted using a model. this analysis was conducted prospectively in using data from october , through by prefecture and by age-class. results the probability of unknown febrile disease with respiratory symptom cluster was estimated as less than % in . discussion the most severe limitation of the present study is that the proposed model cannot be validated. a large outbreak of an unknown febrile disease with respiratory symptoms must be experienced, at which time, practitioners will have to wing it. we expect that no actual cluster of unknown febrile disease with respiratory symptoms will occur, but if it should occur, we hope to detect it. the novel coronavirus covid- outbreak emerged in late , subsequently expanding to , laboratory-confirmed cases and , deaths reported by who as of march [ ] . in japan, excluding cruise ships, cases including asymptomatic cases were confirmed, from which six patients died. however, most have been diagnosed as mild cases as of march , [ ] . the epidemiological linkage of transmission was probably untraceable already in japan as of february , , as it was in wuhan. at that time, cluster identification was necessary, but rapid testing was unavailable. therefore, physicians have been unable to diagnose potential patients especially if they have mild symptoms. therefore, syndromic surveillance is expected to be as useful as traditional surveillance based on diagnosis by physicians. the present study examines prescription surveillance (ps) for detection of some clusters of emerging infectious febrile disease with respiratory symptom including unknown febrile disease with respiratory symptoms [ , ] as syndromic surveillance. in actuality, ps has been operating in japan since . the system provides nationwide syndromic surveillance by the japan medical association, japan pharmaceutical association, and em systems co. ltd. it has been reporting the estimated number of influenza and chicken pox patients and the numbers of patients all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint prescribed antibiotic drugs, antipyretic analgesics (ap), and multi-ingredient cold medications (mic) and antidiarrheal and intestinal drugs, based on prescriptions filled at pharmacies. the system estimates the number of patients every day, as inferred from the number of prescriptions by prefecture by age group. as of the end of january , approximately ten thousand pharmacies were participating, collectively accounting for about % of all pharmacies nationwide. the estimated numbers of patients are presented on the dedicated web site the following morning [ ] . it is the most reliable real-time precision and timely surveillance for influenza [ ] . in japan, national official sentinel surveillance for infectious diseases (nossid) has been operating based on the related law [ ] . for many common pediatric infectious diseases, , sentinel pediatric care facilities, which account for about one-tenth of all pediatric care facilities throughout japan, report the number of cases each week by nossid. for influenza, , internal medicine care facilities have been added. the sentinel reports are regarded as reliable information because they are based on physicians' diagnoses. nevertheless, these reports are published weekly after a ten-day delay following diagnosis. for that reason, nossid probably cannot detect infectious diseases early. moreover, because it is based on diagnoses, it cannot detect emerging or all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint re-emerging diseases that cannot be diagnosed at medical institutions, at least in the initial phase of the outbreak. the earlier study for detection of emerging infectious diseases using ps proposed a model that predicts the number of patients controlling calendar information and activity of common infectious diseases in nossid [ ] . when it found unexplained increases in some type of drug, it reports an aberration. the model in this study is a modified version that addresses ap and mic, corresponding to typical symptoms of covid- patients [ ] . the numbers of patients prescribed other types of drugs were used as additional control subjects. antibiotics are classifiable into five types: penicillin, cephem, macrolide, new all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint quinolone, and others [ ] . to predict the numbers of patients taking those drugs by well-known infectious diseases, we used data for influenza, rs virus infection, pharyngoconjunctival fever, group a streptococcal pharyngitis, gastrointestinal infections, varicella, hand, foot and mouth disease (hfmd), erythema infectiosum, exanthem subitum, pertussis, herpangina, mumps, and mycoplasma pneumonia from nossid. first, from known infectious diseases and calendar information, we predicted the numbers of patients prescribed ap and mic. the dependent variable was the number of patients prescribed drug i on day t. explanatory variables were the nossid reported number of patients of disease j: the latest available data for day t. because nossid publishes data of the prior week on friday at noon, the latest available data are those of two weeks prior on monday-friday, and one week prior on saturday and sunday. furthermore, explanatory variables included dummy variables of the epidemiological week, day of the week, holiday, and the day following a holiday, the day following two consecutive holidays, summer vacation ( - august), and the new year vacation ( - january). moreover, the number of patients prescribed with drugs other than ap and mic were included as explanatory variables. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint next, we calculated the probabilities of the numbers of patients prescribed a certain type of drug in ps comparison to the number predicted by the model. the probability of aberration for the drug was defined as one minus the probabilities of the numbers of patients prescribed a certain type of drug in ps compared with the number predicted by the model. the probability of a cluster with emerging diseases is the product of the probability of aberration in drugs corresponding to symptoms of the emerging diseases. for example, for covid- , because we presume that its typical symptoms are high fever and respiratory symptoms, we can presume that the probability of clusters with unknown febrile disease with respiratory symptoms is the product of the probabilities of aberrations in ap and mic. this analysis, conducted prospectively in , uses data from october , through by prefecture and by age class. age classes were defined as and younger, - , and years and older. we adopted . % as the tentative criterion to detect clusters, which corresponds to a significance level of %. detected clusters of unknown febrile disease with respiratory symptoms based on the tentative criterion and its probability of aberration are shown in the table by age all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint class. there were clusters: of children, of adults, and of elderly people. clusters with probabilities of unknown febrile disease with respiratory symptoms that were higher than . were two: on february among children in mie prefecture and on january among adults in the same prefecture. the figure shows the number of patients with ap or mic among children (upper panel) and adults (lower panel). the arrow indicates the date on which the probabilities of cluster were higher than %. it shows that ap or mic in children or adults were not significantly higher than on other days. results show clusters of unknown febrile disease with respiratory symptoms for which the probabilities were higher than . %. only two clusters were detected for which probabilities were higher than %. therefore, . % was accepted as the criterion for moderate aberration; % was used as the criterion for high aberration. in addition, the lower aberration might be defined as higher than %. however, it is noteworthy that the detected clusters were significantly more numerous in patients with unknown febrile disease with respiratory symptom. they might not be infected covid- . however, they were not with influenza, rs virus or all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint other common pediatric infectious diseases. we can use more real-time daily surveillance of infectious diseases other than ps and nossid, which were used for this study. for example, nursery school absenteeism surveillance system ((n)sassy) has been monitoring the health situation, diagnosed diseases or symptoms, of more than five million students or nursery school kids every day. it is a computerized system comprising a network of information shared among concerned organizations and individuals [ ] [ ] [ ] . we can therefore use (n)sassy as a form of syndromic surveillance in japan. in short, all information related to children's health conditions is integrated in (nursery) schools. it was developed by a research group headed by dr. ohkusa, one author of this paper. it has been funded by the mhlw since . it has retained its copyright to the present day. currently, it is operated by the japanese society of school health. online receipt computer advantage (orca) surveillance data compiled since have been provided for public use [ ] . the data include estimated daily numbers of patients by prefecture or municipality in japan for influenza, hand-foot-and-mouth disease (hfmd), erythema infectiosum (ei), pharyngoconjunctival fever (pcf), respiratory syncytial (rs) virus infection, measles, rubella, and insolation from orca, an electronic medical claim system (http://infect.orca.med.or.jp/) with data produced all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint and then provided freely to medical facilities by the japan medical association. as of the end of september in , orca was used at , medical facilities. orca collected information from about medical facilities as of the end of september, . as inferred from its thousand medical facilities in total, its participation rate was about %. actually, its precision was confirmed for influenza, which was very similar in ps at the nationwide. however precision for other diseases might be insufficient. even so, because it has other information that is not included in ps, integration of orca surveillance and/or (n)sassy might contribute to improvement of detection of covid- or other emerging infectious diseases. the most severe limitation of the present study is that the validity of the proposed model has not been verified. verifying it requires that we experience a large outbreak of covid- . with such a system, practitioners would have to wing it during its first trial. hopefully we expect to be able to detect any cluster of covid- correctly if such a cluster were to exist. the prior day's data of detected clusters have been published on the internet (http://prescription.orca.med.or.jp/syndromic/kanjyasuikei/index.php) since the beginning of march. to date, fortunately, we have no detection from this survey method. the present study supports the author's opinions, which are unrelated to any stance all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. note: this table presents detected aberrations of clusters with unknown febrile disease with respiratory symptoms based on a tentative criterion: . . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . japan ministry of health, labour and welfare author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the real-time prescription surveillance and its application to monitoring seasonal influenza activity in japan prescription surveillance for early detection system of emerging and reemerging infectious disease outbreaks prescription surveillance evaluation of estimated number of influenza patients from national sentinel surveillance using the national database of electronic medical claims annual incidence rate of infectious diseases estimated from sentinel surveillance data in japan prescription surveillance for early detection system of emerging and reemerging infectious disease outbreaks all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the association among (nursery) school absenteeism surveillance system and incidence of infectious diseases descriptive epidemiology for mycoplasma pneumoniae infection using (nursery) school absenteeism surveillance system, and proposal for countermeasures association of influenza outbreak in each nursery school retrospective evaluation for orca surveillance comparison with prescription surveillance all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the we acknowledge all pharmacies participating in ps. no author has any conflict of interest, financial or otherwise, to declare in relation to this study. or policy of professionally affiliated bodies.all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint the right-hand side. arrows show the detection date on which the probability of the cluster was greater than . . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity.the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- - jxvngvz authors: kunii, osamu title: the okinawa infectious diseases initiative date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: jxvngvz at the kyushu–okinawa group of eight summit in , japan announced the okinawa infectious diseases initiative (idi) and pledged to spend us$ billion over a five year period to combat infectious and parasitic diseases in developing countries. the idi has exceeded expectations, spending more than us$ billion over four years. the idi is a unique initiative with its own philosophical basis and specifically tailored interventions and measures that helped to initiate worldwide political and financial commitments in the fight against infectious diseases. notably, it promoted partnerships among stakeholders and emphasized comprehensive and inter-sectoral approaches (i.e. coordination and collaboration between health and other sectors). it helped to create a new vision of what is possible in the global effort against communicable diseases and has been instrumental in shaping the changing environments of development assistance, poverty reduction and other trends to reduce the impact of infectious and parasitic diseases. the okinawa infectious diseases initiative osamu kunii institute of tropical medicine, nagasaki university, sakamoto - - , nagasaki - , japan at the kyushu-okinawa group of eight summit in , japan announced the okinawa infectious diseases initiative (idi) and pledged to spend us$ billion over a five year period to combat infectious and parasitic diseases in developing countries. the idi has exceeded expectations, spending more than us$ billion over four years. the idi is a unique initiative with its own philosophical basis and specifically tailored interventions and measures that helped to initiate worldwide political and financial commitments in the fight against infectious diseases. notably, it promoted partnerships among stakeholders and emphasized comprehensive and intersectoral approaches (i.e. coordination and collaboration between health and other sectors). it helped to create a new vision of what is possible in the global effort against communicable diseases and has been instrumental in shaping the changing environments of development assistance, poverty reduction and other trends to reduce the impact of infectious and parasitic diseases. since the group of seven (g ) summit in denver, usa, world leaders have made major commitments and agreed to collaborate to combat infectious and parasitic diseases [ ] . at the kyushu-okinawa group of eight (g ) summit in july , infectious diseases were on the main agenda for the first time, and major progress was made in securing stronger commitments from all g nations [ ] . the commitments included an agreement to establish new partnerships to help maximize the impact of health and medical interventions, and recognition of the need to expedite the mobilization of additional resources. one of the outcomes of this new-found political appreciation of the urgent need to tackle infectious diseases was the establishment in january of the global fund to fight aids, tuberculosis and malaria (gfatm: http://www. theglobalfund.org/) [ ] . japan remained the top overseas aid donor throughout the s and is still the second biggest after the usa [ ] . even before the denver summit, the japanese government was committed to fighting infectious diseases. in , together with the usa, japan launched the common agenda for cooperation in global perspective [ ] to address global problems such as increasing environmental damage, overpopulation and damage from both natural and manmade disasters. this was followed, in , by the global issues initiatives on population and aids [ ] . japan underscored its undertaking to boost parasite control efforts by launching the global parasite control initiative, also known as the hashimoto initiative (hi), at the g summit in birmingham, uk [ ] . the subsequent okinawa infectious diseases initiative (idi) was designed using the successes and challenges experienced during hi activities, expanding targets and strategies towards broader infectious disease control and prevention, and deploying a broad spectrum of interventions. in this article, i review the idi, focusing on its philosophy, components, activities and achievements. the idi incorporated the following basic philosophy, which greatly influenced the manner in which the programme was designed and implemented. infectious and parasitic diseases as a central issue in economic and social development infectious and parasitic diseases not only threaten the lives of individuals in developing countries but also are an impediment to the social and economic development of those nations [ ] [ ] [ ] , particularly affecting the poor [ ] . the risk of infection in developing countries is increased by several factors, including high population growth rates, poverty, gender disparities, fragile medical systems, inadequate preventive care and treatment services, lack of safe water supply, and malnutrition [ ] [ ] [ ] . these factors are compounded because poor health exacerbates poverty [ ] . fighting infectious and parasitic diseases should be a central part of all development programmes and a crucial element in efforts to reduce poverty [ ] . global partnership and community-based action infectious and parasitic diseases should be viewed as a global issue that requires collective approaches based on international, multisector partnerships [ ] . effective measures to tackle these diseases also require action at the community level based on the concept of primary health care [ ] . as such, it is also important to incorporate measures against infectious and parasitic diseases in all community-level development programmes. the experiences of japan in public health activities and its future role following world war ii, japan developed a public health centre system, trained public health workers extensively, promoted measures for maternal and child health care and enhanced health care services in schools [ , ] . these steps contributed to the rapid reduction in infant mortality rates [ ] . japan also mounted major initiatives to eliminate infectious and parasitic diseases nationwide; for example, by linking public health activities with measures to control tuberculosis (tb), japan substantially reduced the number of tb-related deaths [ ] . okinawa, which comprises the southernmost islands of japan, has a history of successful eradication of malaria, filariasis and other parasitic diseases through active public health measures, including community participation and mobilization [ ] . drawing upon these experiences, japan has provided experts, technical assistance and capacity development programmes to developing countries through the idi by modification and application of the methods and technology that have proved so successful in japan. strengthening the health sector in developing countries the most important philosophy of japanese development assistance is to support the self-help efforts of developing countries. japan supports the development and implementation of health sector plans and/or infectiousdisease-specific intervention programmes designated by the countries themselves. through infrastructure and institution building, in addition to provision of technical assistance, japan supports and facilitates the development of health systems and sector reforms, thereby reinforcing health development planning and programmes, building capacity and helping to ensure sustainability of infectious disease control. the idi has given high priority to supporting human resources development, which is the most crucial component in making health systems function, and to creating quality services and high performance in infectious disease control. japan has provided several training programmes and courses in response to local needs in both developing countries and in japan aimed at individuals from all levels (e.g. from policy makers to field site personnel). to tackle a formidable global issue, japan has consolidated partnerships with various stakeholders, including the united nations (un: http://www.un.org) and other multilateral organizations, donor agencies, nongovernmental organizations (ngos) and civil organizations such as private nonprofit organizations and community groups. japan has also promoted partnerships with developing nations that have already shown progress and in which high-quality human resources in infectious disease control already exist. these partnerships have been developed into a cooperative network in developing countries that facilitates sharing of knowledge, skills and expertise among the cooperating countries. for decades, japan has promoted research activities in developing countries that are designed to create new, appropriate technology and quality clinical and laboratory work [ , ] . therefore, the promotion of research activities became one of the key components of the idi. however, because of japanese official development assistance budget constraints (i.e. reduced aid to developing countries) and the increasing trend of targeting worldwide assistance towards poverty reduction [ ] , there has been a priority shift away from scientific research and towards an operational focus, with the goal of providing rapid and direct support to those most in need. consequently, support for research has declined. promotion of public health at the community level japan has paid special attention to the improvement of basic sanitation, clean water, basic education and primary health care within communities, concentrating on interventions that lead to the reduction of infectious diseases. this infrastructure support, ostensibly assistance to boost primary and community health care, has emphasized the need for comprehensive, integrated approaches and has included the construction of health centres, the distribution of medical and laboratory equipment and supplies, the training of community personnel and the facilitation of community participation. major achievements of the idi the formation of the idi stimulated an increase in worldwide political and financial commitment for the fight against infectious diseases [ ] . according to the g performance assessments conducted by the g information between and the start of , japan spent us$ . billion on the idi. idi spending is classified under two categories: direct assistance (us$ . billion) and indirect assistance (us$ . billion). direct assistance involves projects and interventions that directly affect disease prevention. these include treatment and control (i.e. the provision of medicine), diagnostics and vaccines, technical assistance for disease control and training a workforce for disease control. indirect assistance involves projects and interventions that indirectly influence infectious disease control. these include clean water and sanitation, provision of basic education and renovation of health facilities. with regard to the geographical allocation of the idi inputs (table ) , japan has focused mainly on asia. however, more attention is now being paid to africa because it represents the biggest obstacle to the achievement of the millennium development goals (mdgs: http://www. un.org/millenniumgoals/), which are a series of global development targets set by the un millennium assembly. in the direct assistance section, % of spending was allocated to hiv/aids-related projects, whereas < % was allocated to the control of malaria and other parasitic diseases. this reflects the political commitment worldwide and especially the efforts that have been prevailing in recent years to combat hiv/aids [ , ] . japan had a japan has put great emphasis on the promotion of public health and the improvement of underlying social and economic conditions that enable infectious diseases to flourish and spread. these include illiteracy, lack of potable water and sanitation, and inadequate access to basic health services. major importance has been assigned to water supply projects, which facilitate and promote infectious and parasitic disease control and improve the socioeconomic status of communities through reduction of the time and labour required for drawing water [ ] . japanese leadership in this respect has placed japan as the top global donor in the sector, having contributed to more than one-third of water supply and sanitation projects in the developing world to provide > million people with access to safe drinking water and basic sanitation in the past five years [ ] (http://www.mofa.go.jp/mofaj/ gaiko/oda/index.html). the idi also provided extensive support to basic education, not only through the building of schools and the training of teachers but also through equipping schools with basic sanitation and a clean water supply, and developing school-based programmes (e.g. deworming, hygiene education and hiv/aids awareness and education). for discussion of japanese initiatives in school health, see ref. [ ] . the creation of new and multifaceted partnerships has been integrated throughout all idi activities. first, japan has provided small grants to local, international and japanese ngos in > countries. second, japan has cemented and extended its partnerships with other donors, in particular with the usa. japan signed the japan-usaid partnership for global health document in june and sent joint project-formulation missions to nigeria, nepal, honduras and other countries to initiate collaborative efforts to combat infectious diseases. third, japan has strengthened partnerships with the world health organization (who: http://www.who.int), united nations children's fund (unicef: http://www.unicef.org) and other un agencies in various programmes such as the roll back examples japan has provided diagnostic kits, equipment, voluntary counselling, testing centres, training of laboratory technicians and clinicians, and other types of support to control hiv/aids in many hiv-epidemic countries. for vaccine-preventable diseases, the idi has supplied vaccines with a cold chain and provided technical assistance for surveillance and laboratory and clinic management. in support of the global malaria programme and rbm, japan has donated anti-malarial drugs, diagnostic equipment and long-lasting insecticide-treated bed nets, and offered technical assistance and training for laboratory and clinical management, especially in sub-saharan africa and the greater mekong subregion to combat lymphatic filariasis in the pacific region, japan has contributed to pacelf by offering the know-how learned from japanese experience in eliminating this disease in the s. the idi has also provided diethylcarbamazine and immunochromatographic test cards and deployed the japan overseas cooperation volunteers for this programme [ ] . japan has also been one of the largest donors in controlling chagas disease in central america through the provision of equipment and supplies, in addition to technical assistance for programme management and individual interventions, including surveillance, materials for information, education and communication, and insecticide spraying [ ] . in partnership with the who and the carter center (http://www. cartercenter.org), japan has contributed to the guinea worm eradication program by establishing community-based surveillance systems and offering health education in infected villages. the idi also provided indirect assistance to improve water supply systems using new water-filtering equipment. in response to the tremendous impact of severe acute respiratory syndrome (sars) and avian flu on neighbouring asian nations and the urgent need for the containment and control of these disorders, japan provided quick and intensive assistance to china, vietnam and other affected nations. japan provided preventive, diagnostic and curative supplies, in addition to equipment and expertise [ ] . as the largest donor in the western pacific region of the who, the idi has supported the polio eradication programme. this region obtained polio-free status in october [ ] . japan has made the second biggest contribution, after the usa, towards the eradication of guinea worm and continues to support this programme [ ] . the number of people infected with guinea worm worldwide declined by %, from . million cases in to in . good results have also been achieved in chagas disease control in central america. the vector rhodnius prolixus has been eliminated from villages in nine health areas, and the house infestation rate of another vector, triatoma dimidata, has decreased by % following the first large-scale vector control project in guatemala. this project was supported by japan and involved two cycles of residual spraying in > houses in (http://www.paho.org/english/ ad/dpc/cd/dch-jica-pjt.doc). the idi direct assistance programme has provided several projects and interventions, varying from provision of supplies and equipment to technical assistance and training. these were planned and implemented with the recipient governments and development partners according to the needs of the countries. some examples of the idi projects are listed in box . it is difficult to measure the impact of the idi in terms of mortality or morbidity reduction with regard to each infectious disease because the inputs of the idi vary according to the needs of each country. in addition, the contribution by idi is just a part of all the efforts and interventions made by the recipient countries, donors and other development partners. however, there are some examples of the idi having made a major contribution and having shown considerable impact (box ). since the un adopted the mdgs, global development efforts have been increasingly focused and result orientated [ ] . substantial funding has gone to the control of hiv/aids and several other infectious diseases through the gfatm, the us president's emergency plan for aids relief (pepfar: http://www.pepfar.org) and other initiatives. however, relatively little funding is provided to control several parasitic diseases that are now increasingly being labelled as the neglected diseases [ , ] . although some of the recently formed foundations and agencies such as the carter center focus on the eradication or elimination of individual parasitic diseases [ ] , most of the donors and aid agencies focus their attention on hiv/aids and other high-profile killer diseases [ ] . under these circumstances, japan needs to pay special attention to the neglected diseases. apart from the mdgs, there are other approaches that focus on poverty reduction and that promote coordinated efforts and actions involving the recipient country, donor agencies and other stakeholders; these include poverty reduction strategy papers and sector wide approaches [ , ] . these are characterized by a set of operating principles, including broadening policy dialogue, developing a private sector policy (e.g. health and education, and a common realistic expenditure programme), common monitoring arrangements and additional coordinated procedures for funding and procurement [ , ] . this trend has been gradually preventing a single donor from providing specific projects in developing countries [ ] . rather, it is recommended to enable donors to support overall health planning and financing in the recipient countries [ ] . however, many developing countries still have several specific needs that range from the central government to individual communities and from financial assistance to technical matters. donor agencies should carefully observe the real needs of recipient countries and be aware of the short-and long-term effects of external assistance. infectious and parasitic diseases are more prevalent among the poor; therefore, unless special attention and assistance are given for the prevention, treatment and care of the poor, health inequities will broaden and expand, even in a country undergoing rapid development. japan has upheld the concept of 'human security', which is a human-centred approach that protects and improves the welfare of people most in need and empowers them to cope with adversity [ ] . professionals who work on infectious or parasitic disease control in or for developing countries also need to broaden their views to encompass the more comprehensive and integrated approach that is needed if poverty reduction and sustainable development of communities, nations and the world are to be successfully achieved. true to its long-term commitment, japan announced a new health and development initiative (hdi) in . this will build on the foundations and successes of the idi and will commit a further us$ billion over a five-year period [ ] . the hdi will provide substantial resources to help the global community achieve the mdgs and to help many developing countries improve the overall wellbeing of their populations and, thereby, encourage them to work towards attaining a higher quality of life. a world wide web resource for plasmodium vivax will see the launch of www.vivaxmalaria.com, a site for the plasmodium vivax research community. the website will be divided into five sections. (i) disease: data, graphics and links about the history of p. vivax malaria, its incidence, life cycle, morphology of infected cells, parasite strains and therapeutics. (ii) genomics: describing the p. vivax genome project and related genomics initiatives, and comparative genomics of plasmodium species. (iii) issues: highlighting outstanding questions about p. vivax biology, pathology and epidemiology. (iv) resources: protocols, reagents and resources for p. vivax research. (v) meetings: details of previous and upcoming conferences of interest to p. vivax researchers. a supplementary section will categorize links on the site and other links of interest to the community. for information and to offer suggestions, please contact the webmaster at: webmaster@vivaxmalaria.com collaboration in the fight against infectious diseases g countries accused of ignoring debt relief global plagues and the global fund: challenges in the fight against hiv, tb and malaria japan's official development assistance: recent issues and future directions the united states and japan pursue a common agenda japan/united states collaboration in international hiv prevention and care global parasite control initiative of japan (hashimoto initiative) infectious diseases, non-zero-sum thinking, and the developing world social and economic impact of infectious diseases -united states infectious diseases -new and ancient threats to world health the disease profile of poverty: morbidity and mortality in northern uganda in the context of war, population displacement and hiv/aids factors and determinants of disease emergence emerging zoonoses and pathogens of public health significance -an overview infection control: old problems and new challenges global health inequalities: an international comparison ) health, development and the millennium development goals health is a global issue impact of infections on primary caregreater than expected monitoring motherhood: sociocultural and historical aspects of maternal and child health in japan population strategy trends and regional variations in infant mortality rates in japan epidemiological review of tuberculosis okinawan health sector human resources experiences since the mid- s and their applications to international health; final report. ministry of education ghana/jica/who training course in polio diagnostic procedures and vaccine potency testing international cooperation with islamabad children's hospital. pakistan: the jica project halving global poverty okinawa infectious disease initiative (idi) -summary of mid-term evaluation report the global hiv/aids pandemic, structural inequalities, and the politics of international health the global fund to fight aids, tuberculosis and malaria (gfatm). health policy plan water quality and health in the new millennium: the role of the world health organization guidelines for drinking-water quality international cooperation for the improvement of environmental sanitation by japan reconsidering the underestimated burden caused by neglected tropical diseases donor funding priorities for communicable disease control in the developing world. health policy plan neglected' diseases but unrecognised successes -challenges and opportunities for infectious disease control priorities in global assistance for health, aids, and population new approaches to development cooperation: what can we learn from experience with implementing sector wide approaches? working paper , centre for aid and public expenditure poverty reduction strategy papers pro-poor health policies in poverty reduction strategies. health policy plan the sector-wide approach: a blessing for public health? the sector-wide approach in health: what is it? where is it leading? better health in developing countries: are sector-wide approaches the way of the future? rethinking human security health and development initiative (hdi) -japan's contribution in achieving the health-related mdgs pacific collaboration to eliminate lymphatic filariasis control of chagas disease experience and review of sars control in vietnam and china certification of poliomyelitis eradication. who western pacific region dracunculiasis (guinea worm disease) eradication key: cord- - eof y authors: inoue, hiroyasu; todo, yasuyuki title: the propagation of economic impacts through supply chains: the case of a mega-city lockdown to prevent the spread of covid- date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: eof y this study quantifies the economic effect of a possible lockdown of tokyo to prevent the spread of covid- . the negative effect of such a lockdown may propagate to other regions through supply chains because of supply and demand shortages. applying an agent-based model to the actual supply chains of nearly . million firms in japan, we simulate what would happen to production activities outside tokyo if production activities that are not essential to citizens’ survival in tokyo were shut down for a certain period. we find that if tokyo were locked down for a month, the indirect effect on other regions would be twice as large as the direct effect on tokyo, leading to a total production loss of trillion yen in japan or . % of the country’s annual gdp. although the production that would be shut down in tokyo accounts for % of the total production in japan, the lockdown would result in an % reduction of the daily production in japan after one month. covid- , a novel coronavirus disease, has been spreading worldwide. by march , , the total number of confirmed cases of covid- reached , , whereas the total number of deaths was , [ ] . to prevent the spread of covid- , most countries have implemented unprecedentedly stringent restrictions, such as a shutdown of national borders, limits on public gatherings, and closures of schools, shops, and restaurants. in some cases, whole cities and regions have been locked down. for example, wuhan, the epicenter of the novel coronavirus, was locked down from january to march , , including the closure of all public transport and all companies not essential to citizens' survival, including manufacturing plants, for most of the period (reuters, march , ) . apparently, the lockdown heavily affected wuhan's economy and its population of million people. moreover, because wuhan, known as one of china's "detroits", is a center of the automobile industry and supplies parts and components of automobiles to domestic and foreign plants, the effect of the lockdown propagated to other regions of china and other countries through supply chains. for example, honda, a japanese automobile manufacturer that operates plants in wuhan, reduced the production of automobiles in japan due to a lack of supplies of parts from china in early march (nikkei newspaper, march , ) . recently, many studies have empirically confirmed that economic shocks propagate across regions and countries through supply chains. some studies applying econometric analysis to firm-level data show that firms linked with suppliers and customers in areas affected by natural disasters exhibit decreased performance [ ] [ ] [ ] [ ] . several others have estimated economic impacts of the spread of covid- , incorporating propagation through input-output linkages across sectors and countries /citeoecd , mckibbin , bonadio , guan , mccann . for example, mckibbin and fernando [ ] use a hybrid of dynamic stochastic general equilibrium (dsge) models and computable general equilibrium (cge) models assuming international and inter-sectoral input-output (io) linkages and estimate the effect of the spread of covid- on the world gdp. guan and others /citeguan utilize international io tables at the country-sector level and estimate impacts of lockdowns of heavily infected countries on production in the world economy. according to their estimates, if europe and the united states imposed a lockdown strategy with a strictness measure of % (i.e., % of production in most non-essential sectors are shut down) for months, the world value added would decline by %. by contrast, inoue and todo [ ] apply firm-level data for the actual supply chains in japan, rather than inter-sectoral io tables, to an agent-based model to simulate the production trajectory after the great east japan earthquake in . their results indicate that the propagation effect of an economic shock through supply chains was substantially larger than the direct effect of the earthquake. inoue and todo [ ] also show that complex network characteristics of supply chains, such as scale-free properties and complex loops, aggravate the propagation effect. without any network complexity, i.e., if they assume no firm-level inter-linkages but only inter-industry linkages or assume a randomly determined network with no complexity, they find that the propagation effect is quite small. the analytical framework of inoue and todo [ ] has several advantages. first, although some studies quantitatively find that supply chains are equipped with complex network characteristics /citeborgatti social, battini towards, fujiwara , they do not examine propagation of shocks through supply chains. second, the conclusion of inoue and todo [ ] that the structure of networks significantly influences propagation is consistent with recent findings in the network science literature [ ] [ ] [ ] [ ] [ ] [ ] . for example, diffusion is generally promoted when the degree distribution follows a power law and, hence, there are several hub nodes with a large number of links. in such a network, when a shock reaches a hub node, it spreads quickly to many others. third, although the econometric analysis of /citebarrot , boehm , carvalho , kashiwagi quantitatively confirms propagation through supply chains, their analysis cannot estimate the total effect of a shock that can be obtained from the framework of inoue and todo [ ] . finally, the studies estimating the effect of covid- rely on either a macroeconomic econometric model at the country level [ ] or a general equilibrium model assuming international and inter-sectoral io linkages [ , [ ] [ ] [ ] and thus do not incorporate complex inter-firm linkages. as a result, the estimates of the previous studies may be largely undervalued, as suggested by the findings of inoue and todo [ ] . therefore, this study utilizes the framework of inoue and todo [ ] and examines propagation of the economic effect of lockdown of a city to prevent the spread of covid- to other regions through supply chains. in particular, when a large industrial city connected with other regions through supply chains in a complex manner is locked down, the propagation effect may be quite large. therefore, we focus on the economic effect of a lockdown of tokyo on other regions. specifically, by applying the agent-based model developed in inoue and todo [ ] to actual supply-chain linkages of approximately . million firms in japan, we simulate what would happen to production activities outside tokyo if tokyo were locked down or nonessential production activities in tokyo were shut down for a certain period. tokyo is an appropriate case for the purpose of this study because it is one of the largest cities in the world and a hub for global supply chains and was, in fact, locked down in april and may . using this framework, we can obtain a reasonable estimate of the propagation effect of lockdown across regions that cannot be computed from other studies using econometric approaches or io tables and thus can highlight the impact of the propagation through supply chains. the data used in this study are taken from the company information database and company linkage database collected by tokyo shoko research (tsr), one of the largest credit research companies in japan. the former includes information about the attributes of each firm, including the location, the industry, the sales, and the number of employees, whereas the latter includes major customers and suppliers of each firm. because of data availability, we utilize data for firm attributes and supply chains in . the number of firms in the data is , , , and the number of supply chain links is , , . that is, our data identify major supply chains of most firms in japan, although they lack information about supply chain links with foreign entities. because the transaction value of each supply chain tie is not available in the data, we estimate sales from a particular supplier to each of its customers and consumers using the total sales of the supplier and its customers and the input-output (io) tables for japan for . in this estimation process, we must drop firms without any sales information. accordingly, the number of firms in our further analysis is , , and the number of links is , , . although firms in the tsr data are classified into , industries according to the japan standard industrial classification, we simplify them into industries classified in the io tables. s appendix provides details of the data construction process. in the supply chain data described above, the degree, or the number of links, of firms follows a power-law distribution (see s fig) , as often found in the literature [ ] . the average path length between firms, or the number of steps between them through supply chains, is . . this small average path length indicates that the supply chains have a small-world property, i.e., firms are indirectly and closely connected through supply chains. therefore, we would predict that economic shocks quickly propagate through the supply chains. using the same dataset, previous studies [ , ] find that - % of firms are included in the giant strongly connected component (gscc), in which all firms are indirectly connected to each other through supply chains. the large size of the gscc prominently shows that the network has numerous cycles and a complex nature, which is unlike the common image of a layered supply chain structure. our simulation employs the dynamic agent-based model of inoue and todo [ , ] , an extension of the model of hallegatte [ ] that assumes supply chains at the firm level. in the model, each firm utilizes inputs purchased from other firms to produce an output and sells it to other firms and consumers. supply chains are predetermined and do not change over time in the following two respects. first, each firm utilizes a firm-specific set of input varieties and does not change the input set over time. the variety of inputs is determined by the industry of the producer, and hence, firms in the same industry are assumed to produce the same output. second, each firm is linked with fixed suppliers and customers and cannot be linked with any new firm over time. furthermore, we assume that each firm keeps inventories of each input at a level randomly determined from the poisson distribution. following inoue and todo [ ] , in which parameter values are calibrated from the case of the great east japan earthquake, we assume that firms aim to keep inventories for nine days of production on average. when a lockdown directly or indirectly causes a reduction in the production of particular firms, the supply of products of these firms to their customer firms declines. then, one way for customer firms to maintain the current level of production is to use their inventories of inputs. alternatively, customers can procure input from their other suppliers in the same industry already connected prior to the lockdown if these suppliers have additional production capacity. if the inventories and inputs from substitute suppliers are insufficient, customers have to reduce their production because of a shortage of inputs. in addition, suppliers of the firms directly affected by the lockdown may have to reduce production because of the reduction of demand from affected customers. accordingly, the economic shock propagates both downstream and upstream through supply chains. s appendix provides the details of the model. in the simulation, we assume an extreme case of tokyo's lockdown where all production activities that are not essential to citizens' survival (hereafter referred to as non-essential production activities) in the central part of tokyo ( wards, hereafter simply referred to as tokyo) are shut down for either one day, one week, two weeks, one month, or two months. essential production activities are defined as those in the wholesale, retail, utility, transport, storage, communication, healthcare, and welfare sectors (see s appendix for details). after the lockdown period, all sectors immediately resume production at the same level as in the pre-lockdown period. because the inventory target of each firm is randomly sampled from the poisson distribution (see s appendix), we run thirty simulations with different sets of inventory targets of firms for each lockdown duration and average over the thirty sets of results. it should be noted that our assumption does not exactly match the experience of japan. in practice, the japanese government announced the state of emergency for some prefectures including tokyo from april to may , , and for the whole country from april to may . according to the act on special measures for pandemic influenza and new infectious diseases preparedness and response, the japanese government can declare a state of emergency if the spread of a infectious disease, such as covid- , is rapid and nationwide. in the state of emergency, some economic activities were requested to be closed, and people were requested to reduce their contacts with others by %. although there was no legal punishment for disobeying the requests, strong social pressure in japan led people and businesses to voluntarily restrict their activities to a large extent. as a result, production activities including those in sectors not officially restricted were reported to shrink substantially (mainichi newspaper, may , ). because it is still unclear to what extent economic activities shrank, we assume a complete shutdown of non-essential production activities. in addition, to highlight the propagation of the effect of a lockdown of a mega-city to other regions, we focus on a lockdown of tokyo, rather than the entire economy. when tokyo is locked down, the value-added production of tokyo immediately becomes almost zero. because the daily production of non-essential sectors in tokyo is estimated to be billion yen, or approximately . billion us dollars, the total direct loss of production in tokyo due to the lockdown is billion yen multiplied by the number of days of the lockdown period. table shows the direct production loss in tokyo for each case in the second column. in addition, the third column shows the production loss outside tokyo (which includes essential sectors in tokyo; hereafter, this area is simply called outside tokyo), which is the propagation effect through supply chains. the indirect loss is calculated from a total loss less the direct damage over the duration of the lockdown and the following three weeks. as shown in fig , after the three weeks, the production fully recovers. the total loss is the total gap between the pre-lockdown daily production and the current daily production. the results in table are the averages of the simulations. the results indicate that when tokyo is locked down for only one day, the production loss outside tokyo, though not locked down, is already billion yen, % of the production loss in tokyo. when the lockdown continues for a month, the indirect effect on other regions is twice as large as the direct effect on tokyo, and the estimated total production loss is . trillion yen, or . % of the annual gdp. in fig , each line shows the dynamics of the total daily value added in japan in each case assuming different lockdown durations. when the lockdown continues for a month, the daily value added of japan becomes only approximately one-seventh of that before the lockdown. this result implies that even when the initial production loss in tokyo is small, its propagation effect on other regions can become large as the lockdown continues. fig shows temporal and geographical visualizations of the simulation of a lockdown. a video of the visualization is available (see s video). the reductions in firm production are averaged in municipalities. the red areas indicate that the production in the area is less than or equal to % of firms' capacity on average, whereas the light red and orange areas show firms with a more moderate decline in production. the left figure illustrates that a non-negligible number of areas (firms) distant from tokyo are already affected on the first day of the lockdown. two weeks later, affected areas spread throughout the country, as shown in the right figure. these visualizations indicate that the indirect effect propagates geographically as the lockdown is prolonged. in addition to the benchmark simulations above, we experiment with three alternative sets of simulations. first, we assume that all production activities including essential activities are shut down. then, the daily production loss in tokyo is billion yen, % larger than that in the benchmark simulations ( billion yen). however, we find that the total production loss in japan from a lockdown of tokyo for a month is . trillion yen, only % larger than that in the benchmark ( . trillion yen) (see s table for details) . second, we assume that industrial demand is prioritized over consumer demand so that production activities outside tokyo will be less affected. for example, computers can be used table . the loss of value added because of tokyo lockdowns. this table shows the results from the simulations assuming the shutdown of all non-essential production activities. these results are based on the average of the simulations. (unit: trillion yen). indirect effect on other regions in japan total effect (% of gdp) both by customer firms for production and citizens for consumption. in the benchmark simulation, when the output of a product is not sufficient because of a lockdown, we assume that the limited output is rationed to customer firms and consumers based on their relative demand prior to the lockdown. however, in this alternative simulation, customer firms are prioritized to maximize production in downstream firms (see s appendix for details). then, we find that a one-month lockdown results in a production loss in japan of . trillion yen. because the production loss does not substantially change from the benchmark result ( . trillion yen), we conclude that industry prioritization is not very effective in alleviating the propagation effect of a lockdown. finally, we investigate the assumption of a moderate initial shock. since we only know the addresses of the firms' headquarters, we assign each firm to each headquarters' location in the benchmark and the above alternatives. however, even if the headquarters of a firm is located in tokyo, other business establishments and factories may continue to operate (however, it may not be possible to obtain such detailed trade information between business establishments and factories). to take this situation into consideration, we proportionally assign the production loss to firms that have headquarters in tokyo based on the number of headquarters (i.e., counted individually), business establishments, and factories in tokyo over the number in all areas. this setting may be the other side of the extremely mitigated condition because if the headquarters are closed, the other business establishments and factories probably cannot operate at full capacity. obviously, this assumption shows a much smaller direct shock ( billion yen per day) than the benchmark shock ( billion yen per day). however, if the lockdown is prolonged for a month, the total loss is . trillion yen, a difference of . trillion yen from the benchmark. we conclude that this difference is not large enough to change the interpretations of the benchmark results. the simulation results clearly show that the effect of a lockdown of tokyo quickly propagates to other regions outside tokyo, leading to a substantial effect on the entire japanese economy. this conclusion is consistent with the previous finding that economic shocks due to natural disasters propagate to non-disaster regions and result in a large total loss /citeinoue . although the production of non-essential sectors in tokyo accounts for . % of the total production in japan, a lockdown of tokyo for a month would result in a reduction of the daily production in japan of %, or . trillion yen. in addition, the effect on other regions becomes progressively larger as the duration of the lockdown becomes longer: when the duration doubles, the production loss more than doubles. in the case of a lockdown for one day, the total loss of value added outside tokyo is % of the loss in tokyo. however, when the lockdown continues for a month, the loss outside tokyo is twice as large as the loss in tokyo. this result implies that the effect of a longer lockdown can reach firms that are "farther" from tokyo along supply chains in terms of the network. to alleviate the propagation effect through supply chains, one could limit the shutdown of production activities or prioritize producers' use of goods and services over consumers' use. the left and right panels show the first day and the first weeks of the lockdown, respectively. the reductions are aggregated and averaged over firms in municipalities. the red areas, for example, indicate firms in the areas whose actual production is substantially (more than %) smaller than their capacity before the lockdown on average. however, our results indicate that these measures would not work well, particularly when the duration of a lockdown is long. our analysis provides several policy implications. first, because the overall effect of a lockdown of a mega-city on the entire economy is extremely large when we take into account its propagation effect through supply chains, lockdowns should be considered a measure of last resort. instead, the spread of covid- should be prevented earlier using other means and any lockdown of a mega-city should be avoided. second, because the total effect of a lockdown progressively increases with its duration, a mega-city lockdown, if it cannot be avoided, should be as short as possible. policymakers should be aware that policies to alleviate the propagation effect may not work when the lockdown duration is long. several caveats of this study should be mentioned. first, we assume that firms cannot find any new suppliers when supplies from their suppliers in tokyo are disrupted. second, for simplicity, the model assumes that even service sectors have the inventory mechanism. finally, the model in this study only considers the dynamics of production and ignores possible changes in prices and wages incorporated in the literature [ , ] . these assumptions may be too strong, leading to an overestimation of the propagation effect. however, our main result that the propagation effect is substantial would hold. this research was conducted as part of a project entitled ''dynamics of economy and conceptualization: hiroyasu inoue, yasuyuki todo. coronavirus covid- global cases by the input specificity and the propagation of idiosyncratic shocks in production networks input linkages and the transmission of shocks: firm-level evidence from the tohoku earthquake supply chain disruptions: evidence from the great east japan earthquake. columbia business school research paper international propagation of economic shocks through global supply chains. winpec working paper the global macroeconomic impacts of covid- : seven scenarios. cama workding paper firm-level propagation of shocks through supply-chain networks collective dynamics of 'small-world' networks structural holes and good ideas the spread of behavior in an online social network experiment networks: an introduction network science a simple model of global cascades on random networks the world economy at risk (oecd economic outlook global supply chains in the pandemic global economic footprint of the covid- pandemic covid- and the transmission of shocks through domestic supply chains. central bank of ireland large-scale structure of a nation-wide production network propagation of negative shocks through firm networks: evidence from simulation on comprehensive supply-chain data an adaptive regional input-output model and its application to the assessment of the economic cost of katrina. risk analysis modeling loss-propagation in the global supply network: the dynamic agent-based model acclimate transportation and supply chain resilience in the united republic of tanzania: assessing the supply-chain impacts of disaster-induced transportation disruptions the ministry of internal affairs and communications. the updated input-output tables for japan methodology: hiroyasu inoue.writing -original draft: hiroyasu inoue, yasuyuki todo. key: cord- - cch w m authors: soma, takehisa; nakagomi, osamu; nakagomi, toyoko; mochizuki, masami title: detection of norovirus and sapovirus from diarrheic dogs and cats in japan date: - - journal: microbiol immunol doi: . / - . sha: doc_id: cord_uid: cch w m norovirus (nov) and sapovirus (sav) are important causes of human diarrhea. in this study, between and fecal samples were collected from dogs and cats with diarrhea and examined to determine the prevalence of nov and sav infections in japan. to detect caliciviruses, approximately bases targeting the polymerase gene were amplified using rt‐pcr and subjected to phylogenetic and homology analyses. specific pcr products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as nov, two canine isolates as sav and the remaining eight feline isolates as vesivirus (vev). the three nov isolates were classified into the same clade as that of known canine and feline novs; their homologies ( . – . %) were higher than those with human genogroup iv (giv) novs ( . – . %). the homology of the feline nov isolate with previously reported feline nov isolates was particularly high ( . – . %). regarding sav, the two canine isolates were classified into the same clade as known canine savs and their homologies ( . – . %) were higher than those with other mammal savs ( . – . %). the eight feline vev isolates were assumed to be feline calicivirus. the present study is the first report of the presence of nov‐ and sav‐infected dogs and cats in japan. the findings suggest there are species‐specific circulations of nov and sav among dogs and cats, in japan. nov, sav, vev, lagovirus and nebovirus, belong to the family caliciviridae, which comprises small non-enveloped viruses containing approximately . kb singlestranded rna; both nov and sav are clinically important enteric pathogens in humans ( , ) . although canine caliciviruses belonging to vev have been detected and are widely prevalent in dogs in japan and korea, they have not yet been established as an enteric pathogen for dogs ( ) ( ) ( ) ( ) . on the other hand, fcv, which is a vev, is a well-known respiratory pathogen in cats ( ) . nov is classified into at least five genogroups (gi, gii, giii, giv and gv) based on their vp sequences ( ) . additional genogroups (gvi and gvii) have recently been proposed ( ) ( ) ( ) . gi, gii, giv and gvi are known to infect humans; of these, gii is the most prevalent ( , , , ) . giv and gvi novs have been detected in dogs, whereas giv nov has been detected in a cat and a lion ( , ) . the major hunov genogroup gii was also detected recently in the feces of three dogs ( ) and the roles of canine and feline novs in public health and their clinical impact have been inferentially discussed ( , ) . sav is classified into genogroups (gi-gv) based on vp sequences ( ) . sav has not yet been detected in cats. because sav has been detected in only a few dogs, the relationship between sav of canine and human origin has not yet been elucidated in detail; however, the zoonotic potential of sav of dog origins has been preliminarily discussed ( ) . because of their close relationship with humans as companion animals, dogs and cats have attracted special attention with regard to zoonosis; thus, investigation of their epidemiology is meaningful. in this study, we therefore examined the feces of clinical cases for caliciviruses, compared them with known isolates and discuss their diarrheagenic potential. from september to march , the feces of household dogs and cats with diarrhea were submitted by veterinary clinics throughout japan to a commercial veterinary laboratory (marupi lifetech, osaka, japan) for detection of parvovirus. the fecal samples were suspended in sterile saline, the suspensions centrifuged and nucleic acids isolated from the supernatants using a qiaamp dna mini kit (qiagen, germantown, md, usa) according to the manufacture's instructions, after which they were preserved in a freezer. although this kit is designed for dna purification, rna can also be extracted and purified, according to the attached instruction and a previous study ( ) . using a qiagen one step rt-pcr kit, caliciviruses were detected by rt-pcr with the p d-p d primer pair that has been reported to bind to the polymerase gene of the calicivirus ( , ) . this primer pair is expected to amplify a bp fragment of sav and vev and a bp fragment of nov. two point five ml of the extracted rna was added to ml reaction mixtures containing ml of  buffer (finally . mm mgcl ), . mm of deoxynucleoside triphosphate, u rnase inhibitor (promega, madison, wi, usa), . mm of the primers and . ml of enzyme mix. the rna was reverse transcribed at °c for min, followed by inactivation of reverse transcriptase and denaturation of cdna template at °c for min. the cdna was amplified in sequential cycles of denaturation at °c for min, annealing at °c for min and extension at °c for min, followed by a final extension at °c for min. the pcr products were electrophoresed at v for min in a % agarose gel, after which they were stained with ethidium bromide and visualized under uv illumination. the pcr amplicons were purified using exosap-it, and submitted to direct sequencing of both strands by a dye-terminator cycle sequencing methodology (bio matrix research, chiba, japan). the sequences were aligned with reference strains in the genbank database using clustal w and mega software ( ) . phylogenetic trees were constructed employing the neighborjoining method ( ) , and their homologies were using blast search. to investigate other diarrheagenic viruses in the canine samples, cpv- ( ), ccov-i and -ii ( ), cdv ( ), cadv- ( ) and rv ( ) were examined by pcr. for the feline samples, fplv ( ), cpv- , fcov-i and -ii ( ) and rv were examined. the pcr products ( or bp) amplified with the p d-p d primer pair were detected in four and nine of canine and feline diarrheal samples, respectively. when the nucleic acid sequences of the pcr products of these samples were subjected to phylogenetic analysis, two canine and one feline sample were classified into the clade of nov, two canine samples were classified into the clade of sav, and the other eight feline samples were classified into the clade of vev, as shown in figure . the partial rdrp sequences of each viral genus were compared with the reference strains in the genbank database. as shown in figure , the dog-derived nov isolates (ca/osaka / /jp and ca/aichi / / jp) and cat-derived nov isolate (fe/saitama / / jp) were classified into the same clade as known canov and fenov, showing . - . % homologies. this homology rate is higher than that ( . - . %) with giv hunov, which clusters in a different clade on the phylogenetic tree. in addition, the cat-derived isolate (fe/saitama / /jp) had a very high homology ( . - . %) with known fenov strains (cu e and pistoia ). in contrast, the homologies of the canine isolates in the present study with each previous canov isolate in the same clade were . - . %, whereas those with ca/hku_ca f and ca/hku_ ca f not classified into the same clade had low homologies ( . - . %). two isolates from dogs classified as sav (ca/kyoto / /jp and ca/mie / /jp) clustered into the same clade in the phylogenetic tree as previously isolated casavs in ca, usa (ca/an and ca/an d) (fig. ) . the homologies among these four sav strains of dog origin were . - . %, which are higher than those with sav isolates from other mammals including humans ( . - . %). vevs from the other eight cats were classified as fcv species on the basis of the clustering pattern in the phylogenetic tree (fig. ) . table ). both nov and sav, two isolates of each, were detected in the feces of dogs and one isolate of nov was detected in the feces of a cat. to the best of our knowledge, these are the first such findings in japan and only a few studies have detected casav and fenov in other parts of the world ( , ) . in the present study, giv and gvi novs derived from canines and felines could not be discriminated by phylogenetic analysis, presumably because only a short part of the polymerase gene was analyzed; however, three nov isolates detected in dogs and a cat (ca/osaka / /jp, ca/aichi / /jp and fe/saitama / / jp) were classified into the same clade with known canovs and fenovs in the phylogenetic tree. in addition, excluding the strains isolated from chinese dogs (ca/hku_ca f and ca/hku_ca f) whose genogroups have not yet been identified ( ) , the homologies of these three isolates with known canov and fenov ( . - . %) were higher than those with giv hunov ( . - . %), indicating that the present three isolates form a clade with known canovs and fenovs that differs from that of hunovs in the phylogenetic tree. it has been suggested that nov has been circulating among dogs ( ) ; the findings of the present study support this, as described above. however, the homologies of the two nov isolates with known canovs ranged from . to . % even within the same clade, suggesting the presence of several lineages within the canov genogroups, as described previously ( , ) . although only a small number of fenov strains have been characterized, the homologies observed between the present feline nov isolate (fe/saitama / /jp) and previous cat-and lion-derived isolates (cu e and pistoia ) were extremely high ( . - . %), suggesting the presence of a unique circulation of nov in the felidae that differs from that in dogs. on the other hand, the sav isolates from dogs (ca/ kyoto / /jp and ca/mie / /jp) were classified into the same clade as two canine strains previously reported in the usa and the homologies among these ( . - . %) were markedly higher than those in other mammals ( . - . %), suggesting the presence of a unique circulation of sav in dogs or the canidae. canov, fenov and casav may have an infection cycle in each animal species or family. however, the potential for human infection by some types of nov and sav in dogs and cats cannot be ruled out, because of the following observations: (i) gii nov, which is the most prevalent human nov, has been detected from dogs ( ); (ii) giv and gvi novs, which are the main genogroups in dogs and cats, have been detected in humans ( , ) ; and (iii) antibodies against gvi nov have been detected in human sera ( ) . although a significantly high detection rate of canov in dogs with diarrhea has previously been reported ( ) , epidemiologic data have been insufficient and the pathogenic potential of nov and sav for dogs and cats remains undetermined. in the present study, we were unable to clarify the pathogenicity of novs and savs in dogs and cats, because mixed infections with cpv- or fplv, which are the most virulent causes of diarrhea in dogs and cats, were noted in all five samples in which nov or sav was detected. in previous studies, most dogs infected with canov were approximately - months old ( , ) . a high canov prevalence has been reported in kennels ( ) . all four dogs in which nov and sav were detected in the present study were also months old and purebred, suggesting that canov and casav infections likely also occur in environments where puppies and kittens are ordinarily kept, such as pet shops and breeding colonies. fcv was detected in . % of fecal specimens from cats in the present study. although fcv is a representative pathogen for upper respiratory tract, it has been demonstrated not only in laryngopharyngeal secretions but also in feces ( ) . although fcv, which replicates in the upper respiratory epithelial cells, potentially passes through the alimentary tract and is excreted in the feces, a difference in resistance to bile salts has been observed between fcv isolates from respiratory secretions and feces, suggesting the presence of enterocyte-infecting fcv ( ) . no other diarrheagenic viruses were detected in one fcv-positive sample (fe/tokyo / /jp), and only fcov-i, which generally causes no or mild symptoms of intestinal infections when it infects alone, was detected in three samples (fe/yamaguchi / / jp, fe/saitama / /jp and fe/osaka / /jp). these findings suggest that some type(s) of fcv may be enteric pathogens in cats, but this remains to be elucidated in future studies, including experimental infections. norovirus gastroenteritis zoonotic aspects of infections with noroviruses and sapoviruses a calicivirus isolated from a dog with fatal diarrhoea complete nucleotide sequence, genome organization and phylogenetic analysis of the canine calicivirus molecular and seroepidemiological evidence of canine calicivirus infections in japan seroprevalence of canines calicivirus and canine minute virus in the republic of korea feline 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canine norovirus in dogs from portugal different stabilities to bile among feline calicivirus strains of respiratory and enteric origin no authors have conflicts of interest to declare regarding this study. key: cord- -lq yr authors: venter, zoë title: the interaction between conventional monetary policy and financial stability: chile, colombia, japan, portugal and the uk date: - - journal: comp econ stud doi: . /s - - -w sha: doc_id: cord_uid: lq yr the relationship between monetary policy and financial stability has gained importance in recent years as central bank policy rates neared the zero-lower bound. we use an svar model to study the impact of monetary policy shocks on three proxies for financial stability as well as a proxy for economic growth. monetary policy is represented by policy rates for the emerging market economies and shadow rates for the advanced economies in our paper. our main results show that monetary policy may be used to correct asset mispricing, to control fluctuations in the real business cycle and also to tame credit cycles in the majority of cases. our results also show that for the majority of cases, in line with conventional wisdom, local currencies appreciate following a positive monetary policy shock. monetary policy intervention may indeed be successful in contributing to or achieving financial stability. the results, however, show that monetary policy may not have the ability to maintain or re-establish financial stability in all cases. alternative policy choices such as macroprudential policy tool frameworks which are aimed at targeting the financial system as a whole may be implemented as a means of fortifying the economy. the relationship between monetary policy and financial stability became an important topic in the aftermath of the - global financial crisis (gfc) as central banks lowered their policy rates in an effort to revive the economy and the zerolower bound loomed. traditionally, price stability has been the main objective of central banks and therefore of monetary policy. however, the crisis emphasized the need to implement alternative measures and to expand the scope of monetary policy to include a focus on the maintenance of financial stability while maintaining the primary objective of achieving price stability. kryvstov et al. ( ) discuss the relationship between financial and macroeconomic conditions. they note that financial stability conditions are an important part of monetary policy implementation as financial stability is a precondition for a well-functioning financial system. the purpose of this paper is to study the impact of monetary policy shocks on financial stability for the cases of chile, colombia, japan, portugal and the uk. monetary policy influences the amount of money in circulation in the economy as well as the borrowing cost of money. the countries chosen have autonomous, inflation-targeting central banks, which provides the opportunity for more independent monetary policy decisions. in the case of colombia and chile, this combined with a lack of empirical studies due to limited data availability makes for an interesting case study. the uk dilemma following a tumultuous period of brexit negotiations and uncertainty topped off by growing economic and social uncertainty and loss in the midst of the current covid- pandemic and japan's unending battle with deflation and sluggish growth justifies the study of the uk and japan. lastly, although portuguese monetary policy is determined by the objectives of the european central bank, the effect on credit growth in portugal is noteworthy considering portugal's recent economic recovery following a bailout from the troika formed by the european commission, european central bank and the international monetary fund. the potential impact of the current global pandemic on the newly recovered portuguese economy solidifies the need to study the case of portugal. the five countries are looked at on a case-by-case basis with the impact of monetary policy shocks on financial stability being studied for each individual country case. a decade after the gfc, financial stability and the effective implementation of monetary policy remains a priority for many central banks. studying the relationship between monetary policy and financial stability, in particular, the effect on different areas of potential vulnerability may play an important role in future targeted policy decisions. a number of structural vector autoregressive (svar) analyses have been conducted on the us economy as well as other advanced economies. however, due to shorter time series caused by limited data availability as well as structural reforms in latin american countries, svars have not been used frequently in econometric studies. svar models based on the var approach suggested by sims ( ) have been used frequently in japan, portugal and the uk with four available examples being nakahira ( ) , sousa ( , ) and elbourne ( ) , respectively. in this paper, an svar is employed and impulse response functions are used in an effort to test the impact of monetary policy on financial stability. as in cocriş and nucu ( ) , the impact of short-term interest rates on three proxies for financial stability, namely credit growth, stock prices and the exchange rate, is studied. industrial production is included as a proxy for the real sector and hence, for economic growth. in line with cocriş and nucu ( ) , policy rates are used to represent monetary policy choices in emerging economies. shadow rates are used to represent monetary policy intervention in advanced economies. our main results show that monetary policy may be used to correct asset mispricing due to the inverse relationship between monetary policy intervention and the stock market index for four of the five cases, namely the cases of colombia, japan, portugal and the uk. conventional wisdom suggest that contractionary monetary policy would result in a currency appreciation in countries with a floating exchange rate regime as foreign investment is attracted. the results show that the exchange rate appreciates following a monetary policy shock for the cases of chile, japan and portugal while the currency depreciates for the cases of colombia and the uk. the currency depreciation in the case of colombia could be attributed to the country's reliance on exports as colombia relies heavily on commodity exportation while, in the case of the uk, this depreciation is attributed to uncertainty in the local market. the results also show that monetary policy may be used to limit fluctuations in the real business cycle as can be seen by the inverse relationship between the industrial production index and monetary policy intervention in the majority of cases, namely, for the cases of colombia, japan and portugal. credit growth also has an inverse relationship with monetary policy intervention in the cases of chile and the uk. ergo, monetary policy may be used to tame credit cycles. in the cases of colombia, japan and portugal, credit moves in the same direction as monetary policy intervention. thus, positive monetary policy shock results in an increase in credit. the results show that monetary policy intervention as represented by policy rates and shadow rates may indeed be able to contribute to financial stability. the results show that monetary policy may not have the ability to maintain or re-establish financial stability in all cases. alternative policy choices such as macroprudential policy tools aimed at targeting the financial system as a whole may be useful to reinforce the monetary policy framework. the rest of the paper is as follows: literature review" section surveys existing literature; "methodology and data" section discusses the methodology and the data used; "empirical results" section discusses the empirical results; and "conclusion" section concludes. financial instability is defined as a situation in which the economic performance of an economy is potentially affected by price fluctuations of financial assets (crockett ) . in this paper, three proxies for financial stability are included, the first being credit growth which is looked at in existing literature. for example, jiménez et al. ( ) look at the impact of monetary policy on the supply of credit. they show that tighter monetary policy and weaker economic conditions substantially reduce loan granting, in particular loan granting by banks with lower capital or liquidity ratios. in this paper, we find that tighter monetary policy results in reduced credit growth in chile and the uk and increased credit growth in colombia, japan and portugal. jiménez et al. ( ) also note that the gfc showed that the use of monetary policy rates as a means of supporting credit growth seems advisable. further, allen and gale ( ) , schularick and taylor ( ) , gourinchas and obstfeld ( ) and borio ( ) find that credit growth fluctuations are a common forerunner of financial crises. also, jordà et al. ( ) show that excess credit growth in the period prior to a peak is usually associated with a more severe recession and bernanke and lown ( ) and calvo and reinhart ( ) note that crises are often accompanied by bank credit crunches and halting foreign capital flows. given the importance of credit growth as a harbinger of crises as well as an indicator of the magnitude of crises, the study of the relationship between credit growth and monetary policy intervention as represented by policy rates for emerging economies and shadow rates for advanced economies is of importance. our second proxy for financial stability is the exchange rate, the inclusion of which is substantiated by existing literature. for example, exchange rate depreciation is used as a proxy for financial vulnerability in both eichengreen and gupta ( ) and park et al. ( ) . further, eichenbaum and evans ( ) make use of a var analysis to investigate the effect of shocks (in monetary policy) on exchange rates in the usa and find that contractionary monetary policy results in a long-lasting appreciation in the exchange rate. our results show that tighter monetary policy results in an appreciation of the local currency in chile, japan and portugal. the local currency in the uk and colombia, however, depreciates. dhrymes ( ) look at more economically compatible svar models, they find that foreign interest rates and the real exchange rate are determinants of real output. lastly, cavallari ( ) finds that monetary shocks have no effect on real variables in the long run when considering a svar. in contrast, lee and chinn ( ) extended this model to include exchange rate data and found that monetary policy does indeed influence exchange rates. our final proxy for financial stability is the growth in the stock market which is looked at in earlier literature. granville and mallick ( ) use stock prices, exchange rates and loan-to-deposit ratios as proxies for financial stability and define financial stability in terms of changes in stock prices. asiriyan et al. ( ) consider monetary policy in a bubbly world where low interest rates result in frequent booms and busts in asset prices. both monetary policy and bubbles have expansionary effects on the economy with bubbles having both an overhang effect and a wealth effect. in line with asiriyan et al. ( ), we find that tighter monetary policy results in lower stock prices except in the case of chile. both granville and mallick ( ) and cocriş and nucu ( ) define financial stability in terms of the change in the share price, the exchange rate as the units of local currency per unit of euro and the loan-to-deposit ratio. cocriş and nucu ( ) find that in countries with inflation-targeting schemes, the interest rate instrument facilitates financial stability while, in countries with fixed exchange rates, monetary policy does not encourage financial stability except in the case of bulgaria. lastly, we include industrial production as a proxy for the real business cycle in line with cocriş and nucu ( ) , grilli and roubini ( ) , kim ( ) and kim and roubini ( ) . today, the chilean central bank maintains one of the highest levels of central bank independence globally and maintains a successful inflation-targeting framework. colombia is currently latin america's second largest economy, has a stable currency, an abundance of natural resources, a somewhat stable political system and is seen as one of the greatest success stories globally over the past three decades. chile is the largest producer of copper globally and colombia's biggest export is oil; the currently decreased demand as well as plummeting prices in the midst of the covid- pandemic poses a significant risk to both economies. these two countries have been overlooked in empirical research, much like other latin american countries, for a multitude of reasons including the lack of data availability. the adoption of inflation-targeting regimes in the late s has, however, sparked an interest in the monetary policy mechanism. svar models have been used to study both monetary policy (catão and pagan ; parrado ) and fiscal policy (restrepo and rincon ) in chile and colombia. the number of papers implementing this methodology and focusing on the relationship between monetary policy and financial stability is, however, still somewhat limited. japan, the world's third largest economy, is still toiling against lifelong employment, declining exports, strict immigration policies and stagflation all of which has been further amplified by the current global pandemic; this recently resulted in the country slipping into a recession. earlier literature studying japanese monetary policy using an svar model includes nakahira ( ), schenkelberg and watzka ( ) and yanoy and yoshinoz ( ) . the portuguese recovery in the aftermath of the sovereign debt crisis is arguably the greatest success story in southern europe to date. economically, brexit has worsened investor confidence, resulted in weakened economic growth, higher debt levels and lower living standards. politically, the uk has had three prime ministers in as many years which has resulted in stop-and-start brexit negotiations and political disquietude. the uncommonly restricted economic activity in the midst of the current covid- pandemic, the unbridled pressure on financial resources and the excessive strain on governmental infrastructure will no doubt have a disastrously significant impact on the global economy and in particular, on both the portuguese and the uk economies. svar models have been used to study monetary policy in earlier research focusing on the uk (elbourne ; caglayan et al. ) hurwicz ( ) explains that a structural model is one in which the impact of 'interventions' is predicted. this paper makes use of a svar model to identify the impact of interest rate policy on financial stability. i apply this methodology to the cases of chile, colombia, japan, portugal and the uk. our starting point for the analysis is an svar model of order p (svar(p)). the svar model is as follows: u t is a multivariate white noise process with variance/covariance matrix d and represents the structural innovations, b i and d are (k × k) matrices, c contains k elements where k is the number of variables with i = , …, p. there are k(k − )/ restrictions on b and in the case where b is being lower triangular, there are k(k + )/ unknowns in the matrix. when b is invertible, eq. ( ) is equivalent to eq. ( ) which represents a reducedform var(p): where x t is a (k × ) vector of endogenous variables, is a (k × ) vector of intercepts, l are (k × k) coefficient matrices and t = t , t , … , kt � is an unobservable error term representing the reduced-form innovations with the following properties: ( ) "deliberate policy actions, or changes in the economy or in the nature of known types" (sims ). and where eq. ( ) represents the relationship between the reduced-form shocks and structural shocks where t spans the space of the structural shocks u t . an svar model allows for contemporaneous relationships between the variables and also allows us to study the effect of a shock to one equation while other shocks remain constant. this makes the svar model more appropriate than a reducedform var model in which error terms are correlated, and hence, error terms cannot be decomposed into mutually orthogonal shocks (schenck ) . this follows since, following that b is lower triangular, the cholesky decomposition is used to construct the impulse responses to orthogonal/structural form shocks. these impulse response functions are generated in eviews and confidence intervals of standard deviations based on replications are included in each generated impulse response function. the significance of shocks is interpreted at a % level of significance as each impulse response function includes a % confidence interval. shocks are statistical significant when the confidence interval does not contain zero. the order of the variables may be switched in multiple ways; however, existing literature justifies the ordering of the variables used in this paper. the ordering of the variables is in line with the ordering used by cocriş and nucu ( ) with the authors assuming that a recursive transmission scheme exists under the assumptions that firstly, industrial production is not immediately affected by monetary policy shocks and secondly, loan-to-deposit ratios (credit growth in this paper), stock indices and the exchange rate may be affected immediately by monetary policy shocks. the variables are ordered as above due to the assumption that industrial production affects all other variables contemporaneously. geske and roll ( ) find that changes in real activity, in our case growth in industrial production, lead to changes in money supply, in our case interest rate changes, and in turn, changes in the stock market. the ordering of our variables assumes that the exchange rate is affected contemporaneously by industrial production, interest rates, credit growth and the stock market which is in line with hacker et al. ( ) who find evidence that the exchange rate is granger caused by the nominal interest rate differential. robinson ( ) industrial production innovation short term policy rates innovation credit growth innovation stock price innovation exchange rate innovation i also conclude statistical significance in the cases where the confidence interval does not contain zero for the majority of periods included and also where a single band of the confidence interval is only marginally above or below zero. kim ( ) also finds that stock price movements are driven, to some extent, by interest rate movements. fama ( ) and najand and noronha ( ) show that industrial production is affected by both interest rates and inflation at a lag and, hence, not contemporaneously. argues that financial development follows economic growth. in our case, growth in industrial production has a contemporaneous effect on credit growth. bartov and bodnar ( a, b) show that changes in the dollar affect current abnormal firm returns with a lag, this is in line with angelos ( ) who finds that the volatility of the exchange rate in the us, the uk and japan is largely determined by the volatility of home stock returns. the order of the variables is switched in two robustness checks to confirm the ordering of the primary matrix. the impulse response functions are not affected by the relevant changes in the ordering of the variables in any cases. three unit root analyses are conducted, namely the augmented dickey-fuller (adf) test, the phillips-perron (pp) unit root test and the hylleberg, engle, granger and yoo (hegy). both the pp test and the adf test show that the year-on-year growth rates of all but two cases have an order of integration of i( ), and hence, the growth rates are stationary. the third unit root test, the hegy test, is included to account for the seasonally unadjusted data and the annual differencing used. when looking at the test statistics for frequency , we fail to reject the null hypothesis of the presence of a unit root for the case of the stock market in colombia, credit in portugal and the exchange rate in portugal. when considering the semi-annual frequency, annual frequency as well as the joint significance of the full set of unit roots (seasonal and non-seasonal frequencies), the null hypothesis is rejected in all cases, and therefore, no unit roots are present. in the case of the colombian stock market and the portuguese exchange rate, the presence of the unit root may be a small sample effect. the joint significance of the full set of unit roots is, however, rejected. the presence of unit roots should therefore, not be an issue in our sample. the gfc emphasized the need to focus on both financial stability and price stability as the primary objective of central banks prior to the gfc had been the maintenance of price stability. three proxies for financial stability together with ipi to account for economic growth are included in the analysis with each proxy representing a sector of the economy which could potentially conceal financial stability risks (cocriş and nucu ) . monetary policy is represented by the short-term interest rate and the shadow rate in this paper, short-term interest rates are available from the ifs (for chile) and the fred (colombia). in the case of the advanced economies, the shadow rate [krippner ( [krippner ( , shadow/lower bound framework] is used as a measure of monetary policy intervention. the shadow rate is used in the case of the advanced economies due to the inability of policy rates to fully capture the yang and doong ( ) note that empirical evidence shows that changes in stock prices affect exchange rate movements; however, the impact of changes in the exchange rate on stock market prices is less significant. it is thought that stock markets have a more noticeable effect on exchange rates due to the deregulation of financial markets which has decreased the difficulty of and stimulated capital flows across borders. results available on request. extent of monetary policy intervention in the presence of quantitative easing and other unconventional monetary policy tools. the use of the shadow rate allows one to see the effect of monetary policy tools on factors such as the unemployment rate and industrial production without the limitation of the zero-lower bound on policy rates. the relevant variables are as follows: • credit growth as a proxy for the banking system (bis total credit statistics); • stock index as a proxy for the capital market (federal reserve economic data); • the exchange rate measured as local currency units per unit of us dollar as a proxy for the foreign exchange market (federal reserve economic data), exchange rates quoted as us dollar units per unit of local currency are converted; • industrial production as a proxy for economic growth (federal reserve economic data). ( ) suggest that the akaike information criterion (aic) is appropriate when considering monthly data, the hannan-quinn information criterion (hqic) is appropriate when considering quarterly data with a sample size larger than observations and the schwarz bayesian information criterion (sbic) is appropriate when considering quarterly data with any sample size. the sbic suggests that lags be used for the case of chile while lag is suggested for the case of portugal. all three information criteria suggest that lags be used for the case of colombia while lag is suggested for the case of japan. lastly, the hqic and the sbic suggest lag for the case of the uk. the dataset created contains data for chile between q and q , colombia between q and q , japan between q and q , portugal between q and q and the uk between q and q . breaks exist in the dataset, the number of observations quoted below is based on a balanced svar estimation with the suggested number of lags. the svar and the resulting impulse response functions therefore have the following number of observations. chile has observations, colombia has observations, japan has observations, portugal has observations and the uk has observations. to assess the extent to which the differing sample periods may affect the results, a robustness check, the results of which are not statistically significantly different, limiting the sample period to the period between :q and :q is conducted. figure plots the quarterly central bank policy rate for the five countries as well as the shadow rates for japan, portugal and the uk over the period between and . table summarizes the responses of the individual variables to shocks in the central bank policy rate (chile and colombia) and the shadow rate (japan, portugal and the uk). in three of the five cases (colombia, japan and portugal), industrial production declines following a monetary policy shock while, for chile and the uk, industrial production increases. this result is statistically significant for the case of chile. similarly, the former three cases experience an increase in credit growth following a monetary policy shock while the latter experiences a decline. these results are statistically significant in the case of colombia in the short run and the cases of chile, colombia and portugal in the long run. the level of the stock market index declines in four of the five cases with the level decreasing in the case of chile. these a number of robustness checks are conducted in an effort to validate the results. a robustness check using monthly data (table ) is included as the accuracy of an svar model increases as the number of observations increases. the results show that there is a sign change for the case of industrial production for chile with the sign being positive and statistically significant when considering quarterly data and the sign becoming negative without statistical significance when considering monthly data. both industrial production and the stock market have a sign change for the case of japan, credit has a sign change for the case of portugal and both the exchange rate and credit have a sign change for the uk. neither the results for these cases in the original sample nor the results for these cases in the monthly dataset are statistically significant. lastly, there is a sign change for credit in japan with the sign changing from positive to statistically significant and negative. a second robustness check (table ) using monthly log data shows that the exchange rate depreciated after an interest rate shock in the case of chile and colombia while the exchange rate appreciates in the case of japan, portugal and the uk in line with conventional wisdom. (table ) using quarterly data, however, adjusting the start date and end date of the data to ensure that the period studied remains constant for all cases is included. this robustness check studies the period between :q and :q . the results show that there is a sign change for the uk for industrial production when limiting the time period and there is no sign change for credit growth in any of the cases. the results also show that there is a sign change for japan for the stock market as well as a sign change for japan and the uk for the exchange rate. similarly to the first robustness check, neither the results for these cases in the original sample nor the results for these cases in the limited sample are statistically significant. one may therefore conclude that the difference in sample periods across countries does not affect the results in a statistically significant way. further, two robustness checks (tables , ) using a different ordering of variables and quarterly data are included to check whether a change in the ordering of the variables leads to a change in the results. table presents the results of the case where the position of the short-term policy rates innovation and the industrial production innovation are switched, while table presents the results of the case where the position of the stock price innovation and the exchange rate innovation are switched. there are no sign changes resulting from the change in variable ordering. finally, to add to the robustness of the results, a sixth robustness check (table ) using quarterly data and a bvar model is employed. the bvar model is used as a robustness check to address the concern arising from the small sample size as bvar models have a shrinkage estimator, which adjusts coefficients to match the ( ) is used. the use of the minessota prior and hence a random walk prior is appropriate in this analysis (litterman ) . this is due to the random behavior of macroeconomic time series as well as the flexibility of the restrictions imposed. the results are similar to those of the main results of this paper, with the sign of the change staying the same for the majority of cases with the exception of industrial production for japan and the uk and the exchange rate for the uk. in the remaining cases, the magnitude of the effect of the shock becomes slightly smaller. the financial turmoil caused by the gfc highlighted the importance of financial stability and resulted in the relationship between financial stability and monetary policy becoming an important element when policy mandates were updated in the period after the crisis. in this paper, the impact of monetary policy shocks on financial stability for the cases of chile, colombia, japan, portugal and the uk is analyzed. the five countries chosen have autonomous, inflation-targeting central banks with floating exchange rate regimes in place, which allows us to analyze the impact of monetary policy shocks on five countries with similar policy mandates. the set of countries presents an interesting case with colombia and chile being categorized as emerging economies with a history of limited availability of data, the uk and japan facing political uncertainty and economic uncertainty, respectively, and finally, portugal which experienced the most profound economic growth in the group of countries that received an injection of european funding. an svar model is employed and impulse response functions are used in an effort to test the impact of monetary policy on financial stability. credit growth, the stock market index growth and the exchange rate growth are included as proxies for financial stability while industrial production growth is included as a proxy for the business cycle. as in cocriş and nucu ( ) , the impact of short-term interest rates and shadow rates on three proxies for financial stability, namely credit growth, stock prices and the exchange rate, is studied. industrial production is included as a proxy for the real sector and hence for economic growth. in line with cocriş and nucu ( ) , policy rates are used to represent monetary policy choices in emerging economies. shadow rates are used to represent monetary policy intervention in advanced economies. our main results show that monetary policy may be used to correct asset mispricing due to the inverse relationship between the policy rate and the stock market index for the majority of cases namely, colombia, japan, portugal and the uk. conventional wisdom suggests that a positive monetary policy shock would result in a currency appreciation in countries with a floating exchange rate regime as foreign investment is attracted. the results show that the exchange rate appreciates in both the short run and the long run following a monetary policy shock for three of the five cases, namely the cases of chile, japan and portugal while the currency depreciates for the cases of colombia and the uk. the currency depreciation in the case of colombia could be attributed to colombia's reliance on commodity exports. the currency depreciation in the case of the uk is in line with earlier literature including caglayan et al. ( ) and is attributed to uncertainty in the local market. the results also show that monetary policy may be used to control fluctuations in the real business cycle as can be seen by the inverse relationship between the industrial production index and monetary policy shocks for the cases of colombia, japan and portugal. credit growth also has an inverse relationship with monetary policy shocks for the cases of chile and the uk. consequently, monetary policy may be used to tame credit cycles in some cases. however, in the majority of cases, namely colombia, japan and portugal, there is a positive relationship between credit and monetary policy shocks. monetary policy intervention, as represented by policy rates in the cases of emerging market economies and shadow rates in the cases of advanced economies, may indeed be successful at contributing to or achieving financial stability. however, our results show that monetary policy may not have the ability to maintain or re-establish financial stability in all cases. alternative policy choices such as macroprudential policy tool frameworks which are aimed at targeting the financial system as a whole may be implemented to buttress the economy. see fig. . see fig. . see fig. . see fig. . fig. long run structural impulse response function: the uk. responses of industrial production, the shadow rate, credit, stock market and the exchange rate in the uk to a shadow rate shock. a confidence interval of standard deviations based on replications is included. vertical axes-the deviation from the baseline level. horizontal axes-the number of quarters after the shock. source author's estimates based on datastream and bis statistics, irfs generated in eviews see table . see table . effects of euro area monetary policy on institutional sectors: the case of portugal the macroeconomic effects of fiscal policy in portugal: a bayesian svar analysis the macroeconomic effects of fiscal policy an introduction to financial crises a note on the relation between the equity risk premium and the term structure firm valuation, earnings expectations, and the exchange rate exposure effect firm valuation, earnings expectations and the exchange rate exposure effect the credit crunch monetary policy and financial stability: what role in prevention and recovery monetary policy effects on output and exchange rates: results from us, uk and japan when capital inflows come to a sudden stop: consequences and policy options the credit channel and monetary transmission in brazil and chile: a structural var approach. working papers central bank of chile current account and exchange rate dynamics monetary policy and financial stability: empirical evidence from central and eastern european countries sources of real exchange rate fluctuations: how important are nominal shocks? why is financial stability a goal of public policy structural var, marma and open economy models. columbia university discussion paper - anchoring countercyclical capital buffers: the role of credit aggregates some empirical evidence on the effects of monetary policy shocks on exchange rates tapering talk: the impact of expectations of reduced federal reserve security purchases on emerging markets the uk housing market and the monetary policy transmission mechanism: an svar approach stock returns, real activity, inflation, and money monetary policy's role in exchange rate behaviour the fiscal and monetary linkage between stock returns and inflation stories of the twentieth century for the twenty-first monetary and financial stability in the euro area: pro cyclicality versus trade-off liquidity models in open economies: theory and empirical evidence the relationship between exchange rates and interest rate differentials: a wavelet approach on the structural form of interdependent systems the effects of conventional and unconventional monetary policy on exchange rates a practioner's guide to lag-order selection for vector autorgressions. cepr discussion papers credit supply and monetary policy: identifying the bank balance-sheet channel with loan applications financial crises, credit booms, and external imbalances: years of lessons when credit bites back international transmission of us monetary policy shocks: evidence from var's dollar exchange rate and stock price: evidence from multivariate cointegration and error correction model exchange rate anomalies in the industrial countries: a solution with a structural var approach measuring the stance of monetary policy in zero lower bound environments zero lower bound term structure modelling: a practitioner's guide on the nexus of monetary policy and financial stability: recent developments and research. bank of canada discussion paper current account and the real exchange rate dynamics in the g- countries techniques of forecasting using vector autoregressions. federal reserve bank of minneapolis working paper causal relations among stock returns, inflation, real activity, and interest rates: evidence from japan a structural var analysis of the monetary policy stance in japan determinants of non-performing loans in the southern mediterranean countries capital flows during quantitative easing and aftermath: experiences of asian countries effects of foreign and domestic monetary policy in a small open economy: the case of chile. central bank of chile working papers identifying fiscal policy shocks in chile and colombia the generalisation of the general theory structural vector autoregression models. the stata blog real effects of quantitative easing at the zero lower bound: structural var-based evidence from japan credit booms gone bust: monetary policy, leverage cycles, and financial crises macroeconomics and reality structural var's. econ the effects of monetary policy in a small open economy: the case of portugal price and volatility spillovers between stock prices and exchange rates: empirical evidence from the g- countries japanese monetary policy reaction function and time varying structural vector autoregressions: a monte carlo particle filtering approach acknowledgements i am grateful to nuno sobreira and harald badinger for their useful comments at the infer workshop on new challenges for fiscal policy on an earlier version of this paper. i am also grateful to both anonymous reviewers at the journal of comparative economic studies and to andré teixeira for the useful comments. lastly, i am also grateful to antónio afonso for his useful comments. key: cord- -j jj qff authors: mcdonald, kate title: olympic recoveries date: - - journal: nan doi: . /s sha: doc_id: cord_uid: j jj qff in march , prime minister abe shinzō, the tokyo olympic organising committee, and the international olympic committee postponed the tokyo olympics for one year. the delay is the most prominent consequence of the covid- crisis in japan thus far. but the “corona calamity” (korona ka) is bigger than the olympics. the totality of the disaster is impossible to capture. the very thing that makes it a calamity are the myriad rhythms of crisis that intersect at covid- . if there is a shared theme to be found in these rhythms, it is the question of recovery. when will it happen? what will it look like? and what, exactly, will we recover? in what follows, i share three rhythms of crisis and recovery: national history, the tourism industry, and the parcel delivery industry. in what follows, i share three rhythms of crisis and recovery: national history, the tourism industry, and the parcel delivery industry. other rhythms are possible-of legacies of discrimination and the phenomenon of "corona harassment" (korona hara); of elder care, demographic decline, and alienation; of citizens who persevere in protesting the abuse of power even under a state of emergency. but the rhythms of national history, tourism, and the parcel delivery industry intersect squarely at the olympics. in that sense, they provide a frame for exploring the crisis that is both distinct and open-ended-one node of a vast rhizome of crisis temporalities, rhythms, and events. since the beginning of the japanese government's bid for the summer olympics, prime minister abe shinzōmatched his pitch to the rhythm of national history. "we in japan are true believers in the olympic movement," he told the delegates of the ioc in buenos aires in . i myself am just one example. when i entered college in , i began practicing archery. can you guess why? the year before, in munich, archery returned as an olympic event after a long time. my love for the olympics was already well established . . . when i closed my eyes, vivid scenes from that opening ceremony in tokyo in come back to me. several thousand doves all set free at once. high up in the big blue sky five jet planes making the olympic rings. all amazing to me, only years old. japan has hosted two other olympic games since . in , the country hosted the winter games in sapporo. in , the winter games came to nagano. but abe put the downbeat to the story of on . and with good reason. the games were not only japan's sole previous experience hosting the more prestigious summer olympics. billed as japan's return to international society, the games also showcased the triumph of japan's economic and political recovery from the disaster of war. abe and the tokyo olympic organising committee argued that the summer olympics in tokyo would mirror the historical arc of . faced with a slow-burning recession, labor shortages and demographic decline, and challenges from across the political spectrum (including his own party), abe portrayed the summer olympics as a measure of japan's triumph over the triple disaster and as proof that his economic policies of fiscal expansion, monetary easing, and structural reform-"abenomics"would lead the country to a sustainable social and economic future. indeed, the organising committee referred to the olympics as the "recovery olympics" (fukkōgorin). the tokyo olympics represented the climax of nine years of reconstruction in miyagi, iwate, and fukushima prefectures-the three northeastern prefectures most affected abe shinzo, "abe shinzosori daijin no purezenteeshon ioc sokai" [prime minister abe shinzo's presentation to the ioc general assembly], ann news, september , , https://www.youtube. com/watch?v=zf imlfduu (accessed july , ). by the earthquake, tsunami, and nuclear reactor meltdown. it would allay concerns about the safety and stability of tokyo as a place for business and leisure. indeed, abe's first statement to the ioc in was "tokyo-one of the safest cities in the world, now and in . some may have concerns about fukushima. let me assure you, the situation is under control. it has never done, and will never do, any damage to tokyo." the olympic committee invoked to nurture the plausibility of a "recovery games"-of an olympics that could bring about the end of a disaster and restore japan to a place of prestige in the international arena. some links to the story of were bluntly material. five facilities built for would be reused: yoyogi national gymnasium, nippon budokan, equestrian park, tokyo metropolitan gymnasium, and enoshima yacht harbor. other links infused the official narrative of the games. in the promotional campaign that accompanied the final year of preparation, the tokyo games website proclaimed, "the tokyo games completely transformed japan and, with less than days until tokyo , the country is set for another historic and transformative games." the tokyo organising committee most explicitly articulated the idea that the rhythm of - mirrored that of - in its design of the torch relay. the relay would start in naraha, a seaside town of roughly seven thousand residents that was entirely encompassed by the -kilometer evacuation and exclusion zone following the fukushima daiichi nuclear power plant meltdown. "just as the [ ] tokyo olympic flame transmitted the message of postwar recovery last time, we'd like to fulfill the duty of conveying the message of recovery from stricken areas to the world," said satōmasayuki, chief of the nippon koki saigo plant in fukushima prefecture, which made the torches for and would make them again for . more direct connections between and were programmed into the relay itself. tomihisa shoji, a -year old survivor of the atomic bombing of hiroshima, would run the torch through hiroshima prefecture. tomihisa was born in miyoshi city, the same city in which the final torchbearer of the olympics, sakai yoshinori, was born on august , . concluding the first day of the torch relay in fukushima's minamisoma city would be -year old ueno takayuki, who lost his eight-year old daughter erika, his three-year-old son kotaro, and his parents in the march , , tsunami. as did the prime minister, the organising committee chose its japanese olympic chronology carefully, avoiding facts that could not be synchronized with the rhythm of disaster and recovery. the rhythm of the "recovery olympics" was a pattern of sudden, agentless disasters and epic national recoveries: an atomic bomb that falls from the sky; a tsunami that crashes ashore; a town emptied by a nuclear meltdown. recovery serves as the narrative companion to "hope," which political leaders have increasingly promoted as the authorized emotional response to disaster in postwar abe, "abe shinzosori daijin no purezenteeshon ioc sokai." tokyo organising committee of the olympic and paralympic games, "remembering tokyo ," october , , https://tokyo .org/en/news/rememberingtokyo- (accessed july , ). japan times, april , . asahi shinbun, december , . japan. notably absent from the recovery rhythm are other, less ameliorating emotionsanger, sadness, rage-and other, more critical explanations of cause and effect-structural violence, historical misdeeds-that certainly coexist with the hope for a more glorious future. in one very concrete example, the official media of the tokyo games did not feature the tokyo summer olympics in its chronology of the tokyo games. in fact, the tokyo olympics never took place-it moved to helsinki (and was subsequently canceled) because of threats of a boycott against japan for its invasion of china. but the erasure of from the story of is a product of the current moment, which emphasizes recovery by obscuring the historical question of why there was a need to recover in the first place. by contrast, the organizers of the olympics repeatedly invoked -for them, was a chance to recover from the loss of the games. indeed, contributing to the rebranding of the olympics as a beacon of hope for an increasingly broken world was central to abe's pitch to the ioc. "choose tokyo today," abe declared in , "and you choose a nation that is a passionate, proud, and strong believer in the olympic movement." the subtext, not likely lost on ioc members, was that the protests against corruption, economic inequality, and colonialism that arose around the torch relays for the beijing olympics, the london olympics, and the rio de janeiro olympics were unlikely to take place in japan. on march , , abe declared that the tokyo olympics would be delayed until the summer of . faced with increasing pressure from international athletes and concern from public health officials, the ioc and the tokyo organising committee issued a joint statement to announce the delay. the statement took pains not to disrupt the rhythm of national history. the delay was not the result of a failure on the part of the japanese government. it was a prudent response to a global catastrophe. "[g]reat progress has been made in japan to fight against covid- ," the statement read. but "the unprecedented and unpredictable spread of the outbreak has seen the situation in the rest of the world deteriorating." the ioc and the tokyo organising committee folded the postponement into the rhythm of recovery. the olympic flame will stay lit in japan. the postponed games will be a "recovery games" on a new scale. it will be the "light at the end of the tunnel in which the world finds itself at present." "corona shock" but significant damage has already been done, not just to the rest of the world but to japan as well. inviting comparisons with the "lehman shock" of , the media refers to the economic aspect of the corona calamity as the "corona shock" (korona shokku). the tourism industry has been particularly devasted. despite headlines such as "japan pushed some tourism operators into distress weeks before the delay was announced. the virus was viewed as a "direct attack" on the tourism industry. in early march, for example, shizuoka prefecture reported that nearly half a million guests had canceled hotel reservations in the prefecture between january and the end of february. in addition to border closures, which effectively shut down the international tourism industry, the national government's stay-at-home order extended through golden week, a weeklong collection of national holidays in late april and early may and typically the busiest week of the year for domestic travel. many business hotel operators around the country reported ninety percent declines in sales for march, april, and may. the international tourism industry was the canary in the covid- coal mine. the numbers of foreign tourists visiting japan has skyrocketed in the past eight years. in , the monthly average of inbound tourists to japan was , . in , it was . million. in , nearly thirty-two million people visited japan from other countries. for , the abe government had a target of forty million visitors. the tourism boom is the product not of olympic hype but of the union of abe's general policy of "fiscal easing" with a specific policy of "visa easing" to promote travel from china and asean (association of southeast asian nations) countries to japan. since around , and especially after , the ministry of foreign affairs has worked with china, indonesia, india, thailand, laos, cambodia, malaysia, myanmar, vietnam, and the philippines to simplify the visa process and expand the options for tourists traveling from these countries to japan. the reforms included a special category of multiple-entry visa for foreigners who intended to visit iwate, miyagi, or fukushima prefectures, the three prefectures most affected by the triple disaster. when abe took office, he further deregulated the industry, putting into place more relaxed building codes to promote hotel construction, opening up more landing spots at japan's international airports, and expanding the list of items that could be purchased duty-free. the resulting boom in tourist spending was one of the few success stories of abenomics. the , , in to only , , in . the scale of the disaster is directly related to the policy of visa easing. visits from chinese, korean, and taiwanese travelers now make up roughly two-thirds of japan's foreign arrivals. in february , nearly two months before the postponement of the olympics, visits from asian travelers collapsed. visits from chinese travelers were down percent from the previous february. visits from korea were down percent and from taiwan, percent. hope is in short supply. instead, as matsushima kyoko, a hotel operator in shizuoka prefecture, told the asahi newspaper, there is "an anger i can't vent." unlike the olympic recovery, which takes the form of a singular, triumphant event, tourism's recovery depends on a return to consistency-not just for local operators or the nation, but for the world. for many in the industry, the unnerving thing about this disaster is its incommensurability. there are no projections for when recovery will happen, or what recovery will even look like. "it's an unprecedented national crisis," said doko hiroyuki, owner of the doko dried fish store, which sits outside of the famous tsukiji fish market in tokyo. "with the eastern japan triple disaster, there was a lot of energy behind the feeling of 'let's recover!' but this one looks like it's going to be long term, and accordingly the damage will be big." others in eastern japan likewise compare the corona shock to the triple disaster and find the differences between the two events to be uniquely unsettling. said the owner of one business hotel in sendai, the capital of miyagi prefecture and one of areas hit hard by the economic fallout from the disaster, "with the triple disaster, there was also the recovery demand, so we looked forward to that. but these conditions aren't comparable." in western japan, hotel and tourism operators compared the corona shock to local disasters, such as the kumamoto earthquake or the eruption of mount unzen in . the sense that this recovery has no predictable rhythm, however, is shared. in mabi, a city in okayama prefecture that was wiped out by a mudslide in july , hirai hiroyuki, the owner of hinomaru taxi, compared the two experiences: "after the torrential rains, there was the sensation of recovering day after day. but this time i can't see the end." asynchronicity there is a sense in which envisioning recovery as a return to normal-to the "before times," as we have taken to calling it in my family-is the privilege of those whose lives and livelihoods the political-economic structure served. for those industries that were riven with unsustainable rhythms to begin with, however, recovery suggests revolution rather than return. japan's parcel delivery industry is in an ongoing crisis of labor and public relations. in , the crisis spilled over into the public spotlight. that year, two parcel delivery drivers japan national tourism organization, " visitor arrivals to japan and japanese overseas travelers by month," https://statistics.jnto.go.jp/en/graph/ (accessed june , ). jtb tourism research & consulting co., "japan-bound statistics," https://www.tourism.jp/en/ tourism-database/stats/inbound/#monthly (accessed may , ). asahi shinbun, march , . asahi shinbun, april , . asahi shinbun, april , . asahi shinbun, april , . filed a lawsuit against yamato transport for unpaid overtime. the resulting judgment ordered the company to pay billion yen in unpaid overtime to , employees. the amount of back pay owed was unprecedented-orders of magnitude more than any other judgment that year or previously. as evidence mounted that unpaid overtime was a company strategy for containing costs rather than the unfortunate product of a few bad managers, discussion of parcel delivery working conditions made it on to the floor of the diet. more than the lawsuit or the question and answer session in the diet, however, what fixed the industry's crisis image in the popular imagination was a video of a sagawa express worker that went viral in december . the one-minute, thirtysecond cellphone video shows the driver throwing and kicking boxes that he was unable to deliver up an apartment foyer staircase, only to have them fall back down on him or blow away in the wind. his rage fills the screen. as with tourism, the background to the crisis is a dramatic increase in volume over the past decade. in , parcel delivery services in japan delivered over . billion packages to consumers-five hundred million more packages than were delivered to homes than in and nearly one billion more than in . for the fiscal year that ended in march , this number rose to . billion. amazon has played a large part in this increase. as yokota masuo noted in his undercover reporting on the parcel delivery industry, yamato transport drivers "were busy before amazon. but after yamato began to accept amazon packages in , they were made to work without taking a lunch break." amazon is only part of the crisis. parcel delivery companies have chosen to pass the costs of package delivery on to delivery workers rather than consumers. for example, in its pursuit of market share, yamato transport negotiated a contract with amazon which guaranteed that yamato would lose money on deliveries outside of tokyo. even inside of tokyo, the company would only break even. the company's delivery drivers bore the burden of this strategy in the form of long hours and unpaid overtime. the parcel delivery industry and the abe government are embracing a notion of recovery that coincides with a wider embrace of what matsuoka masahiro and yamate taketo call "de-synchronized services," or, commercial transactions in which neither the buyer nor the seller see each other's faces or interact in person. indeed, the abe government highlights parcel delivery as a key component, and test case, for its plans to reengineer the japanese economy to thrive in a state of precipitous demographic decline. a key element of abenomics is increasing worker productivity. by increasing worker productivity, abe hopes to spark a "virtuous cycle" of economic growth that will obviate the need for increased labor immigration: higher productivity will lead to increased profits, which will lead to increased wages and light inflation, which will lead to increased consumer spending, which will foster further increases in profits, higher wages, and light inflation, which encourages more consumer spending, and so on. de-synchronizing is the heart of the parcel delivery industry's plan to increase worker productivity. to date, the most prominent reform measure is an effort to de-synchronize driver and customer: the installation of parcel delivery boxes on porches and in apartment building lobbies, which reduce redeliveries by allowing drivers to deliver packages regardless of the location of the customer. the olympics further tied de-synchronizing to the success of the nation as a whole. as part of its effort to reduce the number of vehicles moving through tokyo during events, the organising committee targeted home parcel delivery and other transport operations, which rely almost exclusively on trucks for distribution. after a year of requests for traffic mitigation plans with few concrete results, in october , the organising committee succeeded in getting the government to officially request changes to delivery company practices: reduce redeliveries, establish more pickup boxes in apartment lobbies and train stations, and reduce the overall number of deliveries for the duration of the olympics. these requests mirrored what the industry had already been promoting for several years. but this time parcel delivery companies and the government fashioned installing parcel delivery boxes into a material contribution to the success of the tokyo games and a source of national pride. now the corona calamity has transformed de-synchronizing society from a neoliberal economic reform to public health commonsense. the number of parcels shipped and delivered in japan is higher than ever. japan's internet giant rakuten reported a percent increase in total sales at rakuten market (rakuten ichiba) and net super (netto supa) in april compared with april of last year. according to anecdotal reports, parcel delivery drivers are delivering percent more packages than they were before. in response, however, yamato transport and other companies have pursued the same set of reforms, geared toward consumer practices rather than sustainable labor. yamato transport has pressured consumers to install parcel delivery boxes by further reducing the hours that it will redeliver packages and the hours that it will even government of japan, "realizing society . ," https://www.japan.go.jp/abenomics/_userdata/abenomics/pdf/society_ . .pdf (accessed june , ). obe mitsuru, "too much demand? japan's delivery giant thinks so," nikkei asian review, october , , https://asia.nikkei.com/economy/too-much-demand-japan-s-delivery-giantthinks-so (accessed july , ). yamato transport also instituted a "work style reform" (hatarakikata kaikaku). these reforms included a limit on the overall number of packages yamato transport would accept, protected lunch breaks, closer supervision of overtime, and other exploratory measures. but these reforms did not apply to the entire industry or to yamato's network of subcontractors. nihon keizai shinbunsha, takuhai kuraishisu, . asahi shinbun, october , . asahi shinbun, may , . yukita kosuke, yukita no yon koma takuhaibin (blog), may , , https://ameblo.jp/yukitanoyonkoma (accessed july , ). receive requests for redelivery. some companies have lifted the requirement for signatures at deliveries, offering contactless "place and deliver" service (okihai). yamato, amazon, and other delivery companies are establishing even more parcel delivery boxes near convenience stores and train stations and in apartment buildings. panasonic reported that march sales of its "combo-light" parcel delivery box, which debuted in october , were more than double the average of the previous four months. but a recovery revolution predicated on making contactless delivery and limited redelivery palatable by establishing parcel delivery boxes-a revolution that encourages consumers and businesses to expect more rather than less flexibility in parcel delivery-is manifestly not a recovery revolution for parcel delivery workers. the push for ever-more delivery options and ever-faster, ever-cheaper delivery rates is its own rhythm of crisis. it authorizes the parcel delivery companies to celebrate parcel delivery workers' asynchronicity-with their bodies, with their families, and with a society that defines itself by the values and needs of consumers-as the solution to japan's economic future instead of forcing the companies, and the nation, to acknowledge the human costs of consumer-driven culture. indeed, the corona calamity underscores the inadequacy of a rhythm of recovery predicated on the wellbeing of our consumer selves at the expense of our laboring selves. there is no telework for parcel delivery. in the united states, workers at amazon, instacart, and fedex struck on may to protest their working conditions during the pandemic. in japan, parcel delivery workers want to quit. the issue is not simply one of pay. many parcel delivery workers are subcontractors who are paid by the parcel. the increase in volume has increased their pay accordingly. but they are working late into the night with no breaks or vacation. yukita kosuke highlights the unsustainability of this rhythm in his parcel deliverythemed web comic and blog yukita no yon koma takuhaibin (yukita's four-panel express home delivery parcel). on may , just after abe extended the country's emergency declaration for three more weeks, yukita posted a comic called "the thing i want now" (ima hoshi mono). "now is the do-or-die moment! let's keep at it!" says the manager of a parcel delivery office. "it's impossible!" the sweating, exhausted delivery worker tells the manager. "more than wages," he says, "i really just want a break." in his commentary, yukita acknowledges the hopelessness that he encounters among parcel delivery workers: "it is very painful to not be able to see what lies ahead. i hope that you can persevere. i am rooting for you." conclusion hope is in short supply when recovery is conceived of as an event. the abe government and previous japanese administrations have chosen hope because hope is asahi shinbun, april , . asahi shinbun, april , . "sosharu disutansu o mamotta nimotsu uketori anshin anzenna 'okihai' no susume" [package delivery that protects social distancing], asahi shinbun, may , , https://www.asahi.com/ and_m/ / / (accessed july , ). yukita no yon koma takuhaibin (blog), march , . yukita no yon koma takuhaibin, may , . during the corona calamity, we can step away from the rhythm of recovery as event, we can enter into rhythms of anger, frustration, and grief. these emotions have unified japanese labor and citizen protest movements in the past. they continue to do so in the present. even in the context of the olympics, recovery as event held weak appeal to those most impacted by japan's disasters. ueno takayuki, who lost most of his family in the tsunami and had planned to carry the olympic torch on the first day of the relay, told the asahi newspaper that he felt a little resistant to the idea of "recovery" ( fukko). nevertheless, he wanted to show his deceased family acknowledgments i would like to thank albert l. park, max rorty, and one anonymous reviewer for their productive comments organizing the spontaneous: citizen protest in postwar japan asahi shinbun asahi shinbun key: cord- - iv fdof authors: hori, keiko; saito, osamu; hashimoto, shizuka; matsui, takanori; akter, rumana; takeuchi, kazuhiko title: projecting population distribution under depopulation conditions in japan: scenario analysis for future socio-ecological systems date: - - journal: sustain sci doi: . /s - - - sha: doc_id: cord_uid: iv fdof this study develops a projection model of future population distribution on the basis of japan’s current depopulation trend and applies this model to scenario analyses that assume population compactification and dispersion. the model enables a description of population migration at two levels. first, municipal populations are projected using the cohort-component method, and second, the spatial distribution of populations within municipalities is projected at a m grid resolution with the use of the gravity model. based on the japanese depopulation context and the country’s national spatial strategy, the compact scenario predicts the formation of medium-scale regional urban areas (population centers located across japan) and the concentration of people on high-density population areas within municipalities. meanwhile, the dispersed scenario predicts the formation of more but smaller regional urban areas and the dispersion of the population to low-density areas. the simulated population distribution for reveals spatial change in population density and age structure, as well as an abundance of areas that were inhabited in but will be zero population areas by . overlay analysis of future land use maps and the simulated population distribution maps can contribute toward identifying areas where natural capital such as farmland and forest plantation should be managed but where there will be significant population loss by . electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. in contrast to the population growth trend in much of the developing world, aging and depopulation have emerged as two important issues in many developed countries. according to united nations population division (unpd), "unprecedented" aging is a pervasive global phenomenon, and an increasing number of countries are experiencing population decreases because of sustained low fertility or emigration (united nations population division ). among the countries where population shrinkage is expected, japan faces the most serious depopulation and radical demographic transition (eberstadt ; matanle et al. ) . it is expected that by , japan's population will have decreased to million from a population of million in (national institute of population and social security research: ipss ). the causes of depopulation stem from delayed marriage and a decline in birth rate relative to the increment of working women and diversified values in life (cabinet office ) . this trend has been accelerated by the outflow of young people from rural to urban areas, where women are frequently isolated and thus face difficulty in obtaining support for raising children (japan policy council ) . depopulation has expanded from rural and remote areas to suburban and urban areas (matanle et al. ) , and the ministry of land, infrastructure, transport and tourism (mlit) estimates that % of inhabited areas across the country will experience % or greater population loss by (mlit a) . intertwined human societies and ecosystems constitute the interdependent socio-ecological system, in which ecosystems provide necessary services for human well-being, supported by sustainable resource utilization and management by human society (colding and barthel ; duraiappah et al. ) . population change is a significant indirect driver of ecosystem change, impacting the ecosystem through changes in local land use patterns (matanle et al. ; ma ) . the abandonment of land and property is inevitable in areas undergoing depopulation, leading to serious damage to natural capital (the stocks of natural resources consisting of ecosystems) that provides ecosystem services (matanle et al. ; karcagi-kovats and katona-kovacs ) . the ministry of agriculture, forestry and fisheries (maff ) and the ministry of the environment (moe ) reported that the decrease in the number of people who can engage in the primary industries led to increased abandoned farmlands and degraded forests within the depopulated and aging rural communities of japan. such underuse and undermanagement of natural capitals have been recognized as crises that drive biodiversity loss and the decline in ecosystem services (moe ; ohsawa et al. ) . urban shrinkage has also become a significant research agenda for the social and land use sciences (haase et al. ) . however, if abandoned land is managed for rewilding, it is possible that such designed "new nature" can provide various ecosystem services (gross ; navarro and pereira ) . thus, to plan appropriate management strategies to maintain and recreate sustainable socio-ecological systems at the local scale, projections of future population distribution with high spatial resolution are essential. city shrinkage is a multi-layered process caused by various patterns of out-migration. therefore, spatially estimating feasible demographic structures is problematic (gross ; feldhoff ) . matanle ( ) argues that internal migration in japan, leading to both rural and urban shrinkage, can be understood at four levels of movement: inter-regional (sub-national), inter-prefectural, inter-municipal, and intramunicipal. although complexities and uncertainties about social processes exist, an experimental approach should be used to project future population distribution and ecological design requirements (gross ) . scenario analysis which seeks plausible futures with multiple assumptions about demographic and development trends has been widely recognized as an experimental approach to the development of national spatial strategies (daly and kitchin ) . of significant importance to an exploration of the impact on ecosystems is the question of whether a shrinking society will lead to population compactification or dispersion. this question has led to dominant and contradictory theories about sustainable urban forms (holden and norland ) , and can be rephrased as intensive development (or settlement) within a small area or extensive development over a large area (soga et al. ) . as a movement related to compactification or dispersion thus far in japan, the continued outflow of people from rural to urban areas and expansion of urban areas, including suburban areas, have occurred due to industrialization and urbanization during the era of high economic growth after world war ii (feldhoff ; matanle ) . relocation to outside of metropolitan areas has been promoted for industries and population to mitigate constant population loss in rural areas since the s (cabinet office ). thereafter, the necessity of development with compactification has been the subject of debate since the s in response to progressive risks, such as a decline in the working population and intensification of environmental problems (ohashi and ishizaka ; murayama ) . the compact society and the dispersed society have different ecosystem impacts. for example, continuous forested or green spaces can be maintained under compact development, enabling the conservation of local biodiversity (sushinsky et al. ; gagné and fahrig ) . a dispersed society, on the other hand, provides populations with access to nature and opportunities to reconnect with nature, and promotes the utilization of local natural resources through agriculture and forestry (scott et al. ; moe ) . given the different impacts on socio-ecological systems, projecting population distribution under processes of compactification and dispersion is very important. a review of previous research that models future population distribution reveals a common approach to nation-wide spatial population distribution. multiple-step simulation processes, for example, at the state or municipal level and at the grid level, have been applied to express compactification and dispersion. wissen et al. ( ) projected future population distribution in switzerland using four scenarios in two steps: the allocation of municipal population gain and loss, and settlement distribution modeling at m resolution. the u.s. environmental protection agency (epa ) estimated state level populations using the cohort-component model as a first step, and then simulated spatial allocation of housing density using the gravity model at ha resolution. thorn et al. ( ) simulated decadal change in population density in new hampshire for - by incorporating municipal population allocation and the allocation of -m grid cells using cost distance-weighted gravity model. although these studies share some common approaches, none had depopulation trends as a baseline assumption. daly and kitchin ( ) characterized decline-oriented planning as a spatially selective approach to cost-effective redevelopment, unlike the growth-oriented planning of "distribution" under circumstances of quantitative increase. ohashi et al. ( ) argue that population decrease involves quite complex processes meaning that it cannot always be explained by the same simple mechanisms that express population growth. given these arguments, an appropriate method must be used to project spatial population distribution trends under conditions of serious population decline in japan. in the japanese context, ariga and matsuhashi ( ) and matsui et al. ( ) have attempted to project population distribution by applying compact and dispersed scenarios while assuming a fundamental depopulation trend. however, these studies only modeled population distribution within municipalities or prefectures and did not adopt multi-scale simulations, such as the common processes observed in previous studies. as mentioned above, four levels of internal migration (from inter-regional to intra-municipal) have been observed to cause population decline in japan (matanle ) . this suggests that developing a new projection method to express both inter-prefectural or inter-municipal and intra-municipal migration under depopulation trends would make a significant contribution to a more realistic scenario analysis of depopulation in japan. the aim of this study was to develop a population distribution projection model under conditions of depopulation, expressing migration at multiple levels, to conduct scenario analyses that assume compactification and dispersion of japan's population. this study also explored the availability of human labor for the sustainable management of natural capital, such as the maintenance of farmland and secondary forest, and this paper proposes sustainable design strategies for future socio-ecological systems in japan. the developed model presents japan as a "pioneer" country with crucial and ongoing depopulation challenges and aims to provide useful insights for other asia-pacific and european countries experiencing depopulation. to explore plausible natural capital and ecosystem service futures across japan, the pances (predicting and assessing natural capital and ecosystem services through an integrated social-ecological systems approach) project developed four scenarios (pances ; saito et al. ) using the scenario axis method (klooster and asselt ) . these four scenarios were situated on two axes: ( ) future society will have a "natural capital base" that promotes ecosystembased infrastructure development and land management, or a "produced capital base" that depends more on human infrastructures and technologies; and ( ) population concentration will advance toward "urban compactification", with populations concentrated in compact cities and the rewilding of underutilized land, or there will be less concentration, leading to a decentralized society with "dispersed populations" maintaining rural communities. as part of the pances project, matsui et al. ( ) developed a simulation model for population spatialization under the second axis of urban compactification versus dispersed population. this model applied a gravity-based population allocation algorithm at km resolution to express migration within prefectures. to improve the reality of population distribution and spatial resolution, the study presented in this paper adopts a new process to express migration at multiple levels. under full-blown population decline, japan's national land concepts are as follows: "development of national land promoting interaction-led regional revitalization" and "multi-layered and resilient compact and networked structure" (mlit ) . these concepts present a vision of the concentration of infrastructure, services, and industries in specific areas that hold a certain population size throughout japan. it is a vision of regional revitalization and innovation triggered by the active interaction of people, goods, money, and information from different areas, such as between urban areas and rural areas. in particular, this vision seeks to mitigate the continuous net outflow of residents from rural areas to tokyo, where the fertility rate is lowest, as such migration has intensified rural population decline, leading to increased depopulation. measures to realize a multi-layered "compact and networked structure" vary depending on regional scale and characteristics (mlit ) . to stem population flow from rural to metropolitans, especially to the tokyo, osaka, and nagoya metropolitan areas, the promotion of regional urban areas is required. regional urban areas indicate areas outside of metropolitans becoming population or business centers around which vigorous local economic and residential bases can form. the ministry of internal affairs and communications (mic) has already taken some steps to promote such regional centers. for example, regional cities with populations greater than , have been promoted by mic as centers of high level service function and business called "core regional urban areas (mic a)", while cities with populations of more than , and surrounding municipalities have been named "self-support settlement region (mic b)", and proposed as centers of small clusters of settlements. it has been recommended that different scales or types of population centers are formed as regional urban areas based on existing city scales in each region. with regard to population distribution within municipalities, mlit ( ) has proposed the formation of compact cities by concentrating urban functions and settlement within existing urban centers, and developing "small stations" in rural areas that will provide life-services to surrounding communities. in addition, a movement to promote a return to rural living, including in depopulated areas, has also been suggested as an alternative approach to stem depopulation. given these national strategies, this study developed a projection model of japanese population distribution using two projection steps (fig. ) . the first municipality resolution model expressed different formations of population centers as realistic scenarios under depopulation conditions. new migration trends were assumed to occur beyond prefectural boundaries and subnational blocks. in the compact scenario, this study assumed medium-scale (smaller than major metropolitan) regional urban areas with populations of more than , , corresponding to "core regional urban areas" (mic a). in the dispersed scenario, municipalities of more than , were assumed to form smaller but more regional urban areas, corresponding to "self-support settlement region" (mic b). based on the u.s. environmental protection agency (epa the second step of the model reproduced the population distribution at a m grid resolution by population density projections using the arranged gravity model based on previous research (epa ; thorn et al. ; matsui et al. ) . in this study, the compact scenario assumed population concentration in high-density areas within municipalities. in the dispersed scenario, the dispersion or return of people to low-density areas was assumed in the nation-wide simulation. by way of prefectural case study, a further analysis of the formation of small population centers in rural areas to maintain community functions was conducted. in the final step, an overlay analysis of projected population distribution and future land use and land cover (lulc) map was conducted to spatially examine the availability of labor for the management of natural capital, such as the maintenance of farmland and secondary forests. this analysis was conducted so that the projected population maps can contribute to the design of appropriate sustainable socioecological strategies. as with the pances scenarios , population distribution projections were conducted to , based on data from japan's national population census. the municipal population was projected according to sex and -year age groups, using the cohort-component method with -year steps as shown in eqs. ( ) and ( ), referring to regional population projections for japan: - (ipss ): where p t,i,j,k is the population of municipality k ( - , for each city, town, village, special ward, and administrative district of ordinance-designated cities ) in year t ( - ), by -year age group i (i = [age - ]- [over age ]) and sex type j (j = [male] and [female]). ( ≤ s ≤ ) and m t− →t,i− →i,j,k (− ≤ m) express survivorship rates in municipality k and net migration rates to municipality k of -year age group i − from year t − to year t by each sex type j. a minus m value means the outflow of population from municipality k exceeds inflow. c t,k and b t,j,k are, respectively, child-woman ratios (average population of the age group - per woman in the age group - ) and sex ratios for the population of age group [age - ] of municipality k in year t. the baseline municipal population and the parameters to project future population (s, m, c, b) were cited from the population census (statistics bureau of japan: sbj ) and regional population projections for japan: - (ipss . the to parameters (ipss ) were utilized again to project the to municipal population. municipal migration rates were adjusted for each scenario. in the compact scenario, by referring to the requirements and actual examples of a central city of "core regional urban areas" (mic a), municipalities with populations greater than , and daytime/night-time population ratios (sbj ) of more than . were selected as regional urban centers. ordinance-designated cities were judged by the total population of administrative districts constituting the city. in contrast, based on the central city's requirements of "self-support settlement region" (mic b), municipalities located outside of japan's three major metropolitan areas, with populations of more than , , and with daytime/night-time population ratios of more than were selected as regional urban centers in the dispersed scenario. these central cities and their adjacent municipalities (described as municipality group a) were assumed to form regional urban areas, and the migration ratios of these municipalities were enhanced according to the following equation: where ṁ t− →t,i− →i,j,k∈a is adjusted migration rate of -year age group i − to municipality k∈a from year t − to year t by each sex type j. α (α > ) is a parameter for each scenario to determine the strength of population concentration in regional urban areas. increased population flow into group a municipalities was assumed to come from municipalities not included in group a. the migration rates of these other municipalities were decreased according to the following equations: where ṁ t− →t,i− →i,j,k∉a is adjusted migration rate of -year age group i − to municipality k∉a from year t − to year t by each sex type j. β t− →t,i− →i,j (β > ) is a dependent variable calculated by α to express the degree of additional population outflow from the municipalities outside of the regional urban areas. considering the significance of expressing diverse of scenario assumptions, including extreme and plausible population distribution , extreme and middle degree population concentrations into regional urban areas were expressed by adjusting parameter α. table summarizes the municipality resolution scenario assumptions. the grid resolution nation-wide population distribution was projected by allocating projected municipal populations into where p ,i,j,k,l and r ,i,j,k,l express the population of grid l belonging to municipality k and the population ratio of grid l to total population of municipality k in , by -year age group i and sex type j. r ,i,j,k,l was calculated corresponding to each scenario assumption. in the compact scenario, concentration in densely populated areas within municipalities was assumed, and in the dispersed scenario, dispersion to low-density areas within municipalities was assumed. for each municipality, age group, and sex, grids with an expected increased population ratio (grid group [a]) were extracted. grids with a higher ranked population ratio in were selected as grid group [a] for the compact scenario, and those with lower ranked population ratio (grids with accumulated population ratios up to γ( < γ < ) were selected as grid group [a] for the dispersed scenario. γ is a threshold for each scenario to determine which grids are expected to experience an additional inflow of population; outflow of population is assumed in the others. r i,j,k,l∈a was calculated according to the following equation: where δ ( < δ) is a parameter for each scenario to determine the increment degree of population ratio of grids included in group [a] . the population ratios of grids not included in group [a] were evenly decreased according to the following equations such that the population ratio could be totaled to within the municipality: where ε i,j,k ( ≤ ε ≤ ) is a dependent variable expressing the decrement degree of population ratios of grids not included in group [a] for each municipality k, age group i, and sex type j. the parameters for all scenarios in this grid resolution model are summarized in table . with regard to γ, a larger value was set in the compact scenario than in the dispersed scenario. this is because the population is unevenly distributed and concentration to a limited area (ariga and matsuhashi ) and, in general, the cumulative probability of ( ) p ,i,j,k,l = p ,i,j,k * r ,i,j,k,l , ( ) r ,i,j,k,l∈a = r ,i,j,k,l∈a * , r ,i,j,k,l∉a = r ,i,j,k,l∉a * i,j,k , population density shows a bow-shaped curve extending to the lower right [the lorenz curve (lorenz ) ]. the compact scenario had a larger γ threshold because the number of grids in which population ratio increased was less than in the dispersed scenario when the same thresholds were used. consistency between scenarios was maintained by the total number of population ratio with changed distribution [calculated by multiplying γ and (δ − )]. additional processing was conducted at the end of the grid resolution projection to enable comparison with the business as usual (bau) scenario for projected by mlit ( b) . the population of grids projected to be inhabited by very small populations was replaced with using the following equation, based on mlit ( b): where ṗ i,j,k,l,l′ is the population after the replacement process in the -m grid l constituting -km grid l′ (each -km grid consists of four -m grids) belonging to municipality k in , by -year age group i and sex type j. those m grids where the population of all age groups and both sex types were replaced by were considered "zero population grids". after processing, the population of grids not judged to be "zero population grids" were adjusted to fit the total municipal population for . in addition to the nation-wide projection, an additional projection for the dispersed scenario was conducted that focused on ishikawa prefecture. although flat dispersion of the population to low-density areas was expressed in the nation-wide grid resolution model, the japanese government has proposed the formation of "small stations" as central communities to maintain the vital life services in rural areas. under this strategy, population concentration in these small stations is expected to be a probable and realistic trend. according to the cabinet office ( ), small stations should be formulated in each elementary school district or each former municipal area. in the s, s, and s- s, japan experienced dramatic mergers of its municipalities (mic c). the significance of a multi-locational system that maintains functions in former municipal centers has been suggested as a means to prevent drastic population outflow from rural areas (buhnik ) . considering this context, population distribution with the formation of a population center in each former municipal area was projected under the dispersed scenario. this additional model is hereafter referred to as the former municipality model. ishikawa prefecture was targeted for the application of the former municipality model. this prefecture is located on the sea of japan coast, in the middle of honshu, japan's main island (fig. ) . the prefecture's municipalities vary in type (ishikawa prefecture a), and include kanazawa city, with a population of more than , (kanazawa city), and the rural municipalities on the noto peninsula, which have been selected by the food and agriculture organization of the united nations (fao) as a globally important agricultural heritage system (fao). ishikawa prefecture is a case study site of the pances project. although hashimoto et al. ( ) have attempted a scenario analysis of land use and ecosystem services on noto peninsula, population distribution did not fully consider the local demographic situation. given this background, ishikawa prefecture was selected as a case study site to explore the former municipality model. using data, populated grids in ishikawa prefecture were targeted as grids into which future populations could be allocated. the boundary data of former municipalities were obtained from national land numerical information, administrative zones data (mlit ). the municipal boundaries in place in were used as the boundaries of former municipalities (see fig. ). this additional projection was conducted only for the dispersed scenario based on the nation-wide projection of the population at the m grid resolution (p in eq. ( )). this model expressed the concentration of flatly dispersed population in the central areas of former municipalities with no change to the total population of former municipal areas, according to the following equations: where p′ ,i,j,k′,l and r′ ,i,j,k′,l express the population of grid l belonging to former municipality k′ and the population ratio residing in grid l to the total population of former municipality k′ in , by -year age group i and sex type j. as grids with concentrated population were named grid group [a′] in the former municipality model, higher ranked grids in population ratio r′ [grids with a accumulated ( ) p� ,i,j,k � ,l = ∑ l,k � =k � p ,i,j,k � ,l * r� ,i,j,k � ,l k � = , , …k � … , fig. location, overview, and municipal boundaries of ishikawa prefecture population ratio of no more than γ′( < γ′ < )] in were selected for each former municipality. γ′ is the threshold dividing the grids in group [a′] from others within the former municipality, and δ′( < δ) expresses the incremental degree of population ratio of grids included in group [a′] . as γ′ and δ′, . and . were set for both extreme and middle cases. finally, the same replacement process as shown in eq. ( ) was conducted. as argued in "background" , depopulation in areas holding natural capitals, such as agricultural land and forests, causes a decrease in labor to maintain and manage such capitals, which leads to their underuse or abandonment (maff ; moe ) . the locations where such problems are likely to occur are dependent on future population distribution and land use. thus, the study spatially examined the labor availability for natural capital management by overlaying the projected population distribution map and future land use map. lulc maps, developed according to shoyama et al.'s ( ) method, were used to conduct the overlay analysis. the maps correspond to four pances scenarios at the m grid resolution. the data consisted of categories: residential area, paddy field, cropland, other agriculture lands, abandoned farmland, grassland and bush, natural forest, secondary forest, plantation, and others. the lulc maps of the "natural capital-based compact society (nc)" scenario and the "produced capital-based compact society (pc)" scenario were overlaid by the population map of the extreme case compact scenario, while the lulc maps of "natural capital-based dispersed society (nd)" scenario and "produced capital-based dispersed society (pd)" scenario were overlaid by the map of the extreme case dispersed scenario. the land use category occupying the largest area of each -m grid of the population map was identified for each of those grids as their land cover attribute. among the categories on the lulc map, paddy field, cropland, other agriculture lands, grassland and bush, secondary forest, and plantation were considered to be lands in need of human management [hereafter referred to as mnc (managed fig. summarized results of nation-wide population distribution projection natural capital lands)]. across the country, scenario differences for populations living in the grids covered by the mnc were analyzed to provide an overview of changes in labor availability for the management of local natural capital. mnc grids projected to have high levels of depopulation (particularly zero population grids) and aging (more than half of residents aged over ) were spatially identified for ishikawa prefecture. the resulting population distribution and statistical data for each scenario are summarized in fig. . due to limitations of space, only extreme cases are described, with medium-range cases provided as supplemental materials in online appendix . total population resulted in an increment of approximately , in the compact scenario, and a decrement of , in the dispersed scenario. this was derived from differences in original migration rates by type of municipality. the population of each municipality was calculated by eq. ( ). the population of larger municipalities with comparatively higher migration rates increased in the compact scenario, and the population of smaller municipalities with lower migration rates, including minus values, increased in the dispersed scenario. that is the reason why total population under scenarios differed from bau. these differences can be interpreted as the rise and fall of population flow between japan and other countries. as the third row of fig. shows, the compact scenario resulted in more polarized population sizes in municipalities, with an increase in the number of municipalities of less than and those of more than , , while the number of mid-sized municipalities decreased compared to bau. in the dispersed scenario, a slight increase in the number of municipalities with more than , and less than , was observed. in the grid resolution, the number of zero population grids increased by approximately % from bau in the compact scenario and decreased by % in the dispersed scenario. zero population grids tended to appear on the fringes of inhabited area, and were especially prevalent in the hokkaido block and the chugoku block in the compact scenario (fifth row of fig. ). the formation of smaller population centers across japan were reproduced in the dispersed scenario. the number of grids with a population density of less than and more than increased in the compact scenario compared to the bau (sixth row of fig. ). in contrast, under the dispersed scenario, population density could be maintained at the medium level, i.e., with more grids in the - people/grid range compared to bau. the results of statistical analysis of ishikawa prefecture's population distribution, including the outcome of the former municipality model, are summarized in fig. . the number of zero population grids increased by approximately % from bau in the compact scenario and decreased by approximately % in the dispersed scenario. in the former municipality model dispersed scenario, the number of zero population grids was also less than for bau or the compact scenario. only kanazawa city and its adjacent municipalities were selected as the regional urban area to form a population center in the compact scenario, while in the dispersed scenario, nanao city and its adjacent municipalities on noto peninsula (location is shown in fig. b) were also assumed to form regional urban areas. nanao city and its surrounding areas maintained a larger population in the dispersed scenarios compared to the compact scenarios (fourth row of fig. ). in areas indicated by yellow circles, the former municipality model resulted in comparatively higher density grids in each former municipality area. for kanazawa city, urban residential sprawl is a serious issue for city planners (ishikawa prefecture ). thus, the results of progressive concentration in the city center under the compact scenario and former municipality model dispersed scenario were deemed more desirable. the number of grids by population density resulting from the former municipality model was positioned between the compact and dispersed scenarios for nation-wide projection and was close to the bau result (bottom of fig. ) . however, the number of grids with a density between and people/grid was larger than that of the bau, reflecting the maintenance of smaller population centers. based on the nation-wide results of the overlay analysis, fig. shows the number of grids covered by mnc by population density. the legend indicates the two scenarios, namely overlaid population distribution map and lulc map. the reason for the change in the number of grids is due to changes to certain types of land use, such as paddy fields or plantation forests, based on scenario assumptions [see shoyama et al. ( ) for details]. the grids which were intended to hold a population of only - people and be covered by the mnc were concerned to face possible shortages in labor required to maintain mnc. in the compact scenario, the number of such grids was larger than those of other scenarios and resulted in nearly , grids. in the dispersed scenario, there were a greater number of grids covered by mnc with populations of - people, compared to either the compact scenario or bau. as a nation-wide trend, the risk of shortage in labor for the management of natural capitals was deemed higher in the compact scenario compared with the dispersed scenario. an example of the spatial results of overlay analysis is shown here, with a focus on ishikawa prefecture. the overlaid results for natural capital-based scenarios are provided in this section (fig. ) , and the results of produced capitalbased scenarios are provided in online appendix . the maps in fig. show the spatial distribution of land cover types, and the depopulated grids (black dots) and highly aging grids (red and pink dots) covered by mnc. the number of grids with red dots increased in both the compact and fig. the number of grids covered by mnc by population density (nation-wide) dispersed scenarios, but the increment was more dramatic in the compact scenario (third row of fig. ). in the compact scenario, almost all the grids covered by mnc in the noto area (the location is shown in fig. ) were expected to experience serious depopulation and aging. in addition, the different scenarios revealed differences in type of mnc that would be expected to face higher levels of depopulation and aging. for both scenarios, plantation forest was a major mnc dotted by red and pink. however, in the compact scenario, many of the grids covered by paddy field in also face aging, such as around the reclaimed land of ouchigata (shown as a red circle in fig. , noto digital archive corporation). natural capital-based scenarios for the lulc map assumed that naturally produced rice would be preferred and larger areas of paddy field would not be abandoned compared to bau ). ouchigata reclaimed land was also projected to be maintained as paddy fields, but in the compact scenario, more than half of the residents of this area were projected to be over in . this was due to the expected outflow of younger people to the mid-scale regional urban areas, such as to kanazawa city from hakui city or nanao city which ouchigata reclaimed land belongs. the aim of this research was to develop a projection model of population distribution that expressed multi-level migration and assumed a depopulation trend. the model was built integrating cohort-component method with municipal resolution and a gravity model in grid resolution. this research is methodologically significant in three ways. first, this projection model is capable of expressing all four levels of migration (inter-regional, inter-prefectural, inter-municipal, and intra-municipal) that cause depopulation (matanle ) . this is achieved by combining municipal resolution projections to reproduce migration beyond prefectural and sub-national block boundaries, and the grid resolution model. given previous research in japan (see "modeling future population distribution"), this is a significant advance, as the model enables a realistic and complex projection of population distribution for countries experiencing depopulation. moreover, the model expresses population increments and decrements for medium-sized municipalities and grids with medium population densities for each scenario (fig. ) . due to the decrease in pressure to develop land that accompanies population decline, "selective national land utilization" is advocated in japan (mlit ) . this involves combining different development fig. summarized results of overlaying analysis focusing on ishikawa prefecture strategies according to municipal or area characteristics, such as compact city strategies in more populated and accessible areas, and rewilding strategies (gross ) in highly depopulated and aging areas. to select the most appropriate strategy, future dynamic changes of municipalities and grids with medium-scale population density must be projected and considered. this model can provide outcomes which contribute to "selective" national land development design and local development strategies. second, by applying the cohort-component method, the model enables population projection by -year age group. this overcomes one of the limitations of matsui et al. ( ) in which the total population was projected based on pances scenarios. aging has as serious an impact on the socio-ecological system as depopulation, and spatial analysis of the degree of aging (fig. ) by age-specific projection is a novel and necessary approach to understanding current patterns of depopulation and aging. in addition, by adjusting parameters used for the cohort-component method, other alternative scenario analyses can be more easily conducted. due to highly uncertain future population development (gross ) , a greater variety of scenarios must be examined, including the multiple fertility and mortality sets assumed by ipss (ipss ) . this flexibility allows this projection model to expand scenario analysis and to apply analysis to other countries experiencing similar post-growth development pathways and eventual depopulation (eberstadt ; matanle ) . third, overlaying this analysis on future lulc maps is a valuable method for determining coherent socio-ecological systems policies (see "overlaying on the future land use map"). decline-oriented planning is characterized by a spatially selective approach, and a coherent and holistic approach that addresses all economic, social, and environmental issues is essential (daly and kitchin ) . in this research, overlay analysis spatially demonstrated how the consistency of land use as an environmental aspect and population as a social aspect can be realized. further scenario assessments of economic aspects are expected in the future research through the integration of related models, such as surquas (smart urban area relocation model for sustainable quality stock, togawa et al. ) or exss model (a regional input-output model, gomi et al. ) . as other future research agenda, additional projection of future land use harmonized with this study's projected population distribution is also significant. by utilizing the population-projection-assimilated predictive land use modeling (ppap-lm) approach applied by ohashi et al. ( ) , actual land use patterns affected by population dynamics can be projected in order to support more realistic policy-making. furthermore, in reference to previous research (e.g., matsui et al. ) , expanding the target of this projection model to the working population employed in industrial sectors related to natural capital management is also expected to support the development of feasible industrial policies. the study highlighted challenges and proposed necessary interventions to realize a sustainable socio-ecological system under the compact and dispersed population dispersion scenarios. the first potential problem of the compact scenario is that many municipalities are concerned about the loss of necessary daily services due to population decline. for example, the population size of a municipality should exceed to enable the placement probability of book stores or dental clinics to exceed % (mlit ). given the large number of municipalities with populations of less than among scenarios (fig. ) , enhancing the "networks" between municipalities at various levels is more significant in the compact scenario. in this manner, healthy and cultural lives in small municipalities can be maintained. in other words, inter-municipal cooperation, which is a characteristic of decline-oriented planning (daly and kitchin ) , is more significant in a compact society. "networks for a new era" (mlit ), such as advanced information and communications networks that enable remote medical care and education, must be promoted as part of the information and communications policy of japan (mic ). the second expected challenge under the compact scenario is the shortage of labor for the management of natural capitals. the overlay analysis of the grid resolution identified places where mnc should be maintained, such as paddy fields. under the nc scenario, the identified areas, such as the ouchigata reclaimed land, will face severe aging by (fig. ) . to sustain the paddy fields, which are significant wintering sites for migratory birds (noto digital archive corporation), external participants in activities designed to manage mncs are necessary. relevant policies have been formulated to promote such a "related population" (i.e., people who continuously have relationships with a specific region; naito et al. ) . the initiative to call for volunteer workers for agricultural activities from outside ishikawa prefecture is called "work stay at ishikawa" (ishikawa prefecture b). the national policy called "related population creation and expansion project (mic d)" aims to provide financial support for the local development of the creation of a related population. moreover, smart agricultural technologies (maff ) can be considered another essential solution to support limited labor. technologies that make activities to maintain mncs easier and more accessible for anyone, such as power-assisted suits, must be implemented under the nc scenario. "project for accelerating installation of smart agriculture (maff)" is expected to expand as a supportive policy for local efforts to install smart agricultural technologies. as opposed to not only the compact scenario but also bau, the dispersed scenario resulted in a larger population size in areas covered by the mnc (fig. ; less grids with less than people) compared to . in other words, the dispersed scenario cannot be achieved unless a new type of migration opposite of the current trend toward bau becomes a major trend. this challenge remains for the dispersed scenario. the argued major keys to increase the influx of younger migrants from metropolitan areas to small regional urban and rural areas are profitable and attractive employment and attractive living environment, where raising children is easy (cabinet office ). several measures are expected to create employment. for example, promoting the development of industries with a high affinity for localization, such as welfare, health care, interpersonal services, environmental sectors, culture, and agriculture is important. another measure is that financial support can be given for migrants who contribute to activities for local development, such as the "local vitalization cooperator," which has been in operation since (mic e, hiroi ). moreover, advanced measures that overcome the challenges of existing childcare and education support are necessary. as an alternative, a variety of childcare services can be promoted for migrants without relatives in the neighborhood (kukimoto ) . moreover, development of rural communities with social inclusiveness throughout cultural opportunities can be expected as an effective measure (hirata ) . the covid- pandemic in has led to increased awareness of the risks of living in urban areas with large populations, whereas the advantages of a regional dispersed society have been discussed more seriously and widely (moe ; national governors' association ). the rapid spread of teleworking and increased number of metropolitan residents interested in migrating to rural areas have also been reported as part of the impacts of covid- (cabinet office ; nippon institute for research advancement ). this movement can become an opportunity to accelerate the formation of a dispersed scenario. further projection of population distribution reflecting new dispersed settlement styles and industrial structures corresponding to coexistence with covid- is required in future studies. moreover, this study contributes to providing detailed possibilities for the mixing of multiple scenarios. as murayama ( ) argues, there is no universal approach to land use planning for a depopulating and aging society and, in reality, a one-size-fits-all scenario cannot be rolled out across japan. local municipalities and communities need to plan the most appropriate direction for future growth given their unique local characteristics and circumstances and referring to the four quadrants of the pances scenario. the result of the study can support such initiatives through the spatial description of the different potential challenges related to population distribution under different scenarios. in addition, given that many solutions for sustainability are a combination of both nature-based and human/industrial elements (schaubroeck ) , future strategies in any one area must also combine natural capital and produced capitalbased scenarios. one example of this is that smart agriculture technologies are recommended for ouchigata reclaimed land to optimize a natural capital-based compact society. this exploratory analysis can lead the way to actual future strategy design by mixing pances scenario assumptions. this study developed a projection model of future population distribution under current japanese depopulation trends and applied it to a series of scenario analyses that assumed population compactification and dispersion at both national and prefecture scales. the resulting population distribution projection for reveals spatial changes to population density, age structure, and the appearance of zero population areas. by overlaying these data on future lulc maps, population distribution maps can identify locations and types of mnc that will face labor shortages. based on the results, the study proposes several political measures to overcome the challenges of the compact scenario, such as promotion of information and communications networks which enhance inter-municipal cooperation and support to invite external participants to aid in natural capital management. for the realization of the dispersed scenario, promoting industries with a high affinity for localization and developing attractive rural communities for young migrants through diversified childcare services and cultural opportunities are necessary. in conclusion, this study has developed a useful tool to support spatially explicit and practical planning for the management of natural capital at multiple scales to promote sustainable socio-ecological systems under depopulation and aging trends. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. development of formulation method for 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practising the scenario axes technique measuring and assessing urban sprawl: what are the remaining options for future settlement development in switzerland for ? key: cord- -jkaxemmb authors: nakao, mutsuhiro; takeuchi, takeaki; he, peisen; ishikawa, hirono; kumano, hiroaki title: prevention and psychological intervention in depression and stress-related conditions date: journal: asian perspectives and evidence on health promotion and education doi: . / - - - - _ sha: doc_id: cord_uid: jkaxemmb this chapter focuses on depression and stress-related conditions to discuss possible strategies for the prevention or early management of such conditions. health education constitutes the first important strategy, and we outline a school-based educational activity using a case-method approach. we next illustrate the impact of stressful events on psychological health with the results of a survey among chinese individuals conducted after an unexpected epidemic of severe acute respiratory syndrome in . communication plays an important role in the assessment and management services provided by medical practitioners to sick individuals, with very diverse backgrounds and levels of medical knowledge, who consult health care providers with concerns about their health. in this context, we introduce a recent advance in patient–doctor communication. finally, we address the cognitive and behavioral features of those who suffer from depression and psychosocial stress. based on our recent activities and on evidence pertaining to health promotion and education, we emphasize the importance of health education and communication in the prevention of stress-related diseases and the promotion of physical and psychological health. depression constitutes one of the leading causes of disability in the world and is commonly encountered in primary care settings, in the workplace and school, and in the community. upon identification, depressed patients need appropriate management, including pharmacological treatment, sufficient rest, and social support. enlisting the cooperation of psychological professionals, such as registered psychologists, constitutes an important approach to providing psychological support for depressed patients. commonly used guidelines and educational programs are also effective in preventing depression and suicide-related health problems. depression clearly plays an important role in the epidemiology of suicide [ ] ; more than % of the individuals who commit suicide are depressed, and identifying individuals at high risk for depression might reduce the number of suicide attempts. the incidence of depression has increased rapidly in western countries, and the number of japanese individuals diagnosed with this condition has also increased, from ( . per ) to ( . per ), between and . these data may reflect increased use of a standardized method for diagnosing depression, such as the diagnostic and statistical manual of mental disorders, or they may relate to decreased resistance among those suffering from mind/body distress to visiting a psychiatrist. although depression is generally more common in women than in men, the most dramatic increase in the rate of depression in japan occurred among middle-aged men ( - years old); a similar trend was observed among those who committed suicide. an analysis of the relationship between six categories of occupational status and suicide revealed that suicide rates were the highest among the "unemployed" and "employee" groups. therefore, it is important to target middle-aged employees and unemployed people to identify potentially depressed individuals in japan. if all japanese people over years of age completed a questionnaire asking whether they experienced stress in their daily lives, more than % would respond "yes, i experience stress!" [ ] . this is the current situation in japan. illness can result when stressors are too intense and persistent or when specific individuals are vulnerable to particular stressors due to temperamental and/or adaptive problems. psychosocial stress is closely related to mind/body symptoms, and our own study has shown that the report of somatic symptoms is a good predictor of depression and such depression-related symptoms as suicidal ideation among both the disabled and working populations. it is vital to remember that the mind and body are inter-related. modern medicine tends to focus on somatic conditions, but such conditions represent only a part of the whole. medicine should not be limited to the treatment of disease; it is also important to prevent disease, whether at the tertiary or secondary level. the practice of medicine in a hospital requires a trusting relationship between patient and doctor, and both must be aware of the power of the mind-body connection. communication is a key factor in developing this relationship. although medical practitioners are busy and often do not nurture trusting relationships, such relationships are nonetheless a key component of mind/body medicine. effective communication helps promote patient satisfaction and makes a good physician better; however, a sound education, one that includes attendance at lectures and participation in simulations or hands-on training, is critical for the development of excellent physicians. this chapter focuses on depression and stress-related conditions with a view to developing a preventive strategy in association with health education. we have been developing a case-method teaching approach for japanese medical schools, and these recent activities are described in detail. we also present data from a chinese study assessing the impact of stressful events on psychological health. these data underscore the important role played by communication in the assessment and management services provided by medical practitioners to sick individuals, with very diverse backgrounds and levels of medical knowledge, who consult health-care providers with concerns about their health. in this context, we introduce a recent advance in patient-doctor communication. finally, we address the cognitive and behavioral features of those who suffer from depression and psychosocial stress. epidemic in japan an effective suicide prevention strategy should target potentially depressed students, because most individuals who commit suicide suffer from depression [ ] . education about depression and related matters during school may be an effective method for preventing suicide among students in japan. existing teaching styles, however, are ill-suited to address issues related to suicidal depression, due to the complex nature of this condition. extant teaching styles tend to rely on general content and to omit vivid descriptions. we introduce and discuss the applicability of a case-method teaching approach to issues related to suicide and depression in japan, and propose that this method replace traditional pedagogical approaches to these topics. we also refer to the case-method style used at harvard university. case-method teaching is a practical approach to training in the field of social medicine [ ] . this approach involves discussions about a particular case; participating doctors are taught how to deal with situations in which they must reach decisions about public health. the disease-oriented system (dos) has formed the basis of traditional educational systems, adhering to traditional medical teaching methods. although the dos seems to be an effective and reasonable method for obtaining results; it is neither perfect nor optimal in terms of practicality. the problem-based learning (pbl) method was developed to overcome these limitations in the dos method. medical treatment is incomplete when the individual is considered in isolation. indeed, we must consider various ambient and interactive environmental factors in order to adequately understand the etiology of, and recovery from, a disease. in other words, although we emphasize treating the patients and not the diseases, we do not provide our medical students with the practical training required to achieve this goal. figure visually represents the case-method concept, illustrating a more systematic and holistic approach to medicine, in which the scope of learningwhich proceeds through discussion -is wider than that of dos or pbl. through such quasi experience-based studies, medical students can not only acquire knowledge and develop the desired professional attitudes and behaviors but can also come to learn about the psychological and social problems actually experienced by patients. thus, in a way that mimics medical practice, they will be trained to solve problems embedded in actual situations. in addition, the shift from passive to active learning should enhance other abilities, such as communication skills and the practice of continually updating knowledge about state-of-the-art medical technologies. the case-method approach also offers the advantage of using actual cases to learn about the multidimensional issues involved in various aspects of medical practice, such as drawing on knowledge as the basis of actions, understanding the backgrounds of patients, and maintaining awareness of personal motivations and ethical concerns. however, the case method also has its share of problems. table summarizes the advantages and disadvantages of the case-method approach. we now discuss strategies for dealing with these disadvantages. fig. the reach of the disease-oriented system (dos), problem-based learning (pbl), and case methods more broadly, we need to adopt procedures that enable the identification and measurement of problems, the analysis of key determinants, the development of intervention strategies, the selection of an optimal intervention strategy, and the evaluation of outcomes to solve public health problems involving depression or other mental illnesses. based on the need for these procedures, the proposed problem-solving paradigm includes the following phases: ( ) defining the problem, ( ) measuring its magnitude, ( ) understanding key determinants, ( ) developing prevention/intervention strategies, ( ) setting policy/priorities, ( ) identifying the best solution, and ( ) implementing and evaluating the solution. definitions of public health problems must include the characteristics of the target populations and the nature and extent of exposures. one must also be able to understand the issue from a historical perspective and to consider hypothetical situations, the magnitude of the problem, the limitations that constrain potential approaches, and the definitions of technical terms. the definitional process aims at sharpening the skills used to identify potential problems in society. the core issues in this phase involve who defines the problem and how the problem is contextualized in society. using a multidimensional framework, students sometimes define the problem by playing the role of each stakeholder. after the problem has been defined, it is imperative to measure its parameters. this requires access to biostatistics, vital statistics, and demographic data as well as the skills to store, process, manipulate, and report on such information. the magnitude of the problem must be measured in terms of questions such as "why it is a problem?" and "why and how should it be solved?" details about the problem -its prevalence, incidence, economic impact, and effect on human beings -should also be evaluated and measured. further, the measurements need to be performed with reference to an index identified in the definition of the problem. once the problem has been defined and quantified, decisions about addressing it must be made. at this point, it becomes important to understand the key determinants of the problem, such as those related to its biological etiology (host, agent, and vector), its environmental impact, and the sociocultural/behavioral practices of the at-risk population. this step involves understanding the natural history of the disease process and identifying risk factors and at-risk populations. in the context of a clear understanding of the determinants of the public health problem, a number of alternative interventions can be proposed at the cellular/ microbial, individual, familial, communal, and/or population levels. once a wide range of alternatives is identified and the relative merits of each are considered, policies pertaining to a variety of communication, leadership, management, ethical, and financial issues must be developed. after prioritizing the possible solutions, it is necessary to identify the most appropriate one, on the basis of evidence and sound reasoning. these processes involve not only selecting but also directing and establishing possible policies for actual situations. when planners consider all strategies and focus on numerous possible results, decision making can be expedited by relying on such information. after development, the policy must be implemented and evaluated using most of the same quantitative and analytic skills used in the problem-definition and measurement phases. case-method teaching has been practiced at several universities for many years in a subset of courses, generally in the management area. many other courses have used short cases as vignettes or examples of a principle or concept that an instructor wishes to illustrate; however, few nonmanagement courses have used cases as a vehicle for learning through discussion and problem solving. as described by a "founding father" of the harvard business school method of using cases, "a good case is the vehicle by which a chunk of reality is brought into the classroom to be worked over by the class and the instructor." such cases generally do not have solutions but achieve their learning objectives by helping students develop their analytical skills through diagnosing problems and designing alternative solutions as well as by cultivating their decision-making and leadership abilities by allowing them to choose among alternatives and plan implementation strategies. three levels of teaching objectives are relevant to students who are also professionals: level one is the transmission of knowledge through information, concepts, • frameworks, tools, and techniques level two is the development of skills for analyzing, thinking critically, making • judgments and decisions, and executing decisions level three is the development of a professional identity and leadership capa-• bilities accompanied by a set of values, self-awareness, and a capacity for ongoing learning based on these different levels, we discuss the possibility of applying case-method teaching to education about depression and suicide that is designed to reduce the prevalence of suicide among the school-aged population. this section focuses on a study evaluating psychological responses to an actual stressful event. due to an unexpected epidemic of severe acute respiratory syndrome (sars) in china in [ ] , the chinese government and people became aware of the huge impact and destructive power of a public health crisis. in particular, this situation led to intense psychological responses and panic among the chinese people. we studied the psychological status of the population of china during the sars epidemic and analyzed the factors contributing to their responses to inform our efforts in health education and promotion. both quantitative and qualitative methods were adopted. the survey used stratified random sampling based on the severity of the epidemic to select beijing, taiyuan of shanxi province, and harbin of heilongjiang province as the sites for distributing the questionnaires developed by the authors. we also conducted in-person interviews with relevant department leaders. a total of completed questionnaires were received. analysis indicated that . % of the sample expressed some sort of abnormal psychological response. the area least affected by the epidemic, harbin, showed the lowest rate ( . %) of abnormal responses; this was followed by taiyuan city in shanxi province ( . %). beijing was characterized by the highest prevalence of abnormal responses ( . %). abnormally intense psychological responses were associated with the severity of the epidemic and were evidenced by experiences of nervousness, panic, lack of safety, upset, and anxiety (fig. ) . the major reasons behind these feelings included the sars epidemic itself, its mortality rate and unknown nature, the intense and anxious atmosphere created by responses to the epidemic, and the various rumors about the situation. among the most important contributors to these psychological abnormalities were delays in, and interference with, the operation of normal communications channels during the early days of the crisis, as well as the contradictory information emanating from various channels. the tasks involved in education about public health emergencies should not rest on the shoulders of public health education departments alone; instead, such work becomes the responsibility of society as a whole, and the government, mass media, and professional public health departments should play important roles in this process. the sars crisis had a widespread impact on the psychological well-being of the public. timely comprehension of the psychological state of the public and efficient intervention are absolutely necessary when public health emergencies arise. understanding how patients experience illness provides the basis for a therapeutic relationship between patient and physician. this is especially true in cases of patients with mind/body distress. it has long been acknowledged that patients and physicians tend to have different and sometimes conflicting views on health problems and on reasonable expectations for managing such problems. the perception of being understood by a physician provides emotional support for patients, especially at a time of great stress and vulnerability. communication is a major component of medical encounters and a fundamental means for the physician and patient to share perspectives. a growing body of evidence has linked effective communication not only to patient satisfaction and treatment adherence but also to health outcomes, including symptom resolution, functional status, physiological measures, and pain control. recent trends in japanese medicine have drawn increasing attention to the physician's ability to communicate with patients. communication skill is increasingly considered to be a core medical competency that can be taught and assessed in medical education rather than merely a personal trait associated with the physician. patient-physician communication has been studied intensively in western countries, and various instruments have been developed to objectively assess levels of communication in medical encounters. the roter interaction analysis system (rias) is one of the most widely used systems for assessing medical interactions. developed by debra roter, a professor at johns hopkins university, it has contributed to our understanding of the structure of communication in medical encounters and the link between communication and quality of care. the rias has several methodological strengths, and it has been used in many studies in many countries [ ] . the coding approach is tailored to the specific dyadic exchanges that occur in medical encounters. coding categories directly reflect the content and context of the routine dialogue between patients and physicians, including both task-focused and socio-emotional domains. identification and classification of verbal events are coded directly from videotapes or audiotapes, not from transcripts. coding software to help coders work directly from the audio/video data is also available. elimination of the resource-intensive effort necessary for an accurate and full transcription represents a practical advantage offered by the rias. in addition, because coding proceeds directly from video or audiotapes, it is possible to assess the tonal qualities of the interaction. furthermore, the validity and reliability of the rias have been assessed in previous studies, and both have been rated favorably in a comparison study. the list of rias-related studies is available from the rias website (http://riasworks.com). communication units are defined as "utterances," the smallest distinguishable speech segment to which a classification may be assigned. codes are assigned to each of these utterances. approximately mutually exclusive coding categories are used (table ) . table shows the results of an analysis of patient-physician communication occurring during cancer consultations in japan. the details of this study have been previously published [ ] [ ] [ ] . this analysis using the rias shows that a major part of the interactions involved information giving by both physician and patient. on the other hand, striking differences between physicians and patients emerged with respect to several categories. physicians asked nearly twice the number of questions, whereas patients produced twice as many positive utterances. the consultation was largely focused on biomedical topics, and the proportion of psychosocial exchanges was minimal. the regression analyses of patient satisfaction with respect to physician and patient communication behaviors, controlling for the identity of the physician and the characteristics of the patient and consultation [ ] [ ] [ ] , showed that a greater proportion of open-ended questions and a lower proportion of direction were associated with higher patient satisfaction. these communication styles have often been recommended to physicians for use in patient-centered consultations. on the other hand, the perceived emotional responsiveness of physicians was negatively associated with patient satisfaction. in this study, most of the utterances classified as physician emotional responsiveness were statements intended to reassure and encourage patients, such as "there is no need to worry" or "you will be all right." these data suggest that reassurance and encouragement might differ in essential ways from empathy and acceptance. that is, encouraging statements, especially when offered early in the presentation of patient complaints, may function as interruptions, preventing patients from fully describing their concerns. it is also possible that patients felt that they were expected to respond to reassurance from physicians by concluding their remarks even if they still had something to say. among the patient communication behaviors, question asking was associated with lower levels of satisfaction, possibly because the information needs of patients increase when the information provided by physicians is not sufficient or comprehensible. this may lead to greater question asking and lower satisfaction with the consultation. thus, patient question asking might express patient dissatisfaction. although the previous section described the use of the rias in research settings, the rias has also been used as an educational tool. indeed, this measure can be used for individual feedback in that it offers an objective assessment of communications. it also helps in understanding the structure of the entire communication as well as the function of each communication in medical encounters. further, it provides specific examples of each communication category, offering many ways to facilitate talking by patients, demonstrating empathy, and so on. in relation to medical education, further research is needed to explore when and how the key communication skills identified in communication research should be taught as well as to assess whether these skills can be learned. the ways in which patient-centered attitudes and communication skills can be developed and sustained by practitioners all along the continuum of health-care professionals should also be considered. although research in japan remains limited, previous studies have provided evidence that communication in medical encounters can have a significant impact on patient behaviors and outcomes. further research into patient-physician communication is needed to implement patient-centered care in the social and cultural context of japan. depressed individuals think negative, painful, and discouraging thoughts, such as "i am worthless," "i have no hope," and "i will fail again." they tend to think in irrational black-and-white, all-or-nothing terms such as, "if i make a mistake on this project, my career will end." cognitive therapy, developed by beck and colleagues, capitalizes on this facet of depressive syndromes to alleviate symptoms. numerous randomized controlled trials attest to the efficacy of cognitive therapy, which is often combined with behavioral techniques to constitute cognitive behavior therapy (cbt). cbt is a widely used psychological treatment for depression, and we have performed cbt with a variety of patients for more than years [ , ] . cbt often proceeds on the basis of two kinds of assessment procedures, both of which are referred to as "abc analysis" (fig. ) . abc represents an activating event, a belief, and a consequence (including mood and behavior) according to the analysis depicted in the upper section, whereas it represents an antecedent, a behavior, and a consequence according to the analysis depicted in the lower section. the first abc analysis assumes that a distortion in information processing plays an important role in generating problematic emotions and behaviors. on the other hand, the second abc analysis assumes that the contingency connecting one's own behaviors to the responses of others or the self produces the problematic behaviors. our experiences with cbt have identified problems with using this approach to treat depression in japan and have underscored issues that require further consideration to improve the effectiveness of treatment offered in actual clinical settings. one of the major problems with using cbt to treat depression in japan is that attending psychiatrists and physicians do not have sufficient time to implement the treatment. therefore, collaboration with clinical psychologists will be necessary. however, most hospitals and clinics do not employ clinical psychologists with adequate training in using cbt to treat depression and/or in depression itself. thus, we must first establish a functional collaboration among psychiatrists, physicians, and clinical psychologists. thereafter, the following issues should be considered with respect to delivering the actual treatment: ( ) how to implement cbt in the process of the overall treatment, ( ) how to educate and motivate patients with respect to psychological concerns, ( ) how to use various worksheets or monitoring sheets, ( ) how to handle the variability in depressive moods, and ( ) how to terminate treatment and prevent relapse. although the cognitive restructuring component of cbt is easy to understand and is effective, it is not the only active ingredient in this treatment of depression. indeed, any workable treatment program should also include, at the very least, attempts at behavioral activation. various worksheets or monitoring sheets can be used to conduct cbt in japanese clinical settings with limited staff and time resources. the present chapter addressed the importance of prevention, especially with regard to communication and education, by focusing on recent epidemics of depression and stress-related conditions. the patient-doctor relationship is fundamental to the process of medical diagnosis, and effective communication among residents and specialists engaging in health promotion is also central to the maintenance of healthy lifestyles. knowledge of psychological techniques such as cbt is useful when communicating with depressed patients and with any individual experiencing negative mental states. medical education is also essential in providing adequate numbers of well-trained health care professionals who can assess and manage mental health conditions. the ottawa charter for health promotion, introduced by the world health organization in , declared that health promotion goes beyond health care. although further consideration is needed to end the debate pitting medical care against population-level preventive activities, it is clear that health must appear on the agendas of policy makers at every level of government to facilitate awareness of the health consequences of a wide range of decisions [ ] . the responsibility for health promotion should be shared among individuals, community groups, and health professionals, as well as health service institutions and appropriate governmental agencies. we will continue our clinical and research activities in the service of promoting mental health in japan, china, asia, and throughout the world. the suicide epidemic in japan and strategies of depression screening for its prevention the healthy hospital: maximizing the satisfaction of patients, health workers, and the community a proposed approach to suicide prevention in japan: the use of self-perceived symptoms as indicators of depression and suicidal ideation the healthy hospital: maximizing the satisfaction of patients, health workers, and the community awareness of sars and public health emergencies among general publics (in chinese) the roter interaction analysis system (rias): utility and flexibility for analysis of medical interactions physician-patient communication and patient satisfaction in japanese cancer consultations the interaction between physician and patient communication behaviors in japanese cancer consultations and the influence of personal and consultation characteristics the healthy hospital: maximizing the satisfaction of patients, health workers, and the community changes in cerebral glucose utilization in patients with panic disorder treated with cognitive-behavioral therapy treatment of psychosomatic illness: cognitive behavioral therapy the healthy hospital: maximizing the satisfaction of patients, health workers, and the community the content of the sections written by all five authors was based primarily on the symposium, "prevention and psychological intervention in depression and stress-related conditions," held at the first asia-pacific conference on health promotion and education, makuhari, japan, on july , . in addition, the content of the section authored by drs nakao, takeuchi, and ishikawa was partially based on the th teikyo-harvard symposium, itabashi, japan, on june , . key: cord- - u e authors: kasai, takeshi; nakatani, hiroki; takeuchi, tsutomu; crump, andy title: research and control of parasitic diseases in japan: current position and future perspectives date: - - journal: trends parasitol doi: . /j.pt. . . sha: doc_id: cord_uid: u e between and , japan eliminated several major parasitic diseases. in , the japanese hashimoto initiative was the first global programme to target parasitic diseases. thereafter, japan expanded its international cooperation to cover infectious diseases through integrated development programmes to improve health, to alleviate poverty and to help to achieve the millennium development goals of the united nations. parasite control remains a major component of all subsequent operations. opportunities to build upon past successes in order to improve the situation in the developing world – in addition to tackling emerging national threats – are promising. substantial challenges remain and japan has introduced major national reforms to try to overcome them. between and , japan eliminated several major parasitic diseases. in , the japanese hashimoto initiative was the first global programme to target parasitic diseases. thereafter, japan expanded its international cooperation to cover infectious diseases through integrated development programmes to improve health, to alleviate poverty and to help to achieve the millennium development goals of the united nations. parasite control remains a major component of all subsequent operations. opportunities to build upon past successes in order to improve the situation in the developing world -in addition to tackling emerging national threats -are promising. substantial challenges remain and japan has introduced major national reforms to try to overcome them. setting the scene japan occupies a unique position with respect to infectious disease research and control. japan has been confronting problems that are faced by wealthy nations and problems that persistently threaten poor, developing countries because the japanese islands extend from almost the arctic north to the southern tropical okinawa islands and because the nation recently rose quickly from relative poverty to a position among the wealthiest countries. japan also has the invaluable experience of successfully driving the health of its citizens from a postwar low point to the elevated levels seen today. a major factor in this accomplishment was the control of parasitic diseases. today, the country faces new challenges because the profile of parasitic diseases has shifted from the high prevalence of soil-transmitted parasitoses, seen in the s, to epidemics of previously rare diseases and imported and opportunistic infections. novel parasites, especially in immunocompromized hosts, often cause serious manifestations and present problems with diagnosis and treatment. they also necessitate individual clinical interventions rather than the mass control operations that succeeded in the past. for a better understanding of the current position and future prospects it is important to bear past experience in mind. stemming from japanese advances in the s, health-improvement initiatives and control programmes that involve mass screening and treatment were implemented that now enable japanese nationals to enjoy one of the healthiest lifestyles in the world. in the space of two decades, nationwide, interdisciplinary, multisector publichealth campaigns against parasitic diseases led to the control of ascariasis, trichuriasis, hookworm and other soil-transmitted parasitoses and the eradication of other major diseases such as malaria, schistosomiasis and lymphatic filariasis [ ] [ ] [ ] [ ] [ ] [ ] . parasite control proved to be a leading component of successful postwar development in japan. for example, in terms of the causal factors behind the rapid declines in mortality seen between and , the control and decline of parasitic diseases was second only to advances in combating diseases of the circulatory system [ ] . consequently, during the to years after the war, parasite research and parasite-control programmes were priorities. parasitology departments were among the most important in universities and research institutes and there was close integration between research and field control programmes. in , only % of the population had a safe water supply and outbreaks of waterborne diseases were frequent [ ] . enactment of the water supply law in led to the swift introduction of proper water and sanitation systems ( figure ), which had a rapid and notable impact ( table ) . as coverage expanded, water-borne infectious diseases declined markedly. application of knowledge about the environment, hygiene and disease linkages, together with national advances in science, technology and medicine (i.e. vaccines, drugs and diagnostics such as the kato-katz thick smear test for schistosomiasis), proved crucial. the government also cooperated with numerous nongovernmental organizations (ngos) that were established nationwide and were devoted to the control of parasitic diseases. many researchers were also actively involved in field control programmes that operated in the vicinity of their research facilities. collaboration between the education and health sectors was also pivotal. nationwide, schools were one of the few postwar public infrastructures that remained functional. deworming interventions in schools were combined with health education, which was provided by schoolteachers and supported by researchers. those involved in deworming programmes recognized the invaluable nature of linking different local-level resources, especially schools and community health centres. they also experienced the impact of 'community empowerment' [ , ] . the rapid socioeconomic development in japan, of which the traditional 'village living' philosophy was a driving force, saw the nation evolve from being one that received overseas development assistance (oda) in the early- s to donor nation status by the late- s. the initial focus of japan was to concentrate on helping neighbouring countries in asia but in the new millennium and with globalization increasing that view has expanded to encompass a global assistance focus, with special attention currently being shown to africa. the unique oda philosophy in japan, which is based on the overriding concept of self help and human security, contributes to overseas development by exporting its achievements in disease control and in shaping support for disease research and control activities (http:// www.mofa.go.jp/policy/oda/cooperation/anniv /pamphlet/ contents.html). based on its successes in controlling several major parasitic diseases and its desire to help other nations achieve similar success, japan proposed a global parasite-control initiative at the group of seven (g ) summit in denver, usa, which emphasized the necessity of international cooperation to control parasitic diseases. a report entitled 'global parasite control for the st century' submitted at the group of eight (g ) summit in birmingham, uk, specified the intention by japan to help developing countries to improve their ability to control parasitic diseases and also persuaded the g nations to embrace the proposal [ ] [ ] [ ] . the so-called 'hashimoto initiative' (hi), named after its architect and the japanese prime minister at the time, hashimoto, represented an unprecedented step in japanese oda history. in japan, the prime minister or parliament can influence oda policy but, usually, the bureaucracy or civil servants have been the de facto operator of oda policy [ ] . thus, the hi was unique. hashimoto fervently desired to contribute to the health of people in less-developed counties by transferring japanese experience, especially in disease research and control. he envisioned support for japanese experts who engage in such work at centres that were established abroad as a contribution not only to global health but also as a means to maintain the skills and expertise of parasitology researchers in japan. three years after the hi was proposed, and mindful of major changes regarding the global disease-control scene, japan expanded the scope of the target infectious diseases and announced the more expansive okinawa infectious disease initiative (idi) in [ , ] . the idi aimed to provide $ billion over a five year period. parasitic diseases were retained as a primary target in what was planned as a comprehensive and cohesive range of measures to further social and economic development in recipient countries (http://www.mofa.go.jp/mofaj/gaiko/summit/ko_ / outline/jp/overview.html). actual spending exceeded expectations and totalled >$ billion in four years ( figure ). the idi incorporated several major strategies, including promotion of research and of public-health activities. this was the first time that research was clearly defined as a matter for action in a health-related oda policy document. it quickly became apparent, however, that the shortage of experienced personnel in parasitic-disease control was a crucial issue. as a result, plans were put in place for the development of a global network of research institutions to target infectious and parasitic diseases, to promote research on infectious and parasitic diseases in impoverished countries with a focus on benefiting the poor, and promotion of international cooperation in vaccine research and development [ ] . these centres were expected to provide technical support to field control programmes using japanese oda and would mimic what had happened during the successful national control efforts in japan. japan also had a leading role in the development of the global fund to fight aids, tuberculosis and malaria (gfatm: http://www.theglobalfund.org/en/). spurred by the idi, gfatm was established in after discussion at subsequent g summits and a united nations (un: http://www.un.org) general assembly special session on hiv/aids. the then prime minister, koizumi, expressed strong commitment to the global endeavour at a symposium to commemorate the fifth anniversary of the kyushu-okinawa summit and announced an additional contribution of $ million (http://www.mofa.go.jp/policy/ health_c/gfatm/address .html). in the year , all un member states adopted the millennium declaration [ ] , which put poverty reduction at the centre of concerns and incorporated specific, measurable development goals to be achieved by . these became known as the millennium development goals (mdgs) [ ] . the mdgs comprise eight goals, three of which refer explicitly to health. at the high-level forum on health mdgs in asia and the pacific, japan stated that achieving the health mdgs is a crucial step that is necessary to attain all of the eight mdgs (http://www.mofa.go.jp/policy/oda/ category/health/state .html). in response to the adoption of the mdgs, following the completion of the idi in march , japan expanded the idi into a comprehensive, multisectoral health and development initiative (hdi) [ ] , which was designed to continue and enhance active support in the health-related sector through oda with emphasis on accomplishing the mdgs, particularly in africa [ ] . in the hi, school-based deworming of intestinal helminths was chosen as a main focus for international disease-control programmes. the direct beneficial effects of deworming are well documented and include improved nutritional status, increased weight gain [ ] , better micronutrient uptake, improved cognitive development of children [ ] , improved physical fitness and appetite [ ] and increased school attendance [ ] . however, the reason that school-based deworming was selected for particular attention was not only the direct effects but also the indirect socioeconomic and health benefits that would be bestowed on communities [ , ] . the concepts inherent in the hi remain major pillars of the new hdi. japan also adopted a strong research component within the hdi and included 'strengthening research capacity on the ground and paying due respect to local condition' as the underlying basic policy. crucially, this reflects the japanese self-help philosophy. the hdi emphasizes the importance of understanding local culture, traditions and norms towards ill health and seeks to strengthen research capacity at the field level, in particular those research activities that are designed to facilitate and improve project implementation. in japan, the parasitic-disease research and control community underwent some fundamental changes as a result of its success [ ] . after the eradication of key parasitoses from japan and effective control being established over many others, parasitic diseases were no longer viewed as a subject of major concern. consequently, extensive and well-supported research groups began to lose their resources. they became divided into two groups: those that maintained their interest in disease-control programmes and applied research turned their attention to tackling parasitic diseases outside japan, and those which kept their focus on japan and, instead, concentrated on basic laboratory research on other diseases. this exacerbated the disconnect between research and control. the potential threat from infectious diseases, including those that are newly emerging and some that were never eradicated from japan (coupled with increasing international trade and travel), mean that japan must remain continually alert and prepared. this was exemplified by the recent outbreaks of severe acute respiratory syndrome (sars) and avian flu, which focused attention on how japan is able to respond to infectious disease threats to its inhabitants, in addition to maintaining its responsibility to protect its less well-off neighbours. this was indicated by the creation of a regional stockpile of tamiflu to help combat an outbreak of avian flu, should it arise [ ] . thus, the disconnect between research and control of parasitic diseases, coupled with the rapidly shrinking cadre of skills and expertise capable of dealing effectively with parasitic-disease matters [including the relatively sudden disappearance of community-based health centres ( figure )] has been a major concern in japan. consequently, the government has taken substantial measures to rectify the problem. the hi conceptualized the future of japanese parasitic-disease research and control worldwide. to remain a leader within the hdi, parasitic-disease control programmes will continuously need to work with japanese oda policy makers and position themselves as one of the best means of attaining the mdg targets. one major drawback is that the japanese experience, whereby parasitic-disease control contributed substantially to overall social development, remains primarily an anecdotal record and is not documented in health-system research terms. it is, therefore, important to monitor and evaluate ongoing programmes in order to collect the data and present the results in a more scientific manner. the research agenda that is set by parasitic-disease control is not complete [ ] and an additional body of knowledge and action that takes the functioning of the health system into account must complement the disease-control approach. recognition of the importance of health-system research, in conjunction with attaining the mdgs, has been increasing [ ] . the japanese ministry of health, labour and welfare organized a symposium for health-system development in at which the importance of the linkage between the healthsystem research agenda and a disease-specific generated research agenda was confirmed [ ] . it is reported that undernutrition is the underlying cause of a substantial proportion of all child death [ ] and programmes that are aimed at controlling soil-transmitted helminths have already demonstrated their positive impact on nutrition. therefore, it is important to maintain the integration of efforts to control parasitic diseases with all ongoing healthsystem activities and improvement efforts, nationally and internationally. in japan today, parasitoses are less of a public-health dilemma and more a problem for individuals, with the primary difficulty being one of diagnosis rather than treatment. commonly used parasitological tests are not effective for some of the new diseases and many clinicians are untrained or inexperienced in dealing with them [ ] . moreover, the successful control of parasites has led to a vacuum of interest and attention, with parasitology departments having disappeared from medical schools and the number of staff in the departments that remain has declined substantially. in the late- s, of the medical and veterinary schools in japan, only had either a parasitology or tropical medicine department [ ] . japan needs to solve this dearth of national experts in parasitic-disease research and control -it needs to refocus and rejuvenate its research base. the gaps between applied research, field control and basic research also need to be bridged. the japanese research community has historically benefited enormously from working in close collaboration with control programmes to overcome parasitic diseases. since parasitic diseases became rare and of minor importance in japan, the research community has been diluted and crucial experience and knowledge has been condensed, inherited by a declining number of individuals. it is important to establish collaboration and coordination between groups, in the short-term to overcome the problem of the lack of human resources and in the long-term to promote and improve parasitic-disease research and control. it is important that japan is able to interface and work with the international community and particularly with specialists from countries where parasitic-disease control projects take place. research priorities for health and development in wealthy and resource-poor countries are not the same [ ] . basic research in japan might not necessarily focus on needs that are deemed a priority elsewhere. overall, the crucial factor is that the application of medical science in developing countries should be a joint effort between scientific institutions in countries from the south (i.e. resource-poor) and the north (i.e. wealthy) [ ] . irrespective of a national or global focus, there must also be an underlying principle of engaging affected communities themselves and of them having a pivotal role in developing interventions jointly with ngos, industry and local and central health departments. furthermore, it is imperative to ensure that research is cohesively and continually integrated into control initiatives, as was the case in japan and elsewhere. an example is the community-directed treatment system being used by the african programme for onchocerciasis control (http://www.apoc.bf) to combat onchocerciasis in africa [ ] . japan is facing the (re)emergence of several water-and food-borne parasitic diseases and parasitic zoonoses. this is because of increasing international trade, faster transport, demographic changes and increasing immigration and tourism [ ] . in addition, there are now cases of imported diseases such as malaria and cysticercosis [ ] and the northernmost hokkaido island prefecture is experiencing an epidemic of alveolar echinococcus [ ] . as part of a major rejuvenation of research and development work in science and technology, which began in , the government has been taking active steps to boost national capacity in all aspects of disease research and control. the fundamental reforms and almost complete restructuring of the science and technology system in japan has caused a paradigm shift from 'science, technology and society' to 'science and technology for society' [ ] . there has also been an increase in funding that is devoted to parasitic and infectious diseases. between and , $ - million annually was being devoted to parasitology research from the ministry of education, culture, sports, science and technology (mext). overseas collaboration is also expanding, with the national institute for science and technology policy reporting that % of all scientific publications in arose from international collaborations, compared with only % in . in , mext allocated $ million to inaugurate a new programme to intensify research into measures for coping with infectious diseases and to create centres of excellence (coe) nationally and internationally. so far, at least three coe have been created overseas and four in japan, all coordinated through the newly established center of review trends in parasitology vol. no. research network for infectious diseases (http://www. riken.go.jp/engn/index.html). the third japanese science and technology basic plan ( - ) is designed to promote science and technology that provides an immediate, exact solution to socially essential issues, including international bioterrorism and emerging or re-emerging infectious diseases, such as sars and avian flu, that threaten global safety and security. in addition, human resources will be developed in order to respond to new needs and to contribute towards combating infectious diseases and bioterrorism, natural sciences, humanities and social science and the burgeoning bioinformatics and technology disciplines (http://www .cao.go.jp/ cstp/english/basic/index.html). these activities should promote ongoing international collaboration in basic research, including genomics work. in addition, the activities should facilitate the participation of japan-based basic research groups in field control and applied-research programmes in developing countries that are supported by japanese oda and technical cooperation. despite the challenges, the opportunities for further advancements in parasitic research and control are good, with renewed interest and entrepreneurship, the development of new techniques, tools and delivery systems and deployment of substantial amounts of funding. knowledge drives productivity, economic growth and health; however, unless all stakeholders put a concerted effort into resolving the challenges, the opportunities might fade and the unique and privileged acquired knowledge and experiences of japan could be lost. the analysis and opinions presented in this article represent the personal views of the authors and do not represent the official position of the ministry of health, labour and welfare or the government of japan. the authors declare no conflict of interest. filariasis in asian region retrospective review of the progress in parasitology in japan: the past and the present parasitology and parasitic diseases in japan control of parasitoses in japan historical aspects for the control of soiltransmitted helminthiasis increasing parasitic diseases in japan decline in mortality in japan, u. s. a. and the federal republic of germany -the contribution of specific causes of death japan's experiences in public health and medical systems large-scale control of intestinal helminthic infections in japan, with special reference to the activities of japan association of parasite control japan as long longevity country, it all started from parasites hashimoto initiative -keynote speech. g follow-up international symposium: hashimoto initiative -global parasite control for the st century hashimoto initiative -from policy to implementation. g follow-up international symposium: hashimoto initiative -global parasite control for the st century uk pledges money for who malaria initiative the emergence of japan's foreign aid power okinawa infectious disease initiative -japan's initiative: summary of mid-term evaluation report the okinawa infectious diseases initiative school-health-based parasite control initiatives: extending successful japanese policies to asia and africa united nations millennium declaration: resolution adopted by the general assembly. un document number a/res road map towards the implementation of the united nations millennium declaration: report of the secretary-general health and development initiative. ministry of foreign affairs japan's health revolution to be bestowed on weight gain of kenyan school children infected with hookworm. trichuris trichiura and ascaris lumbricoides is improved following once-or twice-yearly treatment with albendazole moderate to heavy infections of trichuris trichiura affect cognitive function in jamaican school children physical fitness, growth and appetite of kenyan school boys with hookworm, trichuris trichiura, and ascaris lumbricoides are improved four months after a single dose of albendazole worms, education and health externalities in kenya keys to success for a school-based malaria control program in primary schools in thailand beyond deworming: the promotion of schoolhealth-based interventions by japan parasitic disease problems and perspectives from the global standpoint of view proactive plan to help combat pandemic influenza in asia the research agenda for improving health policy, systems performance, and service delivery for tuberculosis control: a who perspective overcoming health-system constraints to achieve the millennium development goals ministry of health where and why are million children dying every year? recent state of parasitoses in japan -epidemiology for clinicians the / report on health research demand-driven research and development programmes: their challenges the future of onchocerciasis control in africa the current status of parasitic diseases in japan echinococcus multilocularis in dogs kanisuido no tebiki [handbook for small-scale water supply system key: cord- -kxqh ip authors: sharma, avinash; singh, surjit title: kawasaki disease date: - - journal: pediatric rheumatology doi: . / - - - - _ sha: doc_id: cord_uid: kxqh ip to learn about the epidemiology, aetiopathogenesis, clinical features and differential diagnosis of kawasaki disease (kd) . to learn about the epidemiology, aetiopathogenesis, clinical features and differential diagnosis of kawasaki disease (kd) . to learn about the acute and long-term management of kd a -year-old boy presented with high-grade fever for days. the baby had been irritable and fretful and was refusing feeds. the mother consulted a paediatrician who prescribed amoxicillin. the fever, however, persisted and the baby developed swelling over the dorsum of hands and feet. the mother also noticed a rash over the trunk along with red lips and red tongue. both eyes were con-gested and there was a swelling on the left side of the neck. as the fever was continuing, she sought consultation from another paediatrician who opined that the baby needed hospitalization for further workup. he ordered an infection workup, obtained blood and urine cultures and started intravenous ceftriaxone. the baby continued to run fever. investigations showed a neutrophilic leucocytosis, raised c-reactive protein levels and an elevated erythrocyte sedimentation rate. blood and urine cultures were sterile. at this time a clinical review suggested a possibility of this being kawasaki disease. d echocardiography did not reveal any coronary artery abnormality. the parents were counselled and a decision was taken to administer intravenous immunoglobulin (ivig) g/kg along with aspirin mg/kg/day. the baby showed prompt clinical improvement. the irritability disappeared within a few hours and the fever was passive by the next day. the mother noticed that the skin over fi ngers and toes had started to peel. a repeat haemogram at this time showed thrombocytosis. the dose of aspirin was reduced to mg/kg/day and the baby was discharged with a follow-up appointment after weeks. kawasaki disease (kd) is an acute, self-limiting, childhood medium-vessel vasculitis. initially described in by dr. tomisaku kawasaki in japanese children as an acute mucocutaneous lymph node syndrome [ - ] , kd may lead to coronary artery abnormalities (caas) in up to % of patients if left untreated. it is for this reason that kd is considered a medical emergency. kd is the most common cause of an acquired heart disease in children in developed countries. it is now often considered to be the most common vasculitic disorder in children. the three highest incidence fi gures of kd in the world come from the far east, viz., japan, korea and taiwan. in these countries, not only is the incidence very high, it is continuing to show a rise on a year-toyear basis [ - ] . nationwide surveys conducted in japan since show that there have been three epidemics of kd in japan and also that the incidence of kd has increased by more than two times in the last two decades [ - ] . japan reports the highest incidence of kd in the world -the present fi gure being / , children below the age of years. korea has the second highest incidence at . / , children below fi ve. in taiwan the current incidence is . per , [ ] . based on statistical projections, it is estimated that by , there will be in people living in taiwan with a history of kd. the corresponding fi gure for the usa is in [ - ] . the incidence of kd, however, has remained static in europe as well as north america. in india, anecdotal reports suggest that the recognition of kd has increased signifi cantly over the last two decades, but confi rmatory epidemiologic data on incidence are not available. a hospitalbased study from chandigarh showed that the incidence was . / , children below years, but this fi gure is, in all likelihood, a gross underestimate [ ] . there is no doubt, however, that over the years awareness of kd amongst paediatricians in india has increased signifi cantly. in the years to come, kd may soon replace rheumatic fever to become the leading cause of acquired heart disease in children in india, just as in japan, europe and north america. kd affects the young with % of cases being below years. although it is uncommon in children below months, kd has been reported in neonates as well. in india, however, the median age of occurrence of kd is higher than in japan probably because kd in infancy and young children is being missed in our country. boys seem to be affected more commonly than girls. the male/female ratio at chandigarh has been . : [ , , - ] . fascinating data on seasonal variations in the incidence of kd have emerged, and these have linked the incidence fi gures of kd in some countries (especially japan and the usa) to tropospheric wind patterns emerging from central asia. such associations have, however, not been proven in other countries, as, for instance, canada [ ] . the causation of kd still remains an enigma. there have been various hypotheses and postulates regarding its aetiology [ , ] . infectious : there are multiple factors which suggest that kd could be an infectious illness. occurrence of the disease in childhood and its virtual absence in adulthood suggest that it may be an infectious disease, and exposure in childhood confers protective immunity, thereby protecting the individuals in adulthood. rarity of its occurrence in early infancy suggests that maternal antibodies may have a protective effect. in addition, clustering of cases in some seasons, few reports suggesting occurrence in families or communities and the clinical presentation with fever, rash and lymphadenopathy, also suggests that it might be an infectious illness. however, till date, no infectious agent has been incriminated with certainty. although several micro-organisms (e.g. streptococcus , staphylococcus , epstein -barr virus , parvovirus , coronavirus ) have been linked with kd, infection alone cannot explain the pathogenesis of the disease. superantigen mediated : the presence of excess of t-lymphocytes with vβ in the coronaries, intestinal mucosa and blood in children with kd suggests that the disease might be superantigen mediated. also, some of the clinical features of kd mimic those of toxic shock syndrome which is a superantigen-mediated disorder. environmental : researchers have also suggested that there might be an environmental trigger which causes this illness. seasonal and geographical clustering can be explained on this basis. genetic : recent evidence based on genomewide association studies suggests that a genetic basis of kd seems entirely plausible. evidence for this hypothesis comes from several epidemiologic data which show that children of certain genetic background seem to have a much higher predisposition to kd. for instance, the incidence of kd amongst children of japanese ancestry in hawaii is much higher than that in other races residing there. in fact, the incidence of kd in children of japanese ancestry there is almost similar to the incidence in japanese children residing in japan. however, till date, no single gene has been implicated, and there is no suggestion of it being an inherited disorder. onouchi et al. have shown the association between kd and itpkc functional polymorphisms [ , ] . it appears that kd is triggered in genetically predisposed children on exposure to some infectious or environmental agent. kd is a systemic medium-vessel vasculitic disorder with a striking predilection for the coronary arteries. this preferential involvement of the coronary arteries is unique to this condition. the infl ammation involves primarily the intima and media leading to damage of the internal and external elastic lamina. the affected vessel may subsequently become ectatic or may show aneurysmal dilatation. healing of these vessels may, in due course, result in stenosis and coronary thrombosis. large coronary aneurysms, and especially giant aneurysms, may never regain normal anatomy [ , ] . the clinical correlates of these changes should be borne in mind. on echocardiography, a coronary artery that gets dilated in the acute phase may regain its normal diameter during convalescence. however, intravascular ultrasound studies have shown that the vessel wall may never regain its recoil and elasticity even when echocardiography suggests a normal anatomy. coronary artery involvement can also be studied by doing computerized tomography (ct) angiographic studies ( fig. . ) . coronary vessels affected in kd have a predisposition to develop calcifi cation during the healing phase. this calcifi cation may render the affected vessels unsuitable for procedures such as angioplasty and stent placement later in life. kd can also affect other arteries like the femoral and axillary. these would, however, get involved only if there has been coronary artery . fever : this is often high grade, may exceed °c and lasts for more than days. the fever typically does not respond to antimicrobials and is associated with irritability which may at times be extreme. fever > days plus at least four of the following: • bilateral conjunctival injection • changes in oropharyngeal mucous membranes • changes in peripheral extremities • polymorphous rash • cervical lymphadenopathy illness not explained by any other disease condition hands followed by desquamation which starts from the periungual regions of hands and feet is a characteristic sign. these changes are seen in about % of children with kd. later in the course of disease, beau's lines may develop over the nails which are transverse ridges more easily felt than seen over the nails. . rash : an erythematous, morbilliform, nonpruritic rash can occur which begins from the trunk and involves the perineal area. the rash is transient and fades by itself. the rash of kd can be polymorphous but is never vesicular. reported to be present in more than % of affected children from developed countries, it may be diffi cult to perceive in children with dark skin. . lymphadenopathy : unilateral, tender, cervical lymphadenopathy occurs in about % of children with kd. a diagnosis of kd can be made if a child with fever for more than days fulfi ls four of the aforementioned fi ve criteria. if a child has fever for less than days or has less than four criteria, the presence of coronary artery abnormalities (caas) detected on d echocardiography would also suggest a diagnosis of kd [ ] . the diagnosis of kd can be very challenging and it may test the clinical acumen of even the most astute physician. it must be clearly understood that as there is no gold standard for diagnosis of kd, one does not know the sensitivity and specifi city of the diagnostic criteria for this condition [ ] . from clinical experience, it is clear that if one applies the criteria very strictly, the specifi city would be high, but this would be at the cost of sensitivity. there is no doubt whatsoever that many children with kd may not fulfi l these criteria. further, as the clinical spectrum of kd evolves over a few days, the clinical fi ndings which may have been present during the fi rst few days of the illness may no longer be evident subsequent in the course [ ] . in addition to these characteristic features, some additional fi ndings suggest the possibility of kd. perianal desquamation which appears earlier than the periungual desquamation is also characteristic, and its presence should be looked for in every child having fever for more than days. a transient arthritis may be seen in a quarter of the patients and may, at times, be the presenting feature of kd. reactivation of the bcg scar can be noted in some infants with kd but this is extremely uncommon in our experience. hydrops of the gallbladder may be seen during the acute phase of kd. asymptomatic anterior uveitis is not uncommon during the fi rst few days of the illness. the clinical course in kd is divided into three phases [ , ] : acute febrile phase : this lasts from the onset of illness to - days and is characterized by high-grade fever, irritability, rash, strawberry tongue, red cracked lips and limb changes. signs of myocarditis may appear in this phase, which include tachycardia, s gallop and congestive cardiac failure. subacute phase : this lasts up to - weeks and ends with return of the acute phase reactants to normal. the caas fi rst appear in this phase. convalescent phase : this lasts for months to years during which healing of the vessels occurs with remodelling and scarring. beau's lines occur during this phase. if a child does not fulfi l the criteria and has less than four features, a diagnosis of incomplete kd is proffered. incomplete kd is commonly seen in infants, and ironically this is the group of patients who are at highest risk for the development of caa. the american heart association has suggested an algorithm for identifi cation of children who do not fulfi l the criteria for kd and are suspected to have kd and so have a risk of developing caa [ ] . when a child with kd presents with features that are not usually seen in this condition, a diagnosis of atypical kd is proffered. clinical features which are unusual in kd include, but are not restricted to, nephritis, seizures, acute hepatitis, neurological obtundation or hypertension [ , ] . because of it being an acute febrile illness, the differential diagnosis of kd includes the common infectious illnesses of childhood: scarlet fever measles hhv- and hhv- toxic shock syndrome stevens-johnson syndrome drug hypersensitivity serum sickness systemic-onset juvenile idiopathic arthritis it cannot be overemphasized that there is no pathognomonic laboratory test for kd. however, there are several investigations which may support a clinical diagnosis of kd. a normocytic, normochromic anaemia with polymorphonuclear leucocytosis is usually seen. thrombocytosis is characteristically seen after day of fever, but thrombocytopenia can also occur, especially when there is an accompanying macrophageactivation syndrome. acute phase reactants like the c-reactive protein (crp) and the erythrocyte sedimentation rate are elevated during the acute phase. sterile pyuria may be found which is of urethral origin; therefore, one may not obtain this fi nding if urine is obtained by suprapubic bladder aspiration. pyuria in a sick and febrile child is liable to be misdiagnosed as a urinary tract infection [ , ] . ultrasonography may reveal hydrops of the gallbladder. the gallbladder may sometimes enlarge to the extent that it becomes palpable on physical examination. cerebrospinal fl uid examination is not indicated in kd. if, however, it has been carried out, one may see fi ndings consistent with aseptic meningitis. lipid abnormalities are not unusual in children with kd. serum triglycerides and low-density lipoproteins are increased and high-density lipoproteins are reduced. these abnormalities begin in acute phase and may take several months to normalize. d echocardiography remains the imaging modality of choice for evaluation of coronary artery abnormalities in children with kd. caas include ectasia and/or aneurysms in proximal parts of left main coronary artery, left anterior descending artery, left circumfl ex artery and/or right coronary artery. this examination needs to be carried out by a pediatric cardiologist or a physician who has the requisite training and experience in evaluation of coronary arteries. coronary arteries are said to be dilated if the size is more than . times that of the adjacent segment. coronaries with diameter of more than mm in a child less than years of age and more than mm in older children are said to be dilated. it is preferable to use z-scores for evaluation of coronaries in children. the use of these z-scores makes the follow-up and comparison easier and more reliable. children with kd can also have several other fi ndings on echocardiography. these include a decrease in ejection fraction (suggestive of myocarditis), mild valvular regurgitation, increased brightness of the coronary vessels and pericardial effusion. all of these are indicative of an ongoing infl ammatory process. it is important to diagnose and treat kd expeditiously as delays in therapy can result in serious morbidity and occasional mortality. the mortality rate of acute kd at chandigarh is approximately . %. caas can develop in a quarter of untreated patients with kd. every child suspected to have kd should be admitted and evaluated so that close clinical observation can be carried out. it cannot be overemphasized that clinical fi ndings in kd may change from day to day, and it is of the utmost importance that these be observed and recorded methodically. treatment of kd consists of intravenous immunoglobulin (ivig) and aspirin started within the fi rst - days of illness [ , , - ] . ivig is given as a single intravenous infusion of g/ kg. it is generally administered over - h, but the infusion must be started slowly so as to avoid occurrence of hypersensitivity reactions. ivig has been found to be very effective if given within the fi rst days of onset of illness. in case, however, the child presents late and the acute phase reactants are still elevated or if there are caas on echocardiography, ivig should still be administered. the response to ivig is often dramatic, and the irritability, so characteristic of kd, often disappears within a few hours of initiation of the infusion. fever defervescence usually occurs within - h. administration of ivig can, on occasions, be associated with signifi cant adverse effects like headache and vomiting. these are thought to be secondary to a mild aseptic meningitis which is known to be associated with administration of ivig. these can usually be managed quite easily with symptomatic treatment. ensuring adequate hydration and a slow rate of infusion of ivig is said to decrease the occurrence of these adverse effects [ - ] . aspirin in anti-infl ammatory doses ( - mg/kg/day) is started along with ivig and is continued till the child becomes afebrile. thereafter, aspirin is continued in antiplatelet doses ( - mg/kg/day) till a follow-up echocardiography is done and found to be normal at - weeks of illness. if caas are found in follow-up echocardiography, then aspirin needs to be continued for prolonged periods. additional anticoagulation (with warfarin or low molecular weight heparin) may be required in children with large aneurysms. while therapy with ivig and aspirin has dramatically reduced the occurrence of caas in kd, a small proportion of patients (approximately - %) still go on to develop caas despite seemingly appropriate therapy having been administered. approximately - % of patients with kd either do not respond or have a recrudescence of fever that recurs within h of completion of ivig infusion. such patients are said to have refractory kd. several japanese investigators [ - ] have put forth risk scoring systems for predicting refractory kd. ogata et al. [ , ] showed that children who were predicted to be ivig resistant on the basis of one such scoring system showed earlier defervescence of fever when treated pre-emptively with a combination of ivig and iv methylprednisolone as compared to the group given ivig alone. the occurrence of caas was also less in the former group. miura et al. [ ] showed that iv methylprednisolone, when used along with ivig in children with refractory kd, does lead to faster defervescence. the effect, however, is not long-lasting. further, the incidence of development of caas is not signifi cantly different in two groups, and the occurrence of adverse effects was signifi cantly higher in the group treated with iv methylprednisolone plus ivig. it must be noted that these scoring systems do not seem to yield reliable results in populations other than japanese and therefore may have limited utility in day-to-day clinical practice in other countries. there is no consensus on the best modality for treatment of refractory kd. therapeutic options include a repeat dose of ivig, intravenous pulse methylprednisolone or anti-tnfα agents (e.g. infl iximab). cyclosporin and plasmapheresis have also been used in such circumstances [ , ] . children with kd who have no caa or have only transient coronary artery ectasia require lowdose aspirin ( - mg/kg/day) for initial - weeks. children with a single small coronary artery aneurysm should be given aspirin at least until the disappearance of aneurysm. however, in a child with giant coronary artery aneurysm, or one with multiple coronary aneurysms, long-term antiplatelet therapy along with antithrombotic therapy in the form of oral warfarin or low molecular heparin is mandated. male sex age less than months thrombocytopenia at presentation neutropaenia, hyponatraemia and hypoalbuminaemia prolonged fever or recrudescence of fever failure to respond to ivig a landmark study by kato et al. [ ] showed that up to % of small-to medium-sized aneurysms resolve on follow-up. giant aneurysms (> mm size), however, do not resolve and are associated with signifi cant long-term morbidity. even when the coronary aneurysm appears to have resolved anatomically, functional vessel wall abnormalities are known to persist and may result in myocardial ischaemia/infarction later in life. kd can be associated with signifi cant long-term sequelae. this has been conclusively borne out through recently published long-term follow-up studies carried out at san diego, usa [ ] . it is obvious that kd is no longer considered to be merely a one-time disease of childhood. considering the fact that the incidence of this disease is showing a steady increase in several countries, it is likely to emerge as the commonest cause for acquired heart disease in children the world over. many of these children would grow up to be adults with coronary sequelae. kd, therefore, needs to be considered as a disease of public health importance, and health planners need to be made aware of these facts. acute febrile mucocutaneous syndrome with lymphoid involvement with specifi c desquamation of the fi ngers and toes in children text book of pediatric rheumatology nelson textbook of pediatrics epidemiology of kawasaki disease in asia, europe, and the united states epidemiologic features of kawasaki disease in acute stages in taiwan, - : effect of different case defi nitions in claims data analysis epidemiologic features of kawasaki disease in japan: results of the - nationwide survey changes in epidemic patterns of kawasaki disease in japan results of nationwide epidemiological incidence surveys of kawasaki disease in japan epidemiologic pictures of kawasaki disease in japan: from the nationwide incidence survey in and monitoring the occurrence of kawasaki syndrome in the united states is kawasaki disease incidence rising in chandigarh a comparison of the clinical profi le of kawasaki disease in children from northern india above and below years of age kawasaki disease -an indian perspective tropospheric winds from northeastern china carry the etiologic agent of kawasaki disease from its source to japan a replication study for association of itpkc and casp two-locus analysis in ivig unresponsiveness and coronary artery lesion in kawasaki disease a genome-wide association study identifi es three new risk loci for kawasaki disease diagnosis, treatment, and longterm management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis and kawasaki disease, council on cardiovascular disease in the young kawasaki disease: issues in diagnosis and treatment -a developing country perspective delayed diagnosis of kawasaki syndrome: an analysis of the problem prediction of intravenous immunoglobulin unresponsiveness in patients with kawasaki disease prediction of resistance to intravenous immunoglobulin treatment in patients with kawasaki disease corticosteroid pulse combination therapy for refractory kawasaki disease: a randomized trial the strategy of immune globulin resistant kawasaki disease: a comparative study of additional immune globulin and steroid pulse therapy adverse effects of methylprednisolone pulse therapy in refractory kawasaki disease long-term consequences of kawasaki disease. a -to -year follow-up study of patients acute myocardial ischemia in adults secondary to missed kawasaki disease in childhood . kd is the most common cause of acquired heart disease in children in developed countries. . the incidence of kd varies across the world; the highest has been reported from japan. . there is no gold standard for diagnosis of kd, it is to be considered in a child with prolonged pyrexia, irritability and sequential appearance of clinical signs of kd. . the management is with ivig and aspirin. the latter is used initially in antiinfl ammatory doses initially and then reduced to antiplatelet dose. the duration of treatment depends on the coronary artery involvement. . there are no clear guidelines for resistant kd, most centres would use a second dose of ivig, and other choices are steroids and infl iximab. key: cord- -s tfvtao authors: nan title: oral abstracts date: - - journal: vox sang doi: . /vox. _ sha: doc_id: cord_uid: s tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are billion hbv-infected people including million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was / ( %), whereas that of anti-hbc-negative components was / ( %), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was %, and the rate was higher without anti-hbs( . %) and lower with anti-hbs( . %). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs < miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to . %, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since , we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every th was designated as 'blood donation day' in . to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june , agreements for blood donation had been signed with enterprises and organizations. every year more than thousand donors agree to donate on a regular basis, and so far about thousand donors have been registered in the 'registered donor system'. the campaign 'every th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~ : - platelet products are contaminated with bacteria, and septic transfusion reactions occurred in : - , transfusions. clinical awareness of the danger and a aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since , and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s- system for red cells, demonstrating in vivo recovery after days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted b-h - no abstract available. b-h - how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately documents are in the repository from around countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in . there is one standard with progressively more rigorous steps of achievement as follows: level : minimum quality and operational requirements level : intermediate quality and operational requirements level : full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in while the malawi piloting will be completed in . comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. c-h - diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcΥr and c receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: ( ) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. ( ) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? ( ) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since , at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. d-h - distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in , the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt , to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt as a priority for action in improving quality management and haemovigilance. d-h - the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa- to - and - , which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa- system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa- system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa- and - , respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in , during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in , and the first workshop of the piwp-ar was organized in taipei in . in may , the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of cases that were diagnosed and studied as naitp ( ), ptr ( ) and ptp ( ) in asian platelet immunology laboratories were reviewed. of the cases, were japanese in japan, were chinese in china, were in india, were chinese in taiwan, china, were in south korea, were in thailand, were in kuwait, and case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa- (in kuwait), of anti-hpa- ( in china and in japan), of anti-hpa- (three in japan, one in china and one in taiwan, china), of anti-hpa- (all in japan), of anti-hpa- ( in china and in japan), of anti-hpa- new(antihit a )(in japan), of anti-hpa- (in japan), of anti-hpa- bw (in japan), of anti-hla( in china, in japan, in korea, in thailand, in taiwan, china and in india), of anti-a (in japan) as well as cases of anti-cd (nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities ( in china and in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa- is mainly found in japanese individuals, while anti-cd (nak a ) isoantibody that occurred in cd type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa- antibodies were found in asian population (all in kuwait), while anti-hpa- is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd deficiency is - %. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa- to and hpa- . the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa- , - , - , and - are common among caucasians while hpa- b is extremely rare. in the chinese however, hpa- b is extremely rare while uncommon in malays. hpa- b and hpa- bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa- b is the most common cause for nait in the asian population as compared to alloanti-hpa- a among caucasians. several cases of nait due to anti-hpa- bw have also been reported in asia. in contrast, nait due to anti-hpa- b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes a-s - setting up a patient blood management programme wood e , engelbrecht s , and robinson k transfusion research unit, monash university, melbourne, australia australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial h. massive blood transfusion is generally defined as the replacement bytransfusion of more than units of red cells over h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this ( %) patients received rh d positive platelet transfusions. in ( %) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was . years and included males and females. average number of rh d positive platelets transfused per patient was . units and total number of platelet units transfused was . anti d was detectable in ( . %) patients post transfusion and included one male and three female patients. of the female patients one was a year old child who received units of random donor platelets and second a year old women who presented in the emergency department as a case of trauma. the third female was year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of blood components were issued during the study period. transfusion feedback form were received for ( . %) blood components, as follows: prbc- ( %), platelets- ( . %), ffp - ( . %). patient identification number /wristband check was not done in ( . %) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in ( . %) cases. cross checking of component unit with request form was missed in ( . ) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in ( . %) episodes, monitoring of pulse in ( . %) episodes and patient's temperature was monitored in ( %) episodes. signature of nurse was missing in ( . %) and that of medical officer in ( %) of the form. adverse transfusion reaction was documented in / forms, whereas the transfusion reactions notified at the blood bank during the same period were . of ( . %) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was min for rbc components, min for platelets and min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = . ). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = ), hpa and hna (n = ) and high frequency antigens (hfa, n = , ) within years. specificities are grouped into single multiplex reactions (mpx, n = ) with up to snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached % for k/k, . for kp a . % for jkand . % for fy a/b/x/ . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel* . (r g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g r)null. serology for js a/b of some few kel* . positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were , and , respectively. call failures (no result) were observed in less than % of all mpxs. genotyping for rhd, rhce ( mpx), gypa and b (mnss, mpx) on more than samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately % again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna ( mpx) showed expected results among samples with the exception of hna- a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping ( mpx), currently, more than , blood donors were analyzed, and delivered: kk, kp(a+bÀ), js(a+bÀ), kel +kel +, lu(a+bÀ), lu +lu À, yt(aÀb+), co(aÀb+), kn (aÀb+), lw(a+b+), and > others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aÀb+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the - tcc ( s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about % of the total cost for serological methods, and about % for molecular methods. the cost of labour is about % for serological methods and < % for molecular. equipment cost contributes to < % of the cost using serological methods and about % using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman â snp genotyping assays in -well plates at ll final reaction volume, on a lightcy-clerii real-time instrument. identification of the cc polymorphism was by probes targeted to the c>g and t>c of the rhce allele while probes targeted to c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with of the ( %) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee ( / , . %), fya-/fyb+ ( / , . %), ss ( / , . %). the co allele responsible for co b was identified in the heterozygous state in four of evaluable samples giving an allele frequency of . % in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd . compared to usd . for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october , in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected donors aged < years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table . table : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of : according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy c> t. the results were confirmed in . % of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a -> log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, % plasma/ % intersol, or pbs. each sample ( . ml) was treated with lm amotosalen and j/cm uva (n = ). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample ( . ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from to bp was evaluated over cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in % plasma/ % inter-sol ( ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in . log to > . log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > % plasma > % plasma. exposure of platelets to amotosalen and uva showed an average of . log to . log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day- post-collection (n = ). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at °c with agitation and samples were taken on day and post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, % v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; mg/ml) for h at °c/ % co . protein secretion was inhibited using brefeldin ( lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd + /cd + ) and neutrophils (cd + / cd + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for days significantly increased monocyte mip- b expression ( . -fold; p = . ), whereas exposure to day platelets did not result in any significant differences in mip- b expression. as expected, lps stimulation significantly increased monocyte production of both il- ( . -to . -fold) and mip- b ( . -to . -fold). however, lps-induced monocyte il- production was significantly reduced by exposure to prt-treated or untreated platelets stored for days (prt-treated: . -fold, untreated: . -fold; p < . ) and days (prt-treated: -fold, untreated: . -fold; p < . ). furthermore, il- production was significantly lower following exposure to day prt-treated platelets compared to untreated platelets (p = . ); however there was no significant difference following exposure to day platelets. lps-induced mip- b production was not significantly differentfollowing exposure to either day or day prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip- , mcp- , mip- a, il- a, il- b, il- , il- , il- , or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for days results in significant changes in the il- production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of samples ofclinical patients and samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega software. moreover, spss . software was applied to compare the difference between these two groups. results: of clinical patients, hcv genotype a, b, a, a, b, a and n was ( . %), ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %), respectively. of blood donors, hcv genotype a, b, a, a, b, a and n was ( . %), ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %), respectively. the proportion of hcv b was higher in clinical patients than in blood donors(v = . , p = . e- ), while the proportion of a and a subtypes were higher in blood donors than in clinical patients(v = . , p = . ; v = . , p = . e- ). one possible cause was the transmission modes varied with different hcv subtypes. hcv b is more related with blood transfusion while a and a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from , the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about years and hcv a was transmitted into guangdong later than b. conclusions: in guangdong province, hcv b and a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv b, a and a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in - . in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were cases in - . among them, patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of patients ( , , , , patients in , , , , , respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells b-pl the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past years has seen the discovery of five new blood groups. at the isbt meeting in , fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd -deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg and abcb respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt . this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim . the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd whose function in complement regulation is well-understood, resulted in production of an alloanti-cd , demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. c-s - teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in , an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with % of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h n ) virus in , isolation of novel bat-associated reoviruses in , emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h n ) virus is evolving, and there has been a recent report of human infection with avian influenza a(h n ) virus as well. additionally, there are imported eids such as influenza a(h n ) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. % of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in to designate june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv- . this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from . % to . % in the general population, but is relatively low in blood donor population ( . %). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from < to iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high ( , - , cp/ml and , - , , iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may to december , donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was . days (table) . eleven ( %) of these were male donors, and the median age was years (range - years). the pcr for hav rna was positive in all of depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of blood components (rbc units, pc units and ffp units) were delivered to hospitals. twenty six products were transfused to patients, and the rest blood components ( rbcs, pc, and ffps) were recalled immediately and discarded. twelve recipients ( %) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were and years old developed symptoms on and days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were s. in korea, most people under years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. c-s - cable rt , pistorius c , andersson m , maponga t , lopez t , preiser w and tedder r hav seroprevalence was highest in the black donors ( %), lower in coloured donors ( %) and lowest white donors ( %). all were hav igm negative. there was an age-related increase from . % ( / ) in those - years old to % ( / ) in those > years. although no active hev infection was identified by pcr on pooled samples, % of donors were hev igg positive. rates were highest in whites at %, followed by coloured at % and lowest in black at %. again prevalence increased with age from . % in those - years to . % in those > years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t c and t c) and prec/bcp region (mutations including a t/g a, g a, t a, c t, t a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of samples ( . %)were subgenotyped successfully. the predominant subgenotypes were b , c , d and a which accounted for . %, . %, . %, and . % respectively. deletions and mutations (t c and t c) in pres region were found in ( / , . %) samples. five of these samples ( . % of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < . ). mutations in polymerase region were found in samples ( %, / ), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in samples ( . %, / ). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = . ). more a t/g a mutations were found in c than b genotype while the opposite was observed for g a mutation (p's < . ). conclusions: subgenotype b was the most frequent strain circulating in hbv infected chinese blood donors, followed by c . hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a t/g a mutations was higher in genotype c than in genotype b while the opposite is the case for g a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. c-s - a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least % of tested products meet or exceed the required platelet count. in practice, this means that up to % of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day , , , , and . assay of mp was by flow cytometry using mab to label cd a, cd , cd , and cd e. thrombin generation (tg) and cd b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day . results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day , red cell mp (rmp) were increased by . at day , and by . by day . small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally < % as many as rmp. levels of pmp rose rapidly from day and peaked at day . lmp began rising at days, increasing to . at day and . at day . emp changed little over days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was < % as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets ( - , per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd b expression increased from day to day in both lr and nlr pc, but in lr pc, it was only - % magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd ligand (scd l, scd ) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd l. method: the pmps were isolated by centrifugation of the platelet-free plasma from apheresis platelet concentrates (a-plts) at , g for h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd l during days storage (correlation was significant at the . level); pmn respiratory burst are declined by removing pmp with . lm pvdf membrane filtration or depletion pmp with cd monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from -day stored a-plts compared with -day stored a-plts (p < . ). conclusion: platelet-derived microparticles carry concentrated cd l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of whole blood donors and platelet donors with vary donation history from to times (mean at . ae . ) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g : - time, g : - times and g : > times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g : . ae . , g : . ae . and g : . ae . ng/ml respectly, p = . ) and mean platelet volume (mpv) was decrease (g : . ae . , g : . ae . and g : . ae . fl respectly, p = . ) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = . , p < . ). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either -or -days old; n = each) were pooled and equally split to obtain matched pairs (day ). one unit was washed with . % saline by repeated centrifugation then resuspended in ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce units of paediatric-sized washed or unwashed rccs. all units were stored at - °c and samples were taken on days , , , , and of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < . considered significant. results: the washing process resulted in reductions in red cell count ( . %), haemoglobin ( . %) and haematocrit ( . %) compared to unwashed rccs. overall, washing and subsequent storage of -and -day old rccs significantly reduced the ph (p < . ), lactate production rate (p < . ), and , -diphosphoglycerate concentration (p = . ). although the atp content of the rcc decreased during storage, it was not changed by washing (p = . ). haemolysis in the rccs was increased by the washing process, but remained < . % on day for all products. extracellular potassium was significantly reduced by washing (p < . ), but increased during storage in both washed and unwashed red cells (p < . for both). washing significantly reduced the number of microparticles in the supernatant of both -and -day-old rcc compared to unwashed rccs (p = . and . respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both -and -day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein- , scd p, rantes, anaphylatoxins (c a, c a, and c a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < . for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. d-s - burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically - or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h n and h n virus can cause acute community acquired pneumonia with to % mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than days after symptom onset. during the pandemic of h n influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august to october , , a total of persons were successfully contacted. a total of screening and whole blood donation appointments were made. in the former ( . %) attended screening but only could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, ( . %) had attended and donated. . l ( units) and l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited patients with severe h n infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients ( . %) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group ( . % vs . %; p = . ). convalescent plasma treatment was associated with significantly lower day , , and viral load, compared with the control group (p < . ) and corresponding lower serum levels of il , il , and tnf (p < . ). between and , patients were randomized to receive h-ivig ( patients) or ivig ( patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day and posttreatment viral load when compared to the control (p = . and p = . respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level days after treatment. subgroup multivariate analysis of the patients who received treatment within days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or: . , % ci, . - . ; p = . ]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of . higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b (tgf-b ) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw . mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase- (cox- ). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw . cells were grown in medium supplemented with % of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps ( ng/ ml added after h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox- in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b content ranging from approximately - ng/ml, and ppp the lowest ( - ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox- detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox- synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox- synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. d-s - tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of : ~ individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists rare blood phenotypes that are divided in two categories as shown in table . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since , our blood center has established moabs ( human and murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk- blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than , donors with rare blood phenotypes (category i, ; category ii, ) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since , a total of units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. d-s - the china national rare blood group screening program started in . the program has screened more than , , donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was / in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, . the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent years, units ( unit = ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt ). five cases were reported by daniels et al, (transfusion, ; : ) and two by reid et al, (transfusion ; (suppl) : s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a -year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was . g/dl, another two rbc bags were transfused, and his vital signs became stable. on day , he was transfused one rbc bag without a hemolytic transfusion reaction. on day , after receiving ml of rbcs, the patient vomited and had cola-colored urine (t-bil . mg/dl, ldh u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: + on day , + on day and negative on day . thereafter his anemia improved gradually by iron medication and he was discharged on day . aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at c ( +), by albumin iat ( +), peg-iat ( +), and papain-iat ( +) with all panel cells, and by peg-iat with samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg and igg with a titer of by peg-iat before transfusion rising to on day ; his serum demonstrated hemolysis after day . no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. d-s - background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. ( ) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program ( ) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. ( ) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of . we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a % increase in blood demand from year to with almost % blood being transfused to patients aged > . therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years - . statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of , donations made by , male and , female donors were analyzed. on average . % of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center ( . % vs . %, p < . ). male were more likely to come back for repeated donations than female ( . % vs . %, p < . ). a total of , donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by july . by the end of year , donations (consisting of males and females) were made with more than . % collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in , , donations were collected which already exceeded the planned collection target of , by year . interestingly, same . % donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a -prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about % of people visiting that vicinity were youths aged to ( . %) and young adults aged to ( . %). the main purposes of the visit were for work related activities ( %), attending school ( . %) and shopping ( . %). on weekends, % of these age groups visited the vicinity for leisure activities or to church. another % who visited there were adults age - years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of units of blood a day within the months of its operation (jan ); and now, has a daily average collection of units of blood. a-s - setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of . billion people, blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july , with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december . terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february and till date reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. a-s - background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in . in addition, the allelic frequency of hpdel was calculated to be . by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between and , in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of , patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january and december , were examined. healthy blood donors: a total of , blood donors who visited the japanese red cross blood centers in the tokyo area between june and august were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of mg/dl followed by simplified sandwich elisa with the detection limit of lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the , patients who had experienced nhtrs ( . %). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, ; ffp, ; rbcs, ; mixed, ] . nineteen of them ( %) experienced anaphylactic trs accompanied by severe hypotension and the other patients ( %) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in patients ( %). in addition to the igg antibody, the anti-hp ige antibody was detected in patients ( %). hpdeficient individuals were identified among japanese blood donors with a prevalence of / , ( . %). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp . the anti-hp antibody was not detected in / ( %) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < . ), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion labora- methods: a study on all reports of mislabeled and miscollected specimens from january to december was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during - was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january and december a total of , samples were received for abo grouping. of incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was . % or in samples. of cases resulted from wrong labeling during collection, cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in incidents, labeling was performed away from the bedside, in cases name labels of a different patient were found in the correct patient's medical file, in cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest % had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in under the ministry of health (moh). since its inception in the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january till december were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: ( ) incident reporting ( ) root cause analysis ( ) identification of corrective actions results: during the study period , requests for blood and blood components were received and total of , , blood components were issued within the hospital. a total of , reported errors were analysed. pre-analytical errors comprised a large majority ( , ; . %), most of them being errors of inadequate patient information on request forms ( ; . %) followed by sampling errors ( ; . %). analytical errors comprised ( ; . %) and post analytical errors accounted for ( ; . %) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is % self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from to in the last four financial yearsa % reduction. in addition, order fulfilment has improved from % to %, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. a-s - wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in . it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last years. evidently, blood shortages are largely preventable by adopting a proactive approach. a-s - . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from . in year to . in year . the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology ( . reduced to . ), surgical ( . reduced to . ), orthopedic ( . reduced to . ) and neurology ( . reduced to . ). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. a-s - fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to . and specialized services to . million population) reduced the regional rc inventory by % ( rcs to rcs; optimal hospital inventory index of . ). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at % (average shelf-life of transfused rcs reduced from . to . weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump ( and ) included in table . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by % (average). chronically transfused hematology and renal patients showed the highest reduction on rcu ( - %, p value < . ). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction ( %) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. a-s - no abstract available. a-s - burnouf t , tzeng ys , deng sc , wang ch , tsai jc and chen tm taipei medical university, taipei, taiwan tri-service general hospital, taipei, taiwan tatung university, taipei, taiwan background: approximately % of diabetic patients develop chronic ulcers, and % of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [ ]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged - with nine non-healing lower extremity ulcers (median size of cm ; range - cm ) were enrolled. their median duration of diabetes and ulcer was . years (range - years) and . months (range - months). none of the patients had received conventional skin grafting in the past. prp was prepared from ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating ml of plasma (tgd- ; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after to days with equal volumes ( to ml) of autologous prp and autologous thrombin to form the platelet gel within - s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well ( % healing rate). the interval between skin graft and complete wound healing ranged from to weeks in the eight successful cases. no ulcer recurrence was noted during the - months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated ( °c/ min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over bovine corneas (bcecs) using standard procedures and grown in a dmem-f medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) % fbs, . % dmso, ng/ml rhu-egf, lg/ml insulin, lg/ml transferrin, and nm cholera toxin (termed ' % fbs medium'), or with (ii) . %, %, . %, or % pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in , or -well plates at °c in a controlled atmosphere containing % co , with medium changes every two days. viable cells were counted for days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin , anti-na/k atpase alpha- subunit, anti-zo- and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained - mg/ml total proteins, and a range of approximately - . , . - , . - . , and . - . ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with . - % pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the ' % fbs medium' could be achieved only using % hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f medium supplemented with % hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor and rantes) and cytokines (e.g. interleukin- b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd / cd l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd + t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd + t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor (pf ) and the erythrocyte duffy-antigen molecule, which binds pf and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. c-s - graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor (pf ) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september . only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf /heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the ' t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. c-s - autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd + t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd + t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp + regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. c-s - seguchi s , maeda t , kanaumi y , kawamura s , kodama m , kawai t , okazaki h and miyata s national cerebral and cardiovascular center, osaka, japan the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor (pf ), resulting in the production of anti-pf /heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august and may , patients from hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf /heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the patients, patients were ultimately diagnosed with hit ( . %). optical density values of anti-pf /heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients ( . ae . vs . ae . for igg/a/m, p < . ; . ae . vs . ae . for igg, p < . ). the incidence of thromboembolic events was significantly higher in hit patients ( . %) than that in non-hit patients ( . %; p < . ). among the hit patients, patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within h and the other after days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. c-s - lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, repeat donors were typed for hla-a, -b and hpa- ,- , - , - , - , - and - using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, female)showing platelet refractoriness to random donor platelets [ h corrected count increment (cci) < ml/m , percentage of platelet recovery (ppr) < %] before. patients phenotyped for both hla-a,b and hpa- ,- , - , - , - , - and - . apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show h ppr > %. the mean , h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion , h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in % of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. c-s - xia w , xu x , ye x , fu y , deng j , liu j , ding h , chen y , shao y , wang j , li h and santoso s guangzhou blood center, guangzhou, china department of biotechnology, guangdong food and drug vocational college, guangzhou, china he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from healthy blood donors were typed for cd deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd deficiency. transfection in mammalian cells (hek- t) with cd mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of / blood donors failed to express cd on their platelets surface. in / individuals no cd expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd deficiency (platelets and monocytes) and type ii cd deficiency (platelets only) were approximately . % and . %, respectively. nucleotide sequencing analysis of type i cd deficient individuals revealed eight different mutations; four of them were not described so far. however, - del attgtgcctatt and - delac appeared to be the most common mutations related to type i cd deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor ) as well as in three cases of ptr (patients - ) and one case of fnait (patient ). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd cell lines. in all ptr patients, transfusion with platelets derived from cd negative donors resulted in good increment ( h, ppr > %). table shows the mutations found in these five gpiv defective individuals. conclusions: more than . % of cd type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. c-s - do we really need ffp? the evolving role of pf and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within h of collection (fp ) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp . in addition, the activity profiles of coagulation proteins in fp prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp , if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp , similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from h to up to days, if stored at °c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. c-s - background: in , the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of % of clinical plasma sourced from male only donors. methods: work began in early to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july showed that the blood service were issuing . % male clinical plasma; the group ab rate was . %. the results improved dramatically by november in groups o, a and b ( . %, . % and . % respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march , the results in group ab had improved to . % (chart below) and the directive was given to manufacture male clinical plasma only as routine. january was the first month ever where % of all clinical plasma was sourced from male donors only. in / , . % of clinical plasma was male onlythe . % constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by %, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide ( th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after days of storage at - °c. the new t&b production method for the separation of -ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. c-s - blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is %. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of - mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. c-s - wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p antigen) at nbc or at blood mobiles organized by nbc from november until july were retrieved and analyzed using spss . . results: a total of donors were nat detected and negative for anti-hiv and p antigen (none in , in , in , in , in , in and in introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. - years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar ). multi-stage random cluster sampling approach was followed and college and university students were targeted through university based blood donor organisations (bdos) out of a total bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version . results: a majority ( %) of the students had heard about blood donation through family/friends and a minority ( %) through the internet, although % preferred internet as spare time activity. majority (+ %) of the students had access to the internet and mobile phone. more than % of the respondents had donated blood: % donated for family, relatives or friends, % donated voluntarily as an act of altruism, and % donated voluntarily once and then stopped donating, but % of these respondents still considered themselves as volunteer blood donors. % indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people ( %), % followed friends invitations, in % cases respondents family or friends had received blood transfusions, % followed the example of fellow students. restriction for blood donation: % generally feared donation, % had a specific fear of the needle, % had no confidence in the public (health) sector, % condemned blood selling practice, % had no confidence in the donation procedure (hygiene), % experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. d-s - murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. d-s - managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that - % of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly % of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 's, most operations were performed to correct congenital heart disease. during the 's and 's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is - % and post bypass is > %) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. d-s - the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna- to . hna- , - , and - a alloantibodies have been implicated in the pathogenesis of trali, and especially hna- a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna- , - and - , those against hna- a and hna- a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna- a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna- , - , - , and - ). more recently, the molecular basis of hna- antigen has been elucidated, and stable cell lines expressing hna- antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna- antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. d-s - one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about % of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. d-s - when seven sera from donors were screened for neutrophil specific antibodies, % samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in . % male,~ . % nulliparous and~ . % parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn , also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn , also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein , mzf , is a transcription factor that trans-activates both ccn and ccn , in distinct cell types. for example, we have shown that mzf can stimulate ccn production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf also regulates the synthesis of ccn in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn in stromal fibroblasts, which in turn results in enhanced loading of ccn into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn expression, through mzf- , and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. d-s - background: foxp + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd + cd À t cells were purified from splenocytes of d mice and stimulated by anti-cd /cd in the presence of tgf-b for days, which were termed 'itreg'. and tdcs derived from bone marrow of d mice were induced by gm-csf, il- and tgf-b and harvested after -day cultivation. then, itregs were expanded by tdcs at the ratio : and collected after days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd t proliferation, induction of foxp + tregs from foxp À t cells and suppression of th cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d mice after immunized with bovine type ii collagen (cii) on day and day , termed collagen-induced arthritis (cia). and itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd + th subsets were analyzed. results: after -day induction in vitro, the percent of foxp + itregs increased from . ae . % to . ae . %. and these itregs could be expanded effectively by tdc in additional -day co-incubation, which continued to express higher levers of foxp ( ae . %) and increased . ae . fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd t cells converting to foxp + itreg and resistant to th cell differentiation more effectively. during the -weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il- , il- and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd + foxp +) and a decrease in the percentage of th cells (cd + il- +) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may the world health assembly passed a resolution, wha . , on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in . blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. a-s - background: blood transfusion service of nepal red cross society was established in the year . since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of centers in districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than , units of blood were served to the needy and around % of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. a-s - background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over million living on islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of . %, human immunodeficiency virus (hiv) of . %, and hepatitis c virus (hcv) . %. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of . million donations determined that id-nat could prevent infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $ million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $ million per year (or~ % savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that whole blood units andup to , blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $ million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. a-s - yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur - days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and out of may suffer from severe infection with significant neurological sequelae. in , transfusion-transmitted wnv infections were described. since , all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, transfusion-associated transmissions of wnv have been documented (mmwr ) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that million dengue infections occur annually and approximately . billion people live in dengue endemic countries (who ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be . - / , blood transfusions in the year , assuming a ration of asymptomatic-to-symptomatic infections of : to : . subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in . a-s - australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b , hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv- ), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~ agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between and through to its full implementation across the acute hospitals in oxfordshire in / , and its maintenance and further development since then. there was an improvement from . % to % of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of min to s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last years there have been substantial efforts to minimise or eliminate trali. despite these activities, in the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over , transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. blood) . that hna- a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna- ,- , and - were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna- a, - b, - c were . , . and . , respectively. hna- null allele was found in one sample. the gene frequencies of hna- a and - b were . and . , respectively. hna- a and - b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between to days, and about days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having ≥ symptoms of any kind. we calculated odds ratios (or) and % confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaÀ patients. results: of patients with samples and interviews - days following transfusion % were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaÀ recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. th we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were % and %, respectively. nobody has ever been bitten by kissing bug. % of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna- , - , and - ) were capable to induce trali reaction. among them, hla class ii and hna- a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from nulliparous females, parous females and male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna- and - were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, ; werth et al, ). hna- antibodies were analyzed by antigen capture assay (bayat et al, ). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of donors ( males and females) showed higher prevalence of hla antibodies production in female compared with male donors ( . % vs . %). we found in out of females donors alloantibodies against hla class i ( . %) and hla class ii ( . %). analysis among female additional immunosuppression may be used in patients with low adamts due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in , and expanded in to include all tma. of hospitals currently participate in the registry. of cases in patients were registered from sites; eight were excluded for incomplete data, leaving episodes in patients. results: median age was years ( - years), % were female, % were relapses. of % were idiopathic and % had ≥ identified precipitant, including infection %, autoimmune %, malignancy %, medication %, pregnant/postpartum %, solid organ transplant %, and allograft %. adamts levels were available in cases ( idiopathic and secondary). adamts was < % in / ( %) idiopathic and / ( %) secondary cases. precipitants in cases with adamts < % included infection (n = ), autoimmune (n = ), medication (n = ), and pregnancy (n = ). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in cases (table ) . of / ( %) received pex, by centrifugation in / , membrane filtration in / ( %), and unknown method in / ( %). plasma infusions were used in / cases (without pex in ). data on additional therapy was available for cases (table ) . rituximab was given in / cases with ≥ precipitant, including infection (n = ), non-haematological malignancy (n = ), haematological malignancy (n = ), autoimmune (n = ), medication (n = ), and pregnancy (n = ). death rates were lower in idiopathic than in secondary cases ( % vs %, p = . ). conclusion: our data confirm that very low adamts does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (< months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as > ml/kg of blood components during the surgery. analysis was done using spss software version . median levels were compared between mt and non-mt group using mann whitney test. results: between august to july , pediatric ldlt were performed in a single center in south india. thirty five ( . %) of them were females and ( . %) males; ( %) of the recipients blood group were o positive, ( %) b, ( %) a and ( %) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in , metabolic /hereditary liver disease in , hepatic tumors in and acute liver failure in recipients. of ( . %) patients received prc, ( %) ffp, ( . %) plt and ( . %) cryoprecipitate. sixteen ( . %) patients received massive transfusion (mt) with a median peld score of (À to ) compared to (À to ) recipients without mt (p < . ). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb ( . vs . mg/dl), platelet count ( vs per mm ), fibrinogen ( vs mg/dl), and a higher bilirubin ( . vs . g/dl). transfusion requirements of ffp was higher in acute liver failure ( ae . ml/kg) compared to metabolic liver disease ( . ae . ml/kg) and biliary atresia ( . ae . ml/kg); (p = . ). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. d-s - distler pb , slaper-cortenbach i and ashford p iccbba, san bernardino, united states of america university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:( ) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').( ) hyphenated class names will be replaced ( ) tc and therapeutic terminology will be replaced ( ) modifiers will be replaced with attributes providing the same information ( ) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes. d-s - kordelas l , rebmann v , ludwig a-k , radtke s , beelen dw , giebel b and horn p department of bone marrow transplantation, university hospital essen, essen, germany institute for transfusion medicine, university hospital essen, essen, germany university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a -years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il- , il- and il- was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord- - ovj xg authors: kurita, junko; sugawara, tamie; ohkusa, yasushi title: forecast of the covid- outbreak, collapse of medical facilities, and lockdown effects in tokyo, japan date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ovj xg background: the number of patients of covid- in tokyo has been increasing gradually through the end of march, . object: support for policymaking requires forecasting of the entire course and outcome of the outbreak including the date of collapse of medical facilities if a lockdown is not initiated. moreover, the effects of a lockdown must be considered when choosing to initiate one. method: data of tokyo patients with symptoms during january − march , were used to formulate a susceptible−infected−recovered (sir) model using three age classes and to estimate the basic reproduction number (r ). based on the estimated r , we inferred outbreak outcomes including the date of collapse of medical facilities if a lockdown were not enacted. then we estimate the lockdown effects. results: results suggest r as . , with a % confidence interval of [ . , . ]. collapse of medical facilities can be expected to occur on april if no lockdown occurs. the total number of deaths can be expected to be half a million people. if a lockdown were enacted from april , and if more than % of trips outside the home were restricted voluntarily, then a collapse of medical facilities could be avoided. discussion and conclusion: the estimated r was similar to that found from other studies conducted in china and japan. results demonstrate that a lockdown with reasonable cooperation of residents can avoid a collapse of medical facilities and save . million mortality cases. the initial case of covid- in japan was that of a patient who showed symptoms when returning from wuhan, china on january , . as of march , , cases had been announced as infected in the community of japan, excluding asymptomatic cases, those for which the onset date or age was not reported, those infected abroad, and those infected on a large cruise ship: the diamond princess [ ] . in metropolitan tokyo, which has about million residents, symptomatic cases were identified as of march , . the entire course of the outbreak must be predicted to evaluate the necessary medical resources for policymaking. moreover, one must evaluate, as a worst case scenario, the collapse of medical facilities which can occur when medical needs far exceed the capacity of medical resources. under such circumstances, the case-fatality ratio (cfr) rises considerably. especially, the capacity of intensive-care-unit (icu) facilities is usually not so large. they are expected to be allocated quickly to patients. therefore, escalation of case mortality represents an important concern. to forecast these phenomena, we construct a simple susceptible-infected-recovered (sir) model for tokyo incorporating the necessary medical resources. then we predict whether a collapse of medical facilities occurs, in addition to the magnitude of mortality if a collapse were to occur. we applied a simple sir model [ , , ] with three age classes: children years old or younger, adults - years old, and elderly people years old or older. we assumed some protection of children [ ] : % of children were protected t infection [ ] . the incubation period was assumed to be equal for all people of the three age classes and following the empirical distribution inferred for the outbreak in japan. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint experiences of japanese people living in wuhan until the outbreak provide information related to mild cases because complete laboratory surveillance was conducted for them. during january -february , , japanese people returned to japan from wuhan. each had undergone a test to detect covid- ; of them, were found to be positive for covid- [ ] . of those , japanese people had exhibited mild symptoms; the other showed no symptom. moreover, two japanese residents of wuhan exhibited severe symptoms: one was confirmed as having contracted covid- . the other died, although no fatal case was confirmed as covid- by testing. in addition, two japanese residents of wuhan with mild symptoms were refused re-entry to japan even though they had not been confirmed as infected. if one assumes that the japanese fatal case in wuhan and that the two rejected re-entrants were infected with covid- , then severe cases, mild cases, and asymptomatic cases were found to exist among these japanese residents of wuhan. we therefore apply these proportions of asymptomatic cases to symptomatic cases in the simulation. assuming that the degrees of infectivity among the severe patients and mild patients are equal also for asymptomatic cases, half of the symptomatic cases can be assumed. this assumption about relative infectiousness among asymptomatic cases compared with symptomatic cases was used also in simulation studies for influenza [ ] [ ] [ ] [ ] [ ] . we sought to ascertain r to fit the number of patients during january - march and to minimize the sum of squared residuals among the reported numbers and the fitted values. its % confidence interval (ci) was calculated using , iterations of bootstrapping for the empirical distribution of epidemic curves. contact patterns among children, adults, and elderly people were estimated in an earlier study [ ] . we identified the following contact patterns: the share of children contacting with other children accounted for / of all contacts, contact with adults accounted for / , and contact with elderly people accounted for / ; the share of adults contacting children accounted for / of all all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint contacts; those contacting with other adults were / , and those contacting with elderly people were / ; elderly people contacting with children were / , with adults were / , and with other elderly people were / . we assumed that contact frequencies in the same age class decreased in the same proportion in all age classes if the tokyo metropolitan government (tmg) were to declare a lockdown in tokyo. however, contact frequencies among other age classes were assumed not to be changed by a lockdown because most of the contact with other age classes can be presumed to occur at home. contacts at home will probably be unaffected by a lockdown. experience in japan has revealed the pneumonia incidence in elderly covid- patients as . %. that among adults is . %. we used these ratios to assume ratios of severe cases to symptomatic cases. among children, no pneumonia case has been reported. only pneumonia cases were received hospital treatment. the length of hospitalization was assumed as days. of those severe cases, % were assumed to require the use of an intensive care unit (icu) and respirator for days. the case fatality rate among icu patients was assumed to be % from experience in tokyo up through march , . however, if patients requiring care at an icu or respirator cannot receive it, then we assumed that the cfr among them was %. we define the collapse of medical facilities as circumstances under which the necessary icu bed number becomes greater than % of all existing icu beds. in tokyo, there are icu beds. therefore, if the necessary number of icu beds becomes greater than , medical service at the facilities will collapse. the cfr among patients who need care at an icu but do not receive it is %. we used data of the covid- community outbreak of patients in japan who showed any symptom during january -march , in tokyo. we excluded some patients who had been infected abroad and who returned from abroad and those who were presumed to be infected persons all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint from the diamond princess. they were presumed not to be community-acquired in japan. published information about covid- patients with symptoms from the ministry of labour, health and welfare (mlhw) japan or tmg was usually affected adversely by some delay because of uncertainty during onset to visiting a doctor or in the timing of a physician's suspicion of covid- . therefore, published data of patients must be adjusted at least a few days. to adjust the data, we applied the following regression analysis. we set xt-k|t as the number of patients for whom the onset date was t-k published on day t. the dependent variables are the degree of reporting delay, where k>m for several m and k. here, m denotes the difference of the publishing dates between the two published. date t represents the publishing date of the latest publishing. the explanatory variables were /k, /m, and /km. the degree of reporting delay was estimated as [estimated coefficient of constant term] + [estimated coefficient of /k]/k, when m was sufficiently large and time had passed. therefore, this estimated degree of reporting delay multiplied by the latest published data is expected to be a prediction of the number of patients for whom the onset date was t-k. we used this adjusted number of patients in the latest few days, including those after vec was adopted. we used published data of , , and - march, provided by mlhw [ ] . first, we estimated r . then we predicted the peak date, total number of symptomatic cases and mortality cases, maximum number of newly infected symptomatic cases per day, beds, and icu beds. we also predicted the date of collapse of medical facilities. moreover, we predicted the effects of the lockdown from april and measured whether the medical system would collapse. then for cases of collapse, we inferred the date of collapse. we calculated the % ci through , bootstrapped distributions. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint all information used for this study has been collected under the law of infection control, japan. there is therefore no ethical issue related to this study. during january -march in tokyo, cases among children, cases among adults, and cases among elderly people were identified as community-acquired covid- for whom the onset date was published. figure depicts the empirical distribution of incubation period among cases for which the exposed date and onset date were published by mhlw. its mode and median were six days; the average was . days. table according all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint to the proportion of voluntary restriction of people leaving their home. a collapse of medical facilities could be prevented if more than % of trips outside of the home were restricted. the collapse of medical facilities could e postponed by three months if more than % but fewer than % of trips outside of the home going were restricted. we applied a simple sir model with three age classes including asymptomatic cases and assuming some proportion of children as protected. an earlier study [ ] [ ] [ ] estimated r for covid- as . - . in wuhan. our obtained r of . was very similar. however, one study revealed that r in japan up through february was just . [ ] . their evidence given was the number of secondarily infected people. of primary cases, they found that cases did not infect anyone. nevertheless, their findings must be considered carefully. they specifically examined only those patients who had been infected by the known confirmed cases. however, half of the patients had no link to any known confirmed case. they were probably counted as primary cases but not as secondary cases. they were infected by someone in the community. in fact, until february, there were infected persons in the community. of those, cases were unlinked. therefore, those unlinked cases were actually secondary cases that had not appeared as secondary cases in the figures of an earlier study [ ] . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint one might assume that the primary cases which proceeded to infect the unlinked secondary cases were the same as the primary cases in an earlier study. if so, then r should be . (= . + / ). alternatively, one can infer that the primary cases of the unlinked patients were not identified by a public health center (phc) and that their distribution of the secondary cases was the same as the figure in an early study. that would mean that primary cases infected secondary cases. therefore, the average number of secondary cases conditional on those which infected more than one person was . (= / ). presumably, the unlinked cases were infected by hypothetical primary cases. moreover, these hypothetical cases represented infection by other cases, and so on. therefore, cases infected cases which were not identified by a phc. in total, the average number of secondary cases is expected to be . (=( + )/( + )). therefore, the true number of r is expected to be in the range of . - . . these numbers are slightly smaller than other estimates, but far greater than one. moreover, on april , medical services can be expected to collapse. eventually, the cases ending in mortality will be approximately . million in tokyo if no lockout is conducted. the number of deaths will be about five times greater than the average number of deaths during an equivalent period. results also demonstrate that if a lockdown is initiated on april and if more than % of trips outside the home were restricted voluntarily, then collapse of medical facilities might be avoided. it is noteworthy that no law exists to enforce curfews in japan. therefore, a lockout would ask, not force, residents to avoid leaving their home voluntarily. consequently, cooperation with a lockout must achieve voluntary cooperation to a great degree. evidence related to compliance is all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint scarce because no lockdown has been conducted in japan to date. an exceptional study asked people about restricting movement outside the home if the government asked them to do it [ ] . results showed that . % would comply voluntarily with such a government request. collapse of medical facilities from covid- might be avoided in japan, even with no enforcement of a lockdown, because strong compliance can be expected. our unpublished research suggests that school closure since march decreased contact frequencies among children by %. voluntary event cancellation since february decreased it among adults by %. although a collapse of medical facilities has been postponed, it appears to be unavoidable if we apply these numbers to a lockdown effect. the sir model is too simple to incorporate households, firms or schools. it is a completely mixed model. it therefore ignores some difference inside and outside of those groups. it can adjust contact patterns to mimic some policies including lockdowns or school closures as in the present study. a model highlighting differences inside and outside of those groups is an individual-based model (ibm), which mimics movements and contacts of individuals. it can therefore evaluate behavioral changes of individuals directly [ ] [ ] [ ] [ ] ] . therefore, we must use ibm for evaluation of a lockdown instead of a sir model. no ibm exists for covid- but an ibm exists for pandemic influenza. especially, the most precise ibm, ribm, has been developed in japan using actual data of transportation [ , ] . it indicated that lockdown with % voluntary restriction to going out can reduce prevalence by percentage points for pandemic flu [ ] . therefore, it shared the same result that % voluntary restriction out of trips outside the home can avoid collapse of medical facilities with the present study for covid- . all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint we predicted outcomes of covid- and lockdown effects in tokyo. we estimated a collapse of medical facilities in late april and about . million cases with mortality in tokyo if a lockdown were not applied. we estimated the effects of lockdown enacted from april and reasonable compliance with voluntary restrictions on trips outside the home. such a lockdown might avoid a collapse of medical facilities. however, it is noteworthy that such a lockdown might continue until a vaccine for covid- can be developed. costs of a lockdown would be huge if it were to last for more than a half of year. its cost-effectiveness is expected to represent a concern in this case. this study represents the authors' opinion. it does not reflect any stance of our affiliation. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint note: "collapse of medical facilities" is defined as necessary icu beds greater than % of capacity. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint notes: bars represent the number of patients by incubation period among cases whose exposure date and onset date were published by the ministry of labour, health and welfare, japan. all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/ . / . . . doi: medrxiv preprint japan ministry of health, labour and welfare. press releases of domestic situation preliminary evaluation for as voluntary event cancellation as counter measure to covid- outbreak in japan as of insignificant effect of counter measure for coronavirus infectious disease - in japan japan ministry of health, labour and welfare strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source mitigation strategies for pandemic influenza in the united states strategies for mitigating an influenza pandemic simulation model of pandemic influenza in the whole of japan social contacts, vaccination decisions and influenza in japan from to : a data-driven analysis in the early phase of the outbreak the reproductive number of covid- is higher compared to sars coronavirus all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges closed environments facilitate secondary transmission of coronavirus disease (covid- ) survey of pandemic behavior: to stay at home or not pandemic simulation: application of the mathematical model for infectious disease. gijutsuhyouronsha all rights reserved. no reuse allowed without permission author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the we acknowledge the great efforts of all staff at public health centers, medical institutions, and other facilities who are fighting the spread and destruction associated with covid- . key: cord- - y ioety authors: akiyama, tomohiro; hirata, takamichi; fujimoto, takahiro; hatakeyama, shinnosuke; yamazaki, ryuhei; nomura, tomohiro title: the natural-mineral-based novel nanomaterial ifmc increases intravascular nitric oxide without its intake: implications for covid- and beyond date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: y ioety there are currently no promising therapy strategies for either the treatment or prevention of novel coronavirus disease (covid- ), despite the urgent need. in addition to respiratory diseases, vascular complications are rapidly emerging as a key threat of covid- . existing nitric oxide (no) therapies have been shown to improve the vascular system; however, they have different limitations in terms of safety, usability and availability. in light of this, we hypothesise that a natural-mineral-based novel nanomaterial, which was developed based on no therapy, might be a viable strategy for the treatment and prevention of covid- . the present study examined if it could induce an increase of intravascular no, vasodilation and the consequent increase of blood flow rate and temperature in a living body. the intravascular no concentration in the hepatic portal of rats was increased by . nm over . s on average after its application. an ultrasonic doppler flow meter showed significant increases in the blood flow rate and vessel diameter, but no difference in the blood flow velocity. these were corroborated by measurements of human hand surface temperature. to our knowledge, this result is the first evidence where an increase of intravascular no and vasodilation were induced by bringing a natural-mineral-based nanomaterial into contact with or close to a living body. the precise mechanisms remain a matter for further investigation; however, we may assume that endothelial no synthase, haemoglobin and endothelium-derived hyperpolarising factor are deeply involved in the increase of intravascular no. humanity is facing a great turning point in terms of its evolution. the novel coronavirus disease pandemic caused by severe acute respiratory syndrome-coronavirus (sars-cov- ) has become a global health emergency. according to the covid- situation report published by the world health organization, on may , more than > . million confirmed cases and , deaths across the world had been attributed to covid- [ ] . the explosive rise in the there are currently no promising therapeutic strategies for either the treatment or prevention of covid- , despite the urgent need. while trials on sars-cov- genome-based specific vaccines (e.g., messenger ribonucleic acid (mrna)- by moderna, inc.; an adenovirus type- vector vaccine called ad -ncov by cansino biologics, inc.; a dna-based vaccine called ino- by inovio pharmaceuticals, inc. and beijing advaccine biotechnology co.; an adenoviral vector-based vaccine called chadox ncov- or azd by astrazeneca plc., the university of oxford and its spinout vaccitech ltd.; a lentiviral minigene vaccine called lv-smenp-dc and lentiviral vector modified artificial antigen presenting cells (aapcs) by shenzhen geno-immune medical institute) [ ] and therapeutic antibodies are currently being tested, these solutions are more long-term as they require thorough safety testing (e.g., the estimated study completion date of mrna- is june ). repurposing existing drugs previously designed for other virus infections and pathologies is also being examined as a rapid response measure to the emergent pandemic as the safety of most of these agents has already been tested. the agents can be divided into two categories depending on their target [ ] . one acts directly on the coronavirus, either by inhibiting the crucial viral enzyme responsible for genome replication (rna-dependent rna polymerase inhibitors such as remdesivir, favipiravir, ribavirin, sofosbuvir, galidesivir and tenofovir and viral protease inhibitors such as lopinavir/ritonavir, azithromycin, ivermectin and nelfinavir) or by blocking viral entry to human cells (virus-cell membrane fusion inhibitors such as apn (rhace ), chloroquine/hydroxychloroquine, teicoplanin and umifenovir). however, hydroxychloroquine and azithromycin combination could be lethal to covid- patients [ ] . the other is designed to modulate the human immune system, either by boosting the innate antiviral immune response (immunotherapies using natural killer cells and recombinant interferon) or by alleviating damage induced by dysregulated inflammatory responses (anti-inflammatory therapies using mesenchymal stem cells, intravenous immunoglobulin, anti-c monoclonal antibodies (e.g., eculizumab, siltuximab, tzls- , sarilumab and tocilizumab), anti-vegf-a monoclonal antibody (bevacizumab), anti-tnf monoclonal antibody (adalimumab), sars-cov- -specific neutralising antibodies, thalidomide, methylprednisolone and fingolimod). passive immunotherapy, using recovered patients' plasma, has been recommended for severe and critical cases of covid- , based on the presence of neutralising antibodies against sars-cov- ; however, concerns have been raised related to the potential risk for transfusion-transmitted infection and potential risk for severe disease due to antibody-dependent enhancement [ , ] . various other treatments and prevention strategies are also currently under investigation, including oxygen therapies such as hyperbaric oxygen, nitric oxide (no) and extracorporeal membrane oxygenation, convalescent plasma therapy, anticoagulant therapy, low-dose corticosteroid therapy, ace inhibitors, angiotensin ii type i receptor blockers, statins, antibiotics, polyclonal antibodies and other pharmacological agents. natural compounds including traditional herbal medicines [ , ] and nutritional interventions such as vitamins, omega- fatty acids, selenium, zinc and iron [ ] could also be considered for enhancing host immunity against covid- infection. the application of big data analysis is also emerging to systematically identify the potential agents for drug repurposing against sars-cov- [ ] . both agent efficacy and agent safety, including in terms of adverse drug reactions and drug-drug interactions, await further clinical confirmation. previous studies have proven the potential of several countermeasures, yet large-scale trials with a broader perspective remain necessary to respond to covid- . the present study proposes an additional strategy for the treatment and prevention of covid- by applying no. in the vascular system, no is produced by the endothelium, a single layer of cells that forms the inner lining of all blood vessels [ ] . endothelium-derived no has several different functions, one of which is vascular smooth muscle relaxation, resulting in vasodilation and a consequent decrease in blood pressure and increase in local blood flow [ ] ; these discoveries were awarded the nobel prize in [ ] [ ] [ ] . previous studies treating sars-cov-infected patients with inhaled no have shown pharmacological and antiviral effects of no [ ] [ ] [ ] , suggesting that no inhalation might be effective for covid- patients [ , ] . indeed, two clinical trials of inhaled no are being conducted in covid- patients [ , ] . meanwhile, balneotherapy (hot spring therapy), sauna therapy and thermal therapy (hyperthermia) also reportedly increase endothelial no synthase (enos), thus inducing vascular endothelial no production [ ] [ ] [ ] [ ] [ ] [ ] . however, hyperthermia may cause health issues such as heat stroke and cardiac morbidity. for example, there are , deaths in japan per annum during bathing, which accounts for > % of all sudden out-of-hospital deaths [ ] , while there are countries in which neither such therapies nor no inhalation are accessible. one potential breakthrough in terms of overcoming these existing challenges may be nanomedicine or nanoscience, which have been advancing since the s. different classes of nanomaterials such as polymeric nanoparticles or nanocapsules, liposomes, metallic nanoparticles, metal oxide nanoparticles, silica-based nanoparticles, quantum dots, peptide or carbon nanotubes and up-conversion nanoparticles have been used for controlled no delivery in medical applications [ ] . however, this requires introducing no-releasing nanomaterials into the human body, and the use of nanomaterials has become controversial due to health risks associated with their applications [ ] . meanwhile, a natural-mineral-based novel nanomaterial that is expected to have an effect similar to that of no therapy without its intake into the body has been available since . it was developed from japanese hot springs and named integrated functional mineral crystal (ifmc) by teikoku pharmaceutical co., ltd., japan [ ] . if it were able to sufficiently increase intravascular no, it might present a supportive treatment and prevention option for covid- since it is inexpensive and easy to use (just putting it or spraying it in a solution on the body). here, we demonstrate how the natural-mineral-based novel nanomaterial can induce an increase of intravascular no, vasodilation and the consequent increase in blood flow. the experimental object of this research is the natural-mineral-based novel nanomaterial ifmc by teikoku pharmaceutical co., ltd., osaka, japan [ ] . a solution of ifmc consists of haematite (fe o ), olivine (mg sio and fe sio ), rhodolite (mnco ), zincite (znco ) and additives such as deionized water, ethanol, methylparaben and sodium metabisulphite. the physical characteristics of ifmc were first investigated using a zeta-potential and particle size analyser, as well as scanning electron microscopy (sem) coupled with an energy-dispersive x-ray spectrometer (eds). then, the intravascular no concentration was measured continuously using a no sensor in the hepatic portal of rats to test whether ifmc could induce an increase of intravascular no. since the long-term continuous measurement of intravascular no was impossible, complementary tests were conducted to determine whether ifmc could increase the surface temperature, blood flow rate, velocity and vessel diameter in the human body. all experimental human and animal protocols were respectively reviewed and approved by the medical research ethics committee (approval no. ) and animal experimentation committee (approval no. ) of tokyo city university, japan. all participants provided voluntary written informed consent prior to participation. we conducted a monitoring test prior to the present study. no problem was found in the monitoring results when spraying ifmc solution on the body or when wearing clothes impregnated with ifmc over several months. since the naturally occurring substances that make up ifmc are hot-spring ingredients, which are completely different from chemical substances that may have mutagenic properties, we believe there would be no side effects for small animals or humans, even if they were taken directly. the particle size distribution in the ifmc solution was measured using a zeta-potential and particle size analyser (els z- , otsuka electronics co., ltd., osaka, japan). dynamic light scattering (photon correlation method) and the contin method were used for the measurement. the measured temperature was . • c when the scattering angle was o . the result showed no scattering intensity. the possible reasons for this include: ( ) there were no particles; ( ) the particles were too small to measure (els z- cannot measure particles smaller than . nm); ( ) substances were present in the solution as ions and were therefore not present as minute particles. we think that mineral components exist as ions in ifmc solution since crystals were obtained when it dried up. sem images of ifmc were taken using an ultra-high-resolution cold-field emission scanning electron microscope fe-sem (su , hitachi high-tech co., tokyo, japan), and eds spectra were obtained using an energy-dispersive x-ray spectroscope (aztecenergy x-max , oxford instruments, oxfordshire, u.k.). ifmc was dropped onto a microscope slide with a pipette and desiccated using a hot plate. the dried deposit was then secured to an fe-sem carbon tape. the measurement conditions of fe-sem were twofold: ( ) magnification, × - ; accelerating voltage, kv; ( ) magnification, × , - , ; accelerating voltage, kv. a silicon-drift detector was used to collect fluorescence spectra. a catheter-type no sensor (inc- , inter medical co., ltd., aichi, japan) was used with an integrated working electrode and a reference electrode (diameter, . mm; sensitivity, pa µm − ). the electric current generated by an electrochemical reaction of no was fed into a head amplifier (haec-a, b × /× or × /× , inter medical co., ltd., aichi, japan) and measured using an electrochemical amplifier (imec- , inter medical co., ltd., aichi, japan) with the voltammetry method. imec- / a is currently the most improved measurement system for no, oxygen, dopamine, glutamine acid and vitamin c both in vivo and in vitro [ ] [ ] [ ] [ ] [ ] . female slc-wistar rats ( - weeks) were obtained from japan slc, inc. all rats were housed individually and maintained on an alternating h light/dark cycle at ± • c. after a five day acclimatisation period, the rats were randomly divided into positive (use of ifmc solution; n = rats for three samples) and negative control (placebo, i.e., with normal saline solution; n = rats for six samples), with ad libitum access to drinking water. the small number of samples was due to the necessity of special surgical techniques; if this failed, the rats would quickly die due to heavy bleeding. therefore, the present study limited the number of samples to secure the experiment from potential failures. all rats were anaesthetised using a cocktail of butorphanol tartrate ( . ml), midazolam ( . ml), medetomidine hydrochloride ( . ml) and physiological saline solution ( . ml) injected intraperitoneally at ml kg − body weight and were maintained in their anaesthesia-induced unconscious state throughout the experiment. the no sensor was inserted into the hepatic portal where blood vessels enter and leave the liver (portal vein, hepatic artery), bile ducts, lymph ducts and nerves all pass. this site was chosen because changes in intravascular no concentration can be measured directly and stable measurement of arterial blood flow can also be made without damaging the artery itself. the chests of the rats in both groups were covered by kimwipes r with ml ifmc solution or normal saline solution to test whether ifmc induces an increase of intravascular no. statistical significance was estimated based on the increase of intravascular no concentration. an ultrasonic doppler blood flow meter (qfm- , hadeco inc., kanagawa, japan) was used to measure the blood flow rate, velocity and vessel diameter of the human brachial artery non-invasively. the measurement conditions were as follows: ultrasonic frequency, . mhz (blood flow velocity measurement) and . mhz (blood vessel diameter measurement); ultrasonic output, ≤ mw cm − (peak time average output), ≤ w cm − (peak pulse output), and ≤ w cm − (maximum output); blood flow range, . - ml s − ; blood flow rate, - cm s − ; blood vessel diameter, - mm; depth, - mm; blood flow rate measurement accuracy, ± %. the research participants were four males aged - years. the measurements were conducted times in total. the upper arm of each participant was covered by ifmc-added or ifmc-free textiles ( % polyester) to test whether ifmc could increase the blood flow rate, velocity and vessel diameter of the human body. statistical significance was determined using a two-sided paired t-test. the research participant was a -year-old female who felt coldness of the hands and feet on a regular basis. the measurement was started right after the solution of ifmc was sprayed on the surface of the participant's hand. changes in the hand surface temperature were measured using an auto-range thermo tracer (th mr, nippon avionics co., ltd., tokyo, japan) at min intervals during a total measurement period of min. statistical significance was determined based on pixel values using a two-sided paired t-test. and then gradually decreased. the peak value and duration of the increase were comparable to those of other reports, which used acetylcholine [ ] [ ] [ ] [ ] . the lower peak value and shorter duration of the increase in this study were probably due to the small size of the rats. the minor spikes right before the increase were probably because of the relaxation of the vascular smooth muscle. indeed, a similar spike was observed when the sensor was moved slightly. the result is, to our knowledge, the first evidence of inducing an increase of intravascular no by bringing the natural-mineral-based nanomaterial into contact with or close to a living body without pharmacological intervention or physical intervention (e.g., acupuncture, massage therapy or balneotherapy). figure shows changes in the blood flow rate, velocity and vessel diameter in the brachial artery. the ranges of the systolic and diastolic peaks of each parameter were comparable to the values of another report [ ] . compared with ifmc and placebo applications, significant increases were observed in blood flow rate and vessel diameter, but there was no significant difference in blood flow velocity. statistical significance values of blood flow rate, velocity and vessel diameter between placebo and ifmc applications were . , . and . , respectively. the significant increases in blood flow rate and vessel diameter were due to vasodilation derived from the increase in intravascular no. the minor change in blood flow velocity was due to the improvement of vascular elasticity followed by relaxation of vascular smooth muscle. the result (i.e., the increase of the blood flow rate due to vasodilation without blood flow velocity change) indicates improvement of the neuroimmunoendocrine system [ ] [ ] [ ] . figure shows changes in the surface temperature of a hand (back) observed by thermography. before spraying the ifmc solution, the mean surface temperature of the entire hand (back) was . • c, and that of the fingers was . • c, indicating that the peripheral circulation was poor. after min, the surface temperature decreased due to vaporisation. however, min later, some of the relatively thick blood vessels in the hand began to swell, and after min, almost all of the thick blood vessels on the back of the hand stood out. meanwhile, after min, the temperature of the peripheral blood vessels in the fingertips increased. the surface temperature of the area close to the blood vessels rose. the temperature of the entire hand had increased at min. the clear behavioural transition from thick to thin blood vessels was obviously due to vasodilation. the mean surface temperature of the entire hand (back) was . • c and that of the fingers . • c at min. the differences in the surface temperature of the entire hand and fingers before and min after ifmc application were significant. the increase in the fingers (+ . • c) was larger than that in the entire hand (+ . • c). the difference is evidence of vasodilatation and the increase of blood flow rate in the peripheral blood vessels. the present study confirmed that the natural-mineral-based novel nanomaterial ifmc, with a size of tens of nanometres (figure ), could induce an increase of intravascular no (figure ) , vasodilation (vessel diameter) and blood flow rate in a living body (figure ) , as well as an increase of the surface temperature of a hand including fingers ( figure ). despite the lower peak value and shorter duration of the increase in intravascular no concentration (figure ) due to the small size of the rats, which were comparable to those of other reports [ ] [ ] [ ] [ ] , the increase of human hand surface temperature lasted at least min (figure ) , suggesting that the effect of ifmc was significant. further, the fact that the increase of the mean surface temperature of the fingers (+ . • c) was larger than that of the entire hand (+ . • c) at min after ifmc application ( figure ) suggested that the vasodilatation and increase of blood flow rate in the peripheral blood vessels were caused by no as an endothelium-derived relaxing factor (edrf). this is corroborated by the increase in the diameter of blood vessels in the brachial artery and the increase in blood flow ( figure ) . the results are, to our knowledge, the first evidence of inducing an increase of intravascular no and vasodilation by bringing the natural-mineral-based nanomaterial into contact with or close to a living body, without pharmacological intervention or physical intervention (e.g., acupuncture, massage therapy or balneotherapy). the precise mechanism of the induction of intravascular no remains uncertain; however, there are two possibilities. the primary possibility is no synthesis. it is well known that the activity of enos followed by no production in the vascular wall is regulated by receptor-mediated signalling, such as vascular endothelial growth factor receptor (vegfr ) and acetylcholine (ach), as well as protein partners, including heat shock protein (hsp ), calmodulin (cam) and caveolin- (cav- ) [ ] . the receptor-mediated biological response includes a time lag between actions of chemical mediators (messenger substances) and receptor reaction. in contrast, ifmc caused its effect within min (figures and ) . the quick response indicates the possibility that ifmc principally involves another mechanism, not receptor-mediated action. therefore, we assume haemoglobin (hb) is deeply involved in the phenomenon. it is known that hypoxic vasodilation does not require the synthesis of edrf/no [ ] . hb and no independently fulfil diverse and complex physiological roles, while together, they subtly modulate microvascular perfusion in response to second-by-second changes in local metabolic demand, contributing to hypoxic vasodilation [ ] . among the candidate molecular mechanisms, only s-nitrosohemoglobin (sno-hb) directly fulfils the physiological requirements [ ] . thus, no is transported by red blood cells to microvascular sites of action in protected form as an s-nitrosothiol on the highly conserved hb β- cys residue, invariant in birds and mammals [ ] . meanwhile, no as a vascular edrf is a gaseous signal substance and considered to be freely diffused between vascular endothelial cells and vascular smooth muscle cells. however, no does not freely diffuse between the endothelial cells and smooth muscle cells of blood vessels [ , ] . the oxidation state of hb is harnessed to control how much no reaches the smooth muscle [ ] . hb has the structure of haem iron with four iron porphyrin units combined with a globin protein. hb has the ability to combine with oxygen molecules (o). therefore, hb not only plays a role of transporting oxygen (o ), but also is deeply involved in the control of no transport. it is well known that the transport of o and no in blood is facilitated by the spin transition of iron atoms in hb. the ferrous ions in haem iron are five-coordinated high-spin complexes; however, they become six-coordinated low-spin complexes when oxygen molecules are added. ferrous ions move to the centre of the porphyrin ring by changing the spin state of d-electrons from high to low spin. this movement at the molecular level changes the structure of hb to enhance the affinity or adsorbability of the other three haem-iron complexes to oxygen molecules. in other words, the electronic structure of d-electrons controls the uptake and release of oxygen molecules. therefore, we may form the following hypothesis: the affinity of hb for no might be reduced by bringing ifmc into contact with or close to a living body. in light of this, magnetic evaluation using a magnetic probe such as a positive muon may clarify the mechanism of action by ifmc on hb. indeed, the mixture of hb as an organic compound and ifmc as an inorganic nanomaterial, which contains transition metal compounds exhibiting antiferromagnetism and weak ferromagnetism, may trigger several different types of interactions. one of the potential methods of clarifying the mechanism is to estimate the magnitude of the internal magnetic field that the muon experiences when ifmc is irradiated with muon beams. specifically, we would be able to examine the relation between the spin properties of ifmc through muon experiments using a high-energy accelerator and the spin transition changes of iron atoms involved in the changes in the affinity or adsorbability to the o/no molecule of haem iron in hb. through the experiments, it would be possible to examine ifmc's remote action or action over a distance between atoms and atoms or between atoms and molecules. in the fields of semiconductor engineering and spintronics, there are previous studies claiming that simple antiferromagnetic insulators may transmit spin information over a distance of several tens of micrometres [ ] . the proposed examination might be the world's first experiment to uncover the spin transmission over a much longer distance. meanwhile, the potential benefits of ifmc are probably not limited to the induction of intravascular no. it is known that there are three different endothelium-derived vasorelaxation factors involved in maintaining vascular homoeostasis: prostacyclin (pgi ), no [ ] [ ] [ ] ] and endothelium-derived hyperpolarising factor (edhf) [ ] . they are all derived from vascular endothelium, whereas they differ in their production processes: no is produced from arginine by enos; edhf is synthesised by a variety of stimulants, such as agonists, shear stress and acetylcholine in a ca + /calmodulin-dependent manner [ ] ; and prostacyclin is produced by cyclo-oxygenase and prostacyclin synthase enzymes [ ] . it has been shown that no and edhf fulfil complementary roles. no mediates vascular relaxation of relatively large conduit arteries (e.g., aorta and epicardial coronary arteries), whereas edhf plays an important role in modulating vascular tone in small, resistance arteries (e.g., small mesenteric arteries and coronary microvessels) [ ] . the vascular tone can be regulated by an edhf-like mechanism in the absence of no [ ] . the clear behavioural transition from thick to thin blood vessels (figure ) suggests that ifmc influences not only the induction of intravascular no, but also the production of edhf. in addition, the effect of ifmc on neuroimmunoendocrine system improvement, indicated by the increase of blood flow rate due to vasodilation without blood flow velocity change ( figure ) , is also worthy of further examination. thus, the precise mechanism and other potential benefits of ifmc are possible future research topics. however, the limited findings of the present study provide insight into the treatment and prevention of covid- . the increase of intravascular no (figure ) can be expected to have both pharmacological and antiviral effects. the pharmacological actions of no are twofold: vascular and nonvascular smooth muscle relaxation and regulation of airway function and the pathophysiology of inflammatory airway disease [ ] . no has also been shown to have antiviral actions, such as an inhibitory effect on the synthesis of viral protein and rna, including the replication cycle of sars-cov [ ] [ ] [ ] . in general, the action mechanism of inhaled no has been thought to be pulmonary vasodilation and the consequent improved oxygenation in the blood of the lungs, which kills the virus, as it does not do well in a high-oxygen environment. ignarro further added that no also interacts directly with the virus to kill it and/or inhibit its replication, and no inhalation is more effective as an antiviral agent than simply as a vasodilator in pulmonary circulation [ ] . the proposed natural-mineral-based novel nanomaterial might be added as an supportive treatment and prevention option for covid- , since it is inexpensive and easy to use (just putting it or spraying it in a solution on the body). given its advantages, it has the potential to overcome the limitations of inhaled no therapy, such as the cost and availability of devices and skilled medical staff. the health risks of the existing no-releasing medical nanomaterials might no longer be a matter of concern, since ifmc does not require intake into the human body. it is probable that the increase of intravascular no concentration ( figure ) is not abnormal; however, quantitative examination of the increase and its persistence in the human body is necessary. nevertheless, there is still an urgent need for promising therapy strategies for both the treatment and prevention of covid- . to summarise, our inter-and trans-disciplinary approach revealed that the natural-mineral-based novel nanomaterial ifmc can induce an increase of intravascular no, vasodilation and blood flow rate, as well as an increase of hand surface temperature in a living body. therefore, it might become a supportive treatment and prevention option for covid- . the precise mechanisms remain a matter for further investigation; however, we may assume that enos, hb and edhf are deeply involved in the increase of intravascular no. the remaining research agendas are to ( ) examine if ifmc can induce an increase of intravascular no and improve long-term blood circulation in humans, ( ) clarify how intravascular no is induced by bringing ifmc into contact with or close to a living body, ( ) examine ifmc's remote action or action over a distance between atoms and atoms or between atoms and molecules, ( ) explore other potential benefits of ifmc, ( ) explore the possibility of other natural-mineral-based nanomaterials, ( ) clinically examine whether ifmc is effective in the treatment and prevention of covid- and ( ) explore urgently and collectively a series of promising therapy strategies for both the treatment and prevention of covid- based on larger scale trials from a broader perspective. funding: no specific funding was received from the public, commercial or not-for-profit sectors to carry out the work described in this article. covid- ) situation report- yuval noah harari: the world after coronavirus covid- lockdowns cause global air pollution declines with implications for public health risk covid- causes record decline in global co emissions near-death experience as a probe to explore (disconnected) consciousness near-death experience in survivors of cardiac arrest: a prospective study in the netherlands the psychological correlates of decreased death anxiety after a near-death experience: the role of self-esteem, 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and l-arginine/adma ratio repeated sauna therapy attenuates ventricular remodeling after myocardial infarction in rats by increasing coronary vascularity of noninfarcted myocardium experimental investigation in rats to identify the cause of sudden death during bathing in japan small molecules for great solutions: can nitric oxide-releasing nanomaterials overcome drug resistance in chemotherapy? potential applications and human biosafety of nanomaterials used in nanomedicine targeted increase in cerebral blood flow by transcranial near-infrared laser irradiation real-time monitoring of liver damage during experimental ischaemia-reperfusion using a nitric oxide sensor effect of adenoviral catalase gene transfer on renal ischemia/reperfusion injury in rats oral treatment with herbal formula b alleviates penile toxicity in aging mice with manganism treatment of cardiac disease by inhalation of atmospheric pressure plasma effects of angiotensin ii on no bioavailability evaluated using a catheter-type no sensor evaluation of coronary endothelial function by catheter-type no sensor in high-fat-diet-induced obese dogs measurement of acetylcholine-induced endothelium-derived nitric oxide in aorta using a newly developed catheter-type nitric oxide sensor evaluation of bioavailability of nitric oxide in coronary circulation by direct measurement of plasma nitric oxide concentration flow velocity and flow volume measurements in the extracranial carotid and vertebral arteries in healthy adults: reference data and the effects of age regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action , , , -tetrachlorodibenzo-p-dioxin (tcdd) induces microglial nitric oxide production and subsequent rat primary cortical neuron apoptosis through p /jnk mapk pathway neuronal control of skin function: the skin as a neuroimmunoendocrine organ nitric oxide: what's new to no? hypoxic vasodilation does not require nitric oxide (edrf/no) synthesis how do red blood cells cause hypoxic vasodilation? the sno-hemoglobin paradigm endothelial cell expression of haemoglobin α regulates nitric oxide signalling hemoglobin-mediated nitric oxide signaling. free radic tunable long-distance spin transport in a crystalline antiferromagnetic iron oxide no news is good news acetylcholine releases endothelium-derived hyperpolarizing factor and edrf from rat blood vessels important role of endothelium-derived hyperpolarizing factor in shear stress-induced endothelium-dependent relaxations in the rat mesenteric artery milrinone enhances relaxation to prostacyclin and iloprost in pulmonary arteries isolated from lambs with persistent pulmonary hypertension of the newborn crucial role of nitric oxide synthases system in endothelium-dependent hyperpolarization in mice endothelium-derived hyperpolarizing factor and vascular function acknowledgments: microanalyses (sem and eds) of ifmc were conducted by dr. a. yoshida, ms. e. shindo and mr. n. hamamura (interdisciplinary research center for nano science and technology, tokyo city university, japan). other measurements, including intravascular no concentration of rats, surface temperature, blood flow rate, velocity and vessel diameter of the human body, were carried out by prof. a. mori (graduate school of integrative science and engineering, electrical engineering and chemistry tokyo city university, japan). the authors declare no competing interests. the following abbreviations are used in this manuscript: key: cord- -xg foch authors: tanaka, yoshikazu; sato, yuka; osawa, shuichi; inoue, mai; tanaka, satoka; sasaki, takashi title: suppression of feline coronavirus replication in vitro by cyclosporin a date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: xg foch the feline infectious peritonitis virus (fipv) is a member of the feline coronavirus family that causes fip, which is incurable and fatal in cats. cyclosporin a (csa), an immunosuppressive agent that targets the nuclear factor pathway of activated t-cells (nf-at) to bind cellular cyclophilins (cyp), dose-dependently inhibited fipv replication in vitro. fk (an immunosuppressor of the pathway that binds cellular fk -binding protein (fkbp) but not cyp) did not affect fipv replication. neither cell growth nor viability changed in the presence of either csa or fk , and these factors did not affect the nf-at pathway in fcwf- cells. therefore, csa does not seem to exert inhibitory effects via the nf-at pathway. in conclusion, csa inhibited fipv replication in vitro and further studies are needed to verify the practical value of csa as an anti-fipv treatment in vivo. coronaviruses are single-stranded rna viruses that generally cause respiratory or intestinal infections such as severe acute respiratory syndrome (sars) in humans and transmissible gastroenteritis (tge) in pigs. feline coronaviruses (fcov) have been classified into two biotypes, comprising the ubiquitous feline enteric coronavirus (fecv) and infectious peritonitis virus (fipv). the widely accepted theory in vitro is that fipv arises by mutation of parental fecv in the gastrointestinal tract of infected cats, then systemically spreads and causes fip that is fatal in cats [ , ] . although the mutation sites are not fully understood, some accessory genes are candidates for the site of the critical mutations responsible for fip [ , ] . approximately % of all purebred cats are infected with feline coronavirus and among these, - % develop the classical symptoms of effusive/ wet fip, the non-effusive/dry form of fip, or a combination of the two [ ] . many strategies have been developed to cure fip. interferon ω inhibits fipv in vitro but is ineffective in vivo [ ] . various other immunosuppressants, such as glucocorticoids and cyclophosphamide, have been studied, but although these drugs prolong life, the outcome of fipv infection remains fatal [ ] . thus, an effective vaccine and therapeutic medicine against fipv are still needed. investigators have reported that the immunosuppressant cyclosporin a (csa) can suppress the genomic replication and transcription of several viruses [ ] [ ] [ ] [ ] . cyclophilins (cyp) are cellular factors with high affinity for csa [ ] and comprise a family of cellular peptidyl-prolyl cis-trans isomerases (ppiases) that catalyze the cis-trans interconversion of the amino-terminal of peptide bonds to proline residues, thus facilitating changes in protein conformation as a chaperone protein [ ] . cyclosporin a blocks both the enzymatic activities of cyp that lead to the calcineurin (cn)-nf-at and p-glycoprotein pathways. the capsid protein of human immunodeficiency virus type (hiv- ) possesses a cyclophilin a (cypa) binding site for incorporation into the virion [ , ] . a csa-induced reduction in cypa inhibits transport of the reverse-transcribed viral genome to the nucleus [ ] . cyclophilin b (cypb) is another target of csa that promotes hepatitis c virus (hcv) replication by regulating the rna-binding ability of the hcv ns b protein. in addition, cypa facilitates mouse cytomegalovirus (mcmv) replication and cypb is required for the infectious entry of human papillomavirus type . here, we show that csa inhibits intracellular replication of the fipv genome and viral protein expression in vitro independently of the nf-at pathway. felis catus whole fetus- (fcwf- ; american type culture collection, va, usa) cells were maintained in dulbecco's modified eagle's medium (d-mem, sigma-aldrich, tokyo, japan) supplemented with % fetal bovine serum (jrh, nissui, tokyo, japan). we propagated fipv ( - strain; a gift from dr tsutomu hodatsu, kitasato university, japan) in fcwf- cells and then purified them by linear sucrose gradient ultracentrifugation. we inoculated fcwf- cells with fipv - at a multiplicity of infection (moi) of plaque-forming unit (pfu) per cell to study their effects on fipv infection. after adsorption for h at °c, the medium containing the virus was removed, and the cells were rinsed three times with phosphate-buffered saline [pbs (−)] and incubated with or without various concentrations of csa (sigma-aldrich), cyclosporin h (csh; cosmobio, tokyo, japan) and fk (sigma-aldrich) for h. the cells were then processed for photography. the cells were adsorbed with fipv, rinsed three times with pbs (−), overlaid with dmem containing % fetal bovine serum and % agarose s (nippon gene, toyama, japan). after a -h incubation, the cells were stained with giemsa solution and the plaques were counted. we isolated the nucleocapsid (n) gene of fipv from the plasmid pbrdi (provided by dr peter j. m. rottier, institute of biomembranes, utrecht university, the netherlands) containing the fipv - genome, using the polymerase chain reaction (pcr) with the primers '-acaaggac gacgacgacaaggccacacagggacaacgcg- ' and '-ccggaattcttagttcgtaacctcatcaa- ' for the first amplification. the products of the first amplification were purified by electrophoresis and gel extraction, and then a second pcr proceeded with the purified products and primers containing a flag-tagged sequence: ( '-gccaccatggactacaaggacgacgacgacaag- ' and '-ccggaattcttagttcgtaacctcatcaa- '). the second pcr products were cut with nco i and eco ri and subcloned into the sites between nco i and eco ri of the ptri-ex . vector (novagen, takara bio, kyoto, japan) to express flag-tagged n protein. the fcwf- cells were infected at an moi of pfu per cell and then incubated with or without csa, csh or fk . the medium was removed at h post-infection, and rnaiso-plus (takara bio) was added to the cells for rna preparation according to the manufacturer's protocol. total rna ( ng) was reverse-transcribed using the pri-mescript rt-pcr kit (perfect real time; takara bio). viral cdna were quantified by real-time pcr using the forward and reverse primers to the fipv-n gene ( '-tggccacacagggacaac- ') and ( '-agaac gaccacgtcttttggaa- '), and the taqman probe (fam-gttgca gcacagccagcataaacaa-bhq- ). reaction mixtures were prepared according to the manufacturer's protocol using premix extaq (takara bio), and sequences were amplified using a sequence detection system (applied biosystems, tokyo, japan) under the following cycling conditions: initial denaturation at °c for s and cycles at °c for s and °c for s each. complementary dna to the fipv-n gene was cloned into the ptri-ex . vector, which was serially diluted to provide standards for fipv gene quantification. the viral rna copy number was normalized using the feline β- -microglobulin (β m) gene (genbank; nm_ ). the β m gene derived from fcwf- cells was cloned by pcr amplification using the following primers: fβ m-f '-ggcgcgttttgtggtcttggtc- ' and fβ m-r '-cacttaacgaccttgggctc- '. the amplified pcr products were subcloned into ptac- plasmids (biodynamics laboratory inc. tokyo, japan) to provide standards for the β m gene. we then quantified the feline β m gene by real-time pcr using the forward ( '-cgcgttttgtggtcttggtcttggt- ') and reverse ( '-aaacctgaacctttggagaatgc- ') primers for the β m gene and detected the gene using the taqman probe, tamra-cggactgctctatctgtc ccacctgga-bhq- . luciferase activities were quantified using pgl . [luc p/nfat-re/hygro] (promega, tokyo, japan), prl-sv vectors for the nf-at response assay and the interferon stimulation response was determined using the plasmid pisre-tk hrluc (f) provided by riken bioresource center (tsukuba, japan) and the pgl promoter (promega). both reporter assays proceeded using the dual-luciferase reporter assay system (promega). briefly, the two reporter plasmids were co-transfected into fcwf- cells with or without csa or fk for each assay. recombinant feline ifnα (pbl biomedical laboratories, nj, usa) was added at a concentration of units/ml to the culture medium to evaluate the response to interferon (ifn). total cell lysates were prepared with reporter lysis buffer provided with the dual-luciferase reporter assay system at h before the assay. luciferase activities were quantified in triplicate assays using a lumat lb (berthold technologies, tokyo, japan). the cell membranes were disrupted with cell lysis buffer [ mm tris-hcl, ph . , mm ethylenediamine tetraacetic acid (edta), % np- , . m nacl] including complete mini (roche diagnostics, tokyo, japan) at h after infection. the cell lysates were resolved by electrophoresis on - % bis-tris gels (invitrogen, ca, usa) and western blotted onto immobilon-p membranes (millipore, tokyo, japan). non-specific protein binding was blocked with % non-fat dry milk for h and then the membranes were incubated with anti-feline coronavirus nucleocapsid (n) antibody (fipv - ; mybiosource, ca, usa) or anti-β-actin (sigma-aldrich) for h. antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidaseconjugated anti-mouse igg antibody (promega) followed by an enhanced chemiluminescence substrate (supersignal west femto maximum sensitivity substrate; thermo scientific, tokyo, japan) according to the manufacturer's protocol. we assayed wst- to evaluate cytotoxicity using the cell counting kit- (dojin chemical inc., wako, japan) according to the manufacturer's directions. statistical significance was determined using the student's t test. for all data analyzed, a significance threshold of p < . was assumed. the values are expressed in some figures as means ± standard deviation (sd). we initially assessed the effects of csa on fipv rna replication using cytotoxicity assays. cyclosporin a at concentrations of - . μm (cytotoxic dose , . ± . μm) did not affect fcwf- cell viability ( figure a ) and dose-dependently reduced the numbers of fipv plaques ( figure b) , whereas μm csa was slightly cytotoxic. cyclosporin a blocks both the enzymatic activities of cyp that lead to the calcineurin (cn)-nf-at and the p-glycoprotein pathway. we therefore assessed the effect of various concentrations of fk , which also blocks the nf-at pathway, on cell viability to confirm that csa inhibited fipv through this pathway. the cytopathic inhibitory effects did not significantly differ in the presence or absence of . - μm fk ( figure b ) and cell viability was not affected by μm fk (figure a ). quantitative rt-pcr showed that . - μm csa dose-dependently suppressed fipv rna replication, whereas fk did not exert significant inhibitory effects, except at μm fk (approximately % reduction compared to μm fk , p < . ; figure a ). western blotting showed that csa, but not fk dosedependently decreased fipv-n protein ( figure b) . we then examined whether the suppressive effects of csa on fipv replication depend on the inhibitory nf-at pathway or p-glycoprotein pathway by incubating fipv-infected cells with csh, which specifically blocks the p-glycoprotein pathway. the results show that no inhibition occurred (data not shown). to determine whether the action of csa and fk involves activation of interferon-stimulated gene responses in fcwf- cells, the isre-luciferase reporter plasmid, pisre-tk hrluc (f) and pgl promoter plasmid as a normalization-control plasmid were transfected into fcwf- cells and cultured with feline interferon α, csa or fk . the results of the dual-luciferase assay showed that none of these factors significantly affected luciferase activities at h after transfection. these results indicate that fcwf- cells are unresponsive, even to interferon α ( figure a) . consequently, the action of csa on intracellular fipv replication does not involve the activation of interferonstimulated genes on fcwf- cells. moreover, to evaluate the effects of csa and fk on the calcineurin-nf-at pathway in fcwf- cells, the nf-at luciferase reporter plasmid, pgl . [luc p/nfat-re/hygro] and prl-sv as a normalization-control plasmid were transfected into fcwf- cells that had been incubated with csa or fk . neither csa nor fk affected nf-at luciferase activities in fcwf- cells ( figure b ). these findings show that csa does not influence the nf-at pathway in fcwf- cells and that the inhibition of fipv rna replication by csa is independent of the calcineurin nf-at pathway. we discovered that csa inhibits intracellular fipv replication in vitro. the results of qrt-pcr and western blotting showed that viral proliferation was strikingly inhibited at csa concentrations between . and μm. the results of cell viability assays showed that μm csa was slightly cytotoxic. the inhibitory effects at this concentration should therefore be considered in the light of cytotoxicity. in contrast, immunosuppressive fk did not inhibit fipv replication except at μm according to the qrt-pcr findings. however, the inhibitory effects of . - μm fk did not significantly differ on western blots and the number of plaques did not significantly differ within the same concentration range of fk . these findings might be related to the difference of the analytical detection sensitivity between qrt-pcr and western blot assays. cyclosporin a binds cyclophilins, whereas fk binds cellular fkbp. each complex independently inhibits the phosphatase activity of calcineurin that mediates the nf-at pathway, which is critical to the expression of cytokines and their receptors [ , ] . the results of nf-at reporter assays indicate that neither csa nor fk influenced nf-at activities on fcwf- cells under our experimental conditions. thus, the antiviral activity against fipv is not involved in the suppression of gene responses regulated by nf-at but instead is exerted through distinct mechanisms that are independent of fk . pfefferle et al. recently described that sarscoronavirus nsp overexpression increases signaling through the nf-at pathway and enhances the induction of interleukin [ ] . our data were somewhat inconsistent with these findings. the discrepancy might be explained in part by the fact that their experimental system included the addition of -o-acetylphorbol -myristate (pma) and ionomycin to the culture medium. further knockdown studies of the nf-at gene would clarify its role in fipv proliferation. furthermore, ifnα did not stimulate isre-promoter activities in fcwf- cells under these conditions. these data suggest that the action of csa on the intracellular replication of fipv is independent of the ifn pathway. this cell line might not be responsive to ifnα and would thus be useful for isolating feline coronavirus. cyclophilins have ppiase activity [ , [ ] [ ] [ ] , contribute to the maturation of several proteins, and are involved in cell signaling, mitochondrial function (atp synthesis), molecular chaperone activity, rna splicing, stress response, gene expression, cholesterol transport, and the regulation of kinase activity [ ] [ ] [ ] [ ] . surface plasmon resonance technology and bioinformatics tools have found that the sars coronavirus n protein binds to cypa [ ] [ ] [ ] . therefore, the n protein of fipv probably binds to cyp proteins to regulate viral replication. the roles of cyp in virus replication and the inhibitory effect by csa on hcv and several other viruses have been studied [ ] [ ] [ ] [ ] . the proposed mechanisms of the inhibitory effect of csa mainly involve cypa and cypb in virus replication [ , , ] . based on these earlier findings, we believe that interaction between the fipv genome is likely or that viral and cyp proteins play critical roles in viral replication and transcription. further studies are required to resolve which cyp is critical for viral replication and which viral protein is required to form replication complexes with cyp and/or other cellular proteins. cats with clinically diagnosed fip have very rarely been cured, although several therapeutic strategies have been attempted. some cats treated with prednisolone and phenylalanine mustard or cyclophosphamide have gone into remission [ ] . various immunosuppressants, such as glucocorticoids and cyclophosphamide, may prolong life but do not alter the fatal outcome [ ] . further investigations using ppiase dominant-negative assays and rna interference methods are warranted to clarify the role of csa against ppiase in fipv replication in vitro. in addition, whether csa could be useful as fip treatment in vivo remains to be determined acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis deletions in the a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis a review of feline infectious peritonitis virus infection: - cellular composition and interferongamma expression of the local inflammatory response in feline infectious peritonitis (fip) effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis treatment of cats with feline infectious peritonitis sdz pri , an orally bioavailable human immunodeficiency virus type proteinase inhibitor containing the -aminobenzylstatine moiety mode of action of sdz nim , a nonimmunosuppressive cyclosporin a analog with activity against human immunodeficiency virus (hiv) type : interference with hiv protein-cyclophilin a interactions requirement for cyclophilin a for the replication of vesicular stomatitis virus new jersey serotype cyclophilin b is a functional regulator of hepatitis c virus rna polymerase cyclophilin: a specific cytosolic binding protein for cyclosporin a active site mutants of human cyclophilin a separate peptidyl-prolyl isomerase activity from cyclosporin a binding and calcineurin inhibition sdz - , a new picornavirus capsid-binding agent with potent antiviral activity in vivo efficacy of sdz - , a new picornavirus capsid-binding agent inhibition of human immunodeficiency virus type replication in human cells by debio- , a novel cyclophilin binding agent handschumacher re: calmodulin, cyclophilin, and cyclosporin a the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits an initial step in folding of transferrin within the endoplasmic reticulum cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins a and b overexpression, purification, and characterization of yeast cyclophilins a and b cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins multiple cyclophilins involved in different cellular pathways mediate hcv replication immunophilins: switched on protein binding domains? peptidyl-prolyl cis-trans isomerase is the cyclosporin a-binding protein cyclophilin cyclophilin a-induced alterations of human immunodeficiency virus type ca protein in vitro cyclophilin a is required for an early step in the life cycle of human immunodeficiency virus type before the initiation of reverse transcription cyclophilin a modulates processing of human immunodeficiency virus type p gag: mechanism for antiviral effects of cyclosporin a purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a suppression of feline coronavirus replication in vitro by cyclosporin a this study was supported by a grant-in-aid for scientific research (c) from the japan society for the promotion of science and the strategic research base the authors declare that they have no competing interests. key: cord- -qh cxp authors: tanaka, miho; kodama, satoshi; lee, ilhak; huxtable, richard; chung, yicheng title: forgoing life-sustaining treatment – a comparative analysis of regulations in japan, korea, taiwan, and england date: - - journal: bmc med ethics doi: . /s - - -w sha: doc_id: cord_uid: qh cxp background: regulations on forgoing life-sustaining treatment (lst) have developed in asian countries including japan, korea and taiwan. however, other countries are relatively unaware of these due to the language barrier. this article aims to describe and compare the relevant regulatory frameworks, using the (more familiar) situation in england as a point of reference. we undertook literature reviews to ascertain the legal and regulatory positions on forgoing lst in japan, korea, taiwan, and england. main text: findings from a literature review are first presented to describe the development of the regulatory frameworks surrounding the option of forgoing lst in each country. based on the findings from the four countries, we suggest five ethically important points, reflection on which should help to inform the further development of regulatory frameworks concerning end-of-life care in these countries and beyond. there should be reflection on: ( ) the definition of – and reasons for defining – the ‘terminal stage’ and associated criteria for making such judgements; korea and taiwan limit forgoing lst to patients in this stage, but there are risks associated with defining this too narrowly or broadly; ( ) foregoing lst for patients who are not in this stage, as is allowed in japan and england, because here too there are areas of controversy, including (in england) whether the law in this area does enough to respect the autonomy of (now) incapacitated patients; ( ) whether ‘foregoing’ lst should encompass withholding and withdrawing treatment; this is also an ethically disputed area, particularly in the asian countries we examine; ( ) the family’s role in end-of-life decision-making, particularly as, compared with england, the three asian countries traditionally place a greater emphasis on families and communities than on individuals; and ( ) decision-making with and for those incapacitated patients who lack families, surrogate decision-makers or ads. conclusion: comparison of, and reflection on, the different legal positions that obtain in japan, korea, taiwan, and england should prove informative and we particularly invite reflection on five areas, in the hope the ensuing discussions will help to establish better end-of-life regulatory frameworks in these countries and elsewhere. recently, two critical laws on end-of-life care were enacted in asia. one is the act on decisions on life-sustaining treatment for patients in hospice and palliative care or at the end of life (hereafter, lst decision act), which was enforced in early in korea [ , ] . this act permits the withdrawal of lst from patients at the end of life. the other is the patient right to autonomy act (hereafter praa) enforced in early in taiwan [ ] . much earlier, in , taiwan enacted the hospice palliative care act, which permits withdrawal of treatment from terminally ill patients at their request. the new law allows treatment withdrawal not only from terminally ill patients, but also from any patient who is in an irreversible coma, in a vegetative state, has severe dementia, or otherwise suffers from unbearable pain. in contrast to these two countries, japan has yet to pass a law on forgoing lst, even though the public discussion on the issue has been ongoing for over a decade. in , japan's ministry of health, labour and welfare (hereafter, mhlw) issued the guideline for medical decision-making process in end-of-life (terminal) care (hereafter, the process guideline); however, it does not explicitly permit or prohibit forgoing lst. in , a group of non-partisan diet members proposed a draft bill for respecting the patient's decision in end-of-life care act (provisional name; hereafter, the draft ad bill), which offers immunity to physicians if they withhold or withdraw lst in accordance with the patient's advance directive (hereafter, ad). as of , the proposed draft ad bill has yet to be submitted to the national diet. as portrayed above, regulatory developments on forgoing lst have progressed in these three countries to differing degrees. unfortunately, the nature and specifics of these remain relatively unknown to other countries, partly due to the language barrier. the legislation, guidelines, and deliberation processes are typically posted only in the native language on the websites of health ministries and parliaments of these countries, so the regulatory schemes for end-of-life care are little known beyond the particular jurisdiction, even by neighbouring asian countries. many western countries formulated public policies about forgoing lst in end-of-life care prior to most asian countries. england is one such example, along with the us and other english-speaking countries including australia and canada. looking specifically to the english legal system, professional medical organisations have long published guidance on end-of-life care, including the doctors' trade union the british medical association (hereafter, bma) and the doctors' regulator in england, the general medical council (hereafter, gmc) , , , english law primarily derives from the common law (i.e. the decisions of judges) and acts of parliament. although common law had long governed lst decisions, in the mental capacity act (hereafter, mca) came into force, which covers lst decisions for adult patients who lack "mental capacity" [ ] . comparison of the situations in korea, taiwan and japan with the situation in england is useful as it enables clarification of legal and philosophical issues pertaining to the legalisation of forgoing lst. we believe that the results of such a comparison will be valuable for critical reflection on the relevant laws and policies, in these countries and others. to this end, the present study involved a literature review to examine the laws, guidelines, and newspaper articles relevant to this issue. below, we first summarise the development of regulatory frameworks on forgoing lst in japan, korea, taiwan, and england. we then compare the regulatory frameworks on forgoing lst in each country. finally, we discuss legal and philosophical issues concerning forgoing lst that need to be addressed in order to create better regulatory frameworks for end-of-life care in asian countries and worldwide. development of regulatory frameworks with regard to forgoing lst in japan, korea, taiwan, and england japan in japan, court cases and other incidents highlighting problems associated with lst withdrawal increased the momentum for legislation. while no bill has been submitted to the national diet for deliberation, several ethical guidelines have been created. here, in order to better understand the current situation in japan, we summarise two crucial court cases and one incident regarding the forgoing of lst. the tokai case ( ) in this case, the attending physician withdrew treatment from a -year-old patient suffering from end-stage multiple myeloma at tokai university hospital. the physician then administered sedative drugs to stop the patient from stertorous breathing, followed by potassium chloride, which causes heart failure if injected undiluted. the patient subsequently died from cardiac arrest, and the attending physician was prosecuted for homicide. in march , the in the united states, beginning with california's enactment of the natural death act in , all states had enacted ad-related laws by . england comprises different legal systems; our focus will be on english law i.e. essentially the law of england and wales. see: <https://www.bma.org.uk/> (accessed april ). the general medical council (gmc) in england is an independent organisation and statutory regulator for the medical profession in england. its guidance applies to doctors working in all four uk countries (i.e., england, wales, scotland, and northern ireland). in addition, the gmc helps protect patients and improve medical education and practice. see: <https://www.gmc-uk.org/> (accessed april ). yokohama district court found the attending physician guilty of murder and sentenced him to years in prison with years of suspension [ ] . in the ruling, the court not only set forth admissibility requirements for active euthanasia, but also provided admissibility criteria for forgoing lst in the obiter dictum (table ) . although the tokai case concluded at the district court level without appeal, the decision had a considerable impact on japanese society and influenced subsequent discussions, not only on active euthanasia, but also concerning the forgoing of lst. the kawasaki case ( ) in the case of kawasaki kyodo hospital, the attending physician removed the endotracheal tube from a -year-old patient upon request from the patient's family. the patient was unconscious due to hypoxic brain injury accompanied by status asthmaticus [ ] [ ] [ ] . when the physician could not alleviate the patient's heavy breathing, she ordered an assistant nurse to inject a muscle relaxant, and the patient eventually died. the doctor was later charged with homicide. the most distinctive aspect of the kawasaki case was that the disputed point in the supreme court was the illegality of treatment withdrawal. both the yokohama district court and the tokyo high court in the kawasaki case decided that treatment withdrawal was illegal because two requirements for withdrawing treatment had not been fulfilled, specifically "the physician's duty of care" and "the patient's autonomy", respectively corresponding to the first and second admissibility requirements in the court decision of the tokai case. the physician was accordingly convicted of homicide. in early , the tokyo high court sentenced the physician to year in prison with years of suspension. in , the supreme court ruled to uphold the high court ruling. the imizu incident ( ) ( ) ( ) ( ) ( ) ( ) in the imizu municipal hospital incident, two physicians withdrew artificial ventilators from seven terminally ill patients in their s to s in response to requests either from the patients or their families [ , ] . all patients died following removal of the ventilators. in , the incident made national headlines, with the hospital director apologising that his staff had undertaken "ethically problematic" treatment withdrawal. in , the police referred the physicians to public prosecutors. by the end of , however, the public prosecutors office decided not to prosecute due to insufficient evidence [ ] . both the kawasaki case and the imizu incident fuelled the fears of healthcare professionals about facing legal or social sanctions over forgoing treatment, and clarification of the legal (im)permissibility of forgoing lst became an urgent issue for the government and professional medical associations. in , soon after the high court decision on the kawasaki case and the exposé of the imizu incident, the mhlw issued the process guideline, which addressed the issue of lst withdrawal (details explained below in section ) [ ] . several professional associations also published end-oflife guidelines, including the guideline on end-of-life care in acute care and intensive care (compiled collaboratively by the japanese association for acute medicine, the japanese society of intensive care medicine, and the japanese circulation society) [ ] , the guideline on end-of-life care (japan medical association) [ ] , and the guidelines for decision making process of elderly care: focusing on the use of artificial hydration and nutrition (japan geriatrics society) [ ] . however, even after publication of the mhlw's process guideline and other professional guidelines, healthcare professionals were not sure about the legality of forgoing lst. for example, in , year after the publication of the mhlw's process guideline, the ethics committee at the kameda medical center approved the withdrawal of the artificial ventilator from a patient with motor neurone disease (mnd), but the hospital director chose not to follow the committee's advice due to legal uncertainty [ ] [ ] [ ] . , , this situation led to the drafting of an ad bill in by a group of non- . it is desirable that the patient has declared his or her wishes when forgoing treatment is considered an option. it is permissible to presume the family's will to be the patient's will, provided that the patient's family makes a deliberate decision that takes into consideration the patient's perspective. . treatments that can be forgone include all treatments considered to be curative measures, supportive measures, or lst, e.g., drug administration, chemotherapy, artificial dialysis, artificial ventilator, blood transfusion, and artificial nutrition and hydration (anh). a the term they actually use in the decision is "stop treatments (chiryo chushi)," but because there appears to be no distinction in the decision between withholding treatments and withdrawing them, we interpreted the word "stop" to mean "forgoing" on april , one of the authors (tanaka) was able to view this important report which the hospital had submitted to the shinminato district police on october , by gaining access to the official files of imizu city. als is a rare neurological disease that mainly involves nerve cells (neurons) responsible for controlling voluntary muscle movements that enable actions such as chewing, walking, and talking. currently, there is no cure for als and no effective treatment to halt, or reverse, the progression of the disease. partisan diet members. seven years later, the draft ad bill has yet to be submitted to the national diet. in korea, the boramae hospital case and the severance hospital case were critically important in shaping the public discussion and subsequent legislation on lst withdrawal. the boramae hospital case ( ) the attending physician at boramae hospital removed the artificial ventilator from a -year-old patient, who had been admitted to an intensive-care unit (icu) after a surgical operation due to massive intracranial haemorrhage, following a request from the patient's wife [ , ] . the doctor and the patient's wife were subsequently charged with homicide and prosecuted as joint offenders. the medical team in the hospital had tried to persuade the wife to continue the patient's treatment in the hospital, but she requested that the patient be discharged because of the financial burden of further hospitalisation, and the team decided to discharge the patient. less than min after leaving the hospital, the patient developed respiratory difficulties and died. expecting to be the beneficiary of a free funeral service if police were engaged, the wife reported this as an unexpected death to the police, and it led to a court case. in , the supreme court sentenced the physician to months in prison and years of probation. the court ruled that if the physicians discharged the patient at the request of the patient's wife and were aware that the reason for the wife's discharge request was the death of the patient and that discharge of the patient would lead to his death, then the physicians were guilty as accomplices to murder. this ruling came as a shock to many korean physicians. until then, proxy decisions by the patient's family had been generally accepted as essential grounds on which physicians could make difficult choices regarding treatment. however, the court decision reminded physicians of their duty to act in the best interests of the patient before complying with the will of a (potentially untrustworthy) proxy. however, physicians regarded this decision as requiring them to do their best to prevent the death of the patient [ ] . the severance hospital case ( ) a -year-old female patient at severance hospital suffered hypoxic brain damage due to severe bleeding from the pulmonary artery, which was a complication arising during bronchoscopic biopsy [ ] . she was deemed to be in a persistent vegetative state (pvs) and could not breathe sufficiently on her own. her family asked the hospital to withdraw the artificial ventilator from the patient, but the hospital refused. the family filed a lawsuit and won the case at the supreme court level. this case was different from the boramae hospital case in two respects. first, the patient was thought to be irreversibly dying. second, the court judged the family's proxy decision to be the patient's will. furthermore, the supreme court pointed out the necessity of legislation on forgoing lst that respects the right to pursue happiness, as guaranteed by the korean constitution. along with these court cases, the following significant events happened in korean society [ , ] . in , in response to the boramae trial, the korean association of medical societies (kams) proposed the kams guideline on forgoing lst. in , in response to the severance case, the kams, the korean medical association, and the korean hospital association together established a set of guidelines on forgoing lst. creation of these guidelines attracted the attention of legal and ethical professionals, policymakers, and medical professionals [ ] . several bills on withdrawing lst were submitted to the national diet from to . these developments led the government to set up consultation bodies in and to take measures to facilitate social consensus-building around these topics. the topics included whether or not treatments may be withdrawn only from terminally ill patients, whether lst (including cardiopulmonary resuscitation and artificial ventilation but not anh) may be withdrawn, and whether and to what extent physicians must respect patient autonomy. these developments culminated in the enactment in , and full enforcement in , of the hospice palliative care and lst decision act. taiwan was one of the first asian countries to create a law on forgoing lst. , , it is believed that the practice of "terminal discharge from hospital" played an important role in shaping the public discussion and subsequent legislation concerning lst. in taiwan, the term "terminal prior to this, a group of non-partisan diet members had announced a draft proposal in that would allow withdrawal of lst, but this was only an outline of a bill and was never submitted to the national diet. the outline of the draft ad bill in was made public at the end of . the initial plan allowed only terminally ill patients to withhold lst. in the strict sense, the patient was near pvs (her brain responded to external stimuli, that she has minimal consciousness) but medical professionals expected she would not survive the condition very long. the court recognised the professionals' prediction, so the court mentioned it as "irreversible dying stage". so it ruled that the hospital should respect the patient's preference. ironically she regained the respiration ability, and lived another months after the court decision. other countries include thailand and singapore. thailand enacted the national health act, b.e. in . singapore enacted the advance medical directive act in . discussion of the situation in these countries is beyond the scope of this paper. discharge" refers to the practice of allowing terminally ill patients to spend the last few hours or days with their families at home by being discharged from the hospital of their own will or their family's proxy decision. the legitimacy of such practice was recognised as part of the "discharge at patient's request," which was stipulated in the medical care act in [ , ] . since then the practice of "terminal discharge" has been widely accepted in the clinical scene in taiwan. along with the above development, the hospice movement began to take root in taiwan [ , ] . in , a substantial discussion on the need for hospices and palliative care began, and the first hospice program was set up in , followed by the creation of the first hospice unit in taiwan in [ ] . since then, several hospice wards have been established in both public and private hospitals, and many religious hospice foundations and non-profit hospice organisations have been created. as the hospice movement developed in taiwan, calls for the legalisation of forgoing lst grew amongst healthcare professionals in . the ministry of health and welfare (formerly the department of health; upgraded as mhw in ) was initially reluctant to answer such requests because the officials believed it was too early to discuss such an issue. however, as the hospice movement progressed in taiwan, the mhw indicated in that providing palliative care and withholding lst were within the realm of a doctor's "duty of care" and, therefore, not illegal. following the policy change in mhw, healthcare professionals sought legislation on do not resuscitate (dnr) orders, in order to allow patients to die naturally in hospital [ ] . as a result, the hospice and palliative care act was enacted in , legalising the withholding of lst from terminally ill patients in line with their ads (including a healthcare proxy to legally make healthcare decisions on behalf of patients) or their family's consent. the law was amended several times and now also permits lst withdrawal at the patient's or family's request, as described in more detail in section below. although the act contributes to the development of hospice in taiwan, it was criticised for not fully respecting patient self-determination for two reasons. first, the physician's duty to tell the truth, to disclose a diagnosis, or to obtain consent from the patient is not explicitly stipulated [ ] . second, even after the law was enforced, the patient's family primarily make decisions to withhold or withdraw lst [ ] . in response to these criticisms, the praa was passed at the end of and enforced in january . the law and professional guidance permit lst to be withdrawn or withheld, not only from patients who have mental capacity and have refused such treatment but also from patients who lack mental capacity. the relevant principles were initially enshrined in professional guidance and court rulings, but some have since been set down in the mca. the crucial first ruling in this area was bland, which concerned a patient who lacked the mental capacity to make decisions about his treatment at the relevant time [ , ] . the bland case ( ) anthony bland (then -yearsold) was trapped in a crush at the hillsborough football stadium disaster in sheffield (england) and later diagnosed to be in a pvs. three years passed without any improvement in his condition. he had not indicated his will concerning the treatment in advance. however, the hospital trust and bland's family came to believe that treatment withdrawal was appropriate as they perceived no benefit in treatment continuation. the hospital applied to the court, seeking a declaration that the withdrawal of bland's anh would be lawful. in , the house of lords, the then superior court, ruled that withdrawing lst is an omission, which can be justified if a responsible body of doctors hold that treatment is not in the patient's best interests [ , ] . the court ruled that future such cases involving patients in the pvs should be brought before a judge for a decision as to whether or not lst should (continue to) be provided. after the decision, bland's anh was withdrawn, and he died shortly afterwards. pvs cases involving anh were, therefore, a matter for the judges and the requirement was subsequently extended to patients in the minimally conscious state (mcs) [ ] . however, the (now) superior court, the supreme court, has recently confirmed that there is no requirement to bring such cases to court [ ] . this means that, regardless of the patient's diagnosis or the treatment to be withdrawn, decisions do not require court approval, although difficult or contested situations may still come before the judges. instead, lst decisions are generally a matter for the healthcare professionals, albeit in consultation with the incapacitated patient's loved ones. following the bland case, professional societies issued guidelines on forgoing lst. these included the bma, which published guidance on withholding and withdrawing lst in [ ] . the gmc also published guidance, entitled "withholding and withdrawing -guidance for doctors" in , which set the standards of practice expected of doctors when they consider whether to withdraw or withhold treatments that might prolong a patient's life [ ] . in , the latter was replaced with new guidance, entitled "treatment and care towards the end of life: good practice in decision making." the gmc's current guidance provides a framework for good practice regarding the provision of treatment and care to patients who are likely to die within a year [ ] . there are also more specific guidance documents, focused on (for example) decisions about cpr and, most recently, anh [ , ] . the various professional guidance encompasses not only incapacitated patients, but also patients who have "mental capacity" (or competence). the common law had long defined capacity in functional terms, i.e. the focus was on whether the patient could comprehend and retain relevant information and reach a decision [ ] . the common law had also long recognised the right of a competent adult patient to refuse lst, a position most powerfully confirmed in re b in , in which a ventilator-dependent patient had this unwanted treatment withdrawn at her request [ ] . furthermore, the common law also recognised an adult patient's right to make an ad refusing treatment they might otherwise receive in the future when incapacitated: provided that the patient was competent and informed, and their advance directive applied to the situation that later arose, their refusal had to be respected [ ] . many of these propositions were then enshrined in the mca in , thus replacing the common law [ , ] . as the parliamentary office of science and technology reported, "while healthcare practice had to operate according to these common law judgements, historically it has been guided by more paternalistic concepts of duty of care without a strong legal awareness. outside of hospitals, however, for many carers of people with learning disabilities, dementia or mental illness, concerns were expressed about the lack of legal guidance [ ] ". the mca, therefore, provided a statutory test for mental capacity and generally provided that decisions about patients who lacked capacity were to be made in their "best interests". although there is no strict legal test of best interests, the courts have increasingly emphasised the importance of respecting the patient's wishes and values [ ] . furthermore, the act also placed adsspecifically now labelled "advance decisions to refuse treatment"on a statutory footing, and for the first time provided for surrogate decision-making, through the creation of "lasting powers of attorney" (hereafter lpa). details of the law are explained below. this section compares the regulatory frameworks of the four countries regarding forgoing lst. the foci under comparison include: the legal effectiveness of advance directives; eligibility criteria for medical conditions; the definition of 'terminal phase'; and proxy decision-making by the patient's family. japan in japan, the tokyo high court ruling in the kawasaki case highlighted the need for a law or guidelines on forgoing lst, yet no relevant law has been enacted to date. the mhlw's process guideline issued in has been the most influential document in this area to date. it consists of the main body and commentary, and contains two core elements: ) respecting patient selfdetermination and ) deciding the course of care by the healthcare team (and not by the attending physician alone). the process guideline stipulates that the healthcare team shall make healthcare decisions through repeated discussions with patients and their families, with a particular emphasis on respecting the patient's will. it also specifies that if patients cannot express their will, the healthcare team shall decide the best course of care in light of the family's wishes. the guideline further states that if it is difficult for the healthcare team to decide or reach a consensus with patients or their family, a committee composed of multiple experts should be set up for consultation. in this way, the process guideline puts a significant emphasis on consensus-building among those involved in the patient's end-of-life care. interestingly, the process guideline is silent on the following two points. first, the main body of the process guideline does not define "the terminal stage" or when the patient is considered as terminal or facing the endof-life stage, even though this phrase appears in the very title of the guideline [ ] . the commentary of the guideline does state that there can be different types of terminal stages according to the patient's disease, which could include terminal cancer, chronic disease, cerebrovascular disease, and senility. the healthcare team is instructed to judge when the patient has reached the terminal stage through careful assessment of the patient's condition. however, the guideline does not provide any specific criteria that would define the terminal stage, such as "six months to live". second, it is unclear whether healthcare professionals can avoid criminal or civil liability for forgoing lst if they act according to all of these countries have a national healthcare system covering all citizens. as for differences, the three asian countries have adopted the social insurance scheme for healthcare, for which citizens of these countries must pay some healthcare costs at the time they receive care. meanwhile, england's national healthcare system is run by tax money, and people do not have to pay when they receive medical care. in japan, administrative guidelines with no legal basis have neither direct legal force in courts nor any direct effects on the rights and duties of citizens. the process guideline has no legal basis, and lack penal or administrative sanctions. however, the guideline is widely understood to express the official position of the mhlw concerning end-of-life care, and is therefore often cited in relevant literature as well as in the media. incidentally, in , the mhlw revised the title of the process guideline to "the process guideline for the medical decision-making at the last stage of life (jinsei no saisyu dankai)," because the term "end-of-life (or terminal) care (shumatuski iryo)" was not popular. however, as was the case with the original process guideline, the revised process guideline did not define the phrase "the last stage of life". the mhlw again revised the process guideline in march , both to extend it to home-based care situations, and to include the idea of acp. the guideline. after all, the process guideline is not legally binding because, although the mhlw issues it, it is not supported by any specific law. in korea, the lst decision act was passed in january , and full enforcement of the law began in february . as the name of the act implies, it allows patients with not only cancer but also other medical conditions to receive hospice and palliative care. additionally, it enables people to create ads about withdrawing lst and sets up a national online registry system for storing ads and reviewing them. physicians are granted access to the registry and are therefore able to access an ad, which can inform decision-making. the act also stipulates that a consensus between two or more family members can be regarded as the patient's will when the patient does not have an ad or cannot express his or her wishes. characteristically, the korean act gives a two-fold definition of "end-of-life". "terminal stage", which is used to define the terminal patient, is when a patient is expected to die in several months, and the provision of hospice palliative care is considered appropriate. "the end of life process" refers to situations where a patient's death is considered imminent due to rapid deterioration of his or her condition and the withdrawal of lst is offered as a choice. while clarifying the necessity of hospice care, the korean law is restricted in its application to lst decisions, because an lst decision is only applicable at the very end of a disease process. the act also provides a clause covering a physician's order on lst (life-sustaining treatment plans), which is a physician's order written after consulting a patient about her wishes about end-of-life care. the physician may (or the patient may ask the physician to) inform the patient about diagnosis, end-of-life care options, and lst options, and prepare this document. when a physician places this document in the healthcare record, it takes priority over any existing ad that purports to state the patient's preferences on lst. in a sense, this order resembles the physician order for life-sustaining treatment (polst) used in the various american states. however, the korean document is supposed to be used within hospitals, whereas polst is focused on emergency medical services. as explained above, taiwan has two laws concerning forgoing lst. the hospice and palliative care act allows withdrawing and withholding lst from a terminally ill patient who has a valid ad, which covers the relevant treatment options and designates healthcare proxies; if there is no ad, the patient's family can submit a consent form on their behalf when patients cannot express their will. the phrase "terminally ill patient" refers to a person who is diagnosed by a physician as having an incurable disease or injury and whose death is imminent based on the best available medical evidence. the praa, which was enacted in january and enforced in january , explicitly stipulated that healthcare institutions and physicians are duty-bound to tell patients about their medical conditions, treatments, procedures, medications, and prognoses in order to honour the patient's right to know. it also stipulated that, with a valid ad, lst (including anh) can be withdrawn from not only terminally ill patients but also patients in an irreversible coma, a persistent vegetative state, and from those with severe dementia or other medical conditions specified by the ordinance of the mhw [ ] . although both laws stipulate forgoing lst, the praa is complements the hospice and palliative care act by focusing on a patient's autonomy. as the praa expands the clinical conditions to non-terminally ill patients and includes anh in the range of lst, advance care planning (acp) must be conducted before any ad is made. such legal regulation of acp is new to taiwanese society because, according to the hospice and palliative care act, consensus-building among the involved parties is not a necessary condition to forgo lst. the decision of forgoing lst for terminally ill patients can be made either with a patient's ad or with their families' consent. since taiwan has little experience of acp, this has been a major focus of work since the enactment of praa in : hospital staff are being trained in this area and acp groups, which consist of a physician, a nurse, and a social worker, are being developed. after the enactment of praa in , the development of acp groups in hospital and provision of suitably knowledgeable hospital personnel has been the main tasks of the enforcement in [ ] . in england, there are many legal developments and professional guidance documents pertinent to the issue of forgoing lst, but for the sake of brevity, we focus on the critical legislation and professional guidance. first, incidentally, in , the mhlw revised the title of the process guideline to "the process guideline for the medical decision-making at the last stage of life (jinsei no saisyu dankai)," because the term "end-of-life (or terminal) care (shumatuski iryo)" was not popular. however, as was the case with the original process guideline, the revised process guideline did not define the phrase "the last stage of life". the mhlw again revised the process guideline in march , both to extend it to home-based care situations, and to include the idea of acp. the lst decision act uses the term "advance statement on life-sustaining treatment" for conventional ad. the mca focuses on decisions made about, with and for adults who lack mental capacity. like the preceding common law, the act essentially defines capacity in functional terms [ ] . if a patient has capacity, they may prepare an advance decision to refuse treatment that might otherwise be provided when she or he lacks capacity. such a decision can apply to lst, provided that the decision is written, signed and witnessed, and there is a clear statement to the effect that the decision will apply even if this will put the patient's life at risk [ ] . the mca provides immunity for physicians who withhold or withdraw treatments from their patients if they reasonably believe that an advance decision exists that is valid and applicable to the treatment in question [ ] . however, the act does not require the patient's medical condition to be terminal in order to execute the patient's ad to forgo treatments. the mca also provides for surrogate decisionmaking, through the lpa. the act also empowers the court to appoint a deputy to make decisions, for example, in cases where there is a serious and enduring lack of capacity, such as due to dementia. however, the lpa is the main means by which a patient can appoint someone to decide on his or her behalf. the mca stipulates that an attorney has the power to consent to or refuse lst if the patient has explicitly so empowered the attorney. notably, however, an lpa is required to make decisions in the best interests of the patient; as such, there is always the possibility that an attorney's decision will be challenged on this basis. indeed, for patients who lack an ad or lpa, best interests provide the test as to whether treatmentincluding lstshould be provided. the act enumerates, rather than elaborates, the factors to be balanced in a best interests decision [ ] . the law in this area had evolved considerably since bland, with the courts increasingly emphasising the holistic nature of the assessment, i.e. medical factors, which had been dominant in the ruling, were not the only consideration [ ] . the mca seeks to emphasise the breadth of the test, as have the courtsincluding the supreme court, the now superior english courtin subsequent rulings [ ] . clinicians are required to consult appropriately in determining a patient's best interests. if there is no one close to a patient to provide input into the decision, the act also provides an independent mental capacity advocate (hereafter imca) service, which can support decisionmaking by and for incapacitated patients in their best interests [ , ] . a further recent legal change concerns patients with prolonged disorders of consciousness. bland had required decisions about anh for patients in the pvs to come to court, a requirement which was later extended to patients in the mcs. although that requirement persisted for decades, recent rulings have confirmed that this is not strictly the law: as such, these cases should no longer routinely come to court, although they may do so, for example, if there is disagreement between the relevant parties about what is in the patient's best interests [ ] . the second crucial regulatory source is the gmc guidance on decision-making in end-of-life care. the gmc guidance addresses issues surrounding decisionmaking in end-of-life care and provides decision-making models for patients who can decide as well as those who lack the capacity to do so [ ] . the guideline advises physicians to provide or forgo lst when they judge that these would not be clinically appropriate for a patient. when making such judgments, physicians must weigh the benefits of treatment against the burdens and risks for the patient. furthermore, physicians should carefully take into account not only clinical considerations but also other factors relevant to the circumstances of each patient, including the patient's wishes, values, and feelings. it also emphasises the importance of acp. unlike the mca, the guidance defines the phrase, "approaching the end of life". in this last section, we will discuss the legal and philosophical issues that have emerged from the comparison of regulatory frameworks relating to forgoing lst in japan, korea, taiwan, and england. in order to bring about improvements in these frameworks, particularly concerning end-of-life care, the following five points must be addressed. first, the definition of terminal stage (or phase) can be essential when considering a regulatory framework for forgoing lst. as described in section , the lst decision act in korea and the hospice and palliative care act in taiwan permit patients to forgo treatment only in the terminal stage. it makes the definition of 'terminal stage' significant, not to mention the clinical judgement of when a particular patient is in the terminal stage. the gmc guidance also addresses decision-making for children, including neonates, and young people. further details are provided in the following guidance. the guidance defines the phrase as follows: for the purposes of this guidance, patients are 'approaching the end of life' when they are likely to die within the next months. this includes patients for whom death is imminent (expected within a few hours or days) and those with: (a) advanced, progressive, incurable conditions; (b) general frailty and co-existing conditions that mean they are expected to die within months; (c) existing conditions if they are at risk of dying from a sudden acute crisis in their condition; and (d) life-threatening acute conditions caused by sudden catastrophic events. both korean and taiwanese laws stipulate that a patient is in the terminal stage when his or her condition is diagnosed as irrecoverable and his or her death is imminent with rapid deterioration of the condition. as explained earlier, the korean act distinguishes between two phases of the terminal stage; namely, the terminal stage when patients with irrecoverable conditions are expected to die within several months, and the end-of-life stage when patients are facing imminent death with rapidly worsening conditions. the act only allows the latter patients to elect for withdrawal of lst. meanwhile, the taiwan act interprets the terminal stage more conservatively and considers terminal patients to be those who have been diagnosed as having an incurable disease or injury, and who cannot avoid death shortly based on the best available medical evidence. this definition more or less corresponds to "the end of life stage" in the korean act. regarding the kind of medical conditions to which the law applies, the korean law limits the "terminal stage" to patients with cancer, acquired immune deficiency syndrome (aids), chronic obstructive pulmonary disease (copd), and chronic hepatic disease, as well as other diseases specified by the ordinance of the mhw. patients with these conditions are eligible for hospice palliative care in the terminal stage, while no such limitation exists for patients in the dying stage who are eligible to elect for termination of lst. on the other hand, although there are similar rules about the coverage of palliative care in the taiwanese national health insurance system, the hospice and palliative care act in taiwan does not specify any health conditions for patients in the terminal stage to be eligible for forging lst. in japan, no law stipulates a definition of terminal stage. the mhlw's process guideline avoids providing a definition and only suggests several instances of medical conditions which can be considered as reaching a terminal stage, such as "when a patient's prognosis is predicted to be from a few days to - months in the case of terminal cancer". england also lacks such a stipulation; indeed, lst may be refused, withdrawn or withheld regardless of the patient's life-expectancy. the mca, for example, is not limited to patients in the terminal stage [ ] . the gmc guideline, however, states that patients are "approaching the end of life" when they are likely to die within the next months, which is broader than the korean or taiwanese criteria. we believe that the definition of terminal stage should be neither too narrow nor too wide when specifying the period within which one can choose to withdraw treatment. if the definition is too narrow and the law only applies to patients who are expected to die within a few days or even a few hours, the case for withdrawing lst might not be so compelling, for they will die in a short time in any case. that said, however, there may be clinical (and ethical) reasons for withdrawing treatment, even in such a short period, to ensure that the imposition of treatment does not adversely affect the dying process, and there may also be cultural reasons for doing so, as demonstrated by taiwan's "terminal discharge from hospital". on the other hand, if the definition is too broad, the law might apply to those with incurable diseases such as mnd, end-stage renal disease (esrd), and alzheimer's disease, who may be able to live for a relatively long period if treatmentsuch as artificial ventilation, dialysis, or gastric fistulais continued. however, expanding the definition to include patients with such conditions might give rise to concerns and confusion, particularly as such a definition would potentially include those who are early in the disease trajectory, and therefore not in the "terminal phase" as this is conventionally understood. moreover, patients who are diagnosed with such incurable diseases might feel threatened because the regulatory framework such as law or guidelines would classify them as "terminal patients" who can have their treatments withdrawn. of course, the requirement that the patient must consent might dispel any concerns these patients and their loved ones might havebut we should also be mindful that such patients might feel under pressure to "consent" [ ] . alternatively, a strict definition of the terminal stage could be avoided altogether. if the principle of autonomy or the right of self-determination is considered paramount, then perhaps we may not need to limit forgoing treatment to the terminal stage, as england's mca and taiwan's new law seem to suggest. however, if we adhere to the idea that the patient can choose to have lst withdrawn only at the terminal stage, then a defined set of clinical criteria for "terminal stage" are likely to be required. the revised law deleted these specific names of disease, and only provided the criteria of terminal stage. the revised law was enforced from march, . in the commentary for the process guideline published by the mhlw, the terminal stage is not defined but the following situations are considered as terminal stage: ) a patient's prognosis is predicted to be from a few days to - months in the case of terminal cancer; ) a patient repeats acute exacerbation of chronic disease and falls into poor prognosis; ) a patient will die within months to years as the sequelae of cerebrovascular disease or senile deterioration. likewise, the science council of japan explains in the report that depending on the disease and condition of a patient, terminal stage can be divided into an acute type (e.g., emergency medical care), a subacute type (e.g., cancer), and a chronic type (e.g., senility, vegetative state, and dementia). based on our analysis, one important lesson is that the purpose of defining the terminal stage should be clear. in particular, two relevant objectives for defining the term would be the specification of a period when retreating from aggressive treatments and focusing on palliative care is appropriate, and the determination of a period when the patient or the family can choose to withdraw lst. furthermore, it appears that policymakers and professional societies should be required not only to define the term but also to establish clinical criteria for judging when patients with various conditions are in the terminal stage. forgoing lst for non-terminal patients (e.g., patients with mnd and those in pvs) the second issue is whether to allow patients who are not in the terminal stagebut are suffering from conditions such as mnd or are in a pvsto forgo lst. such patients who are not in the terminal stage can continue living with the assistance of artificial ventilators and anh. our comparison has revealed different positions on withdrawing or withholding lst from these nonterminal patients. the korean act, for example, limits the permissibility of treatment withdrawal to the dying stage, thereby excluding non-terminal patients with pvs or mnd. taiwan's hospice and palliative care act also applies only to terminal patients. however, the newly enacted praa has a broader scope than the preceding act and allows even pvs patients to forgo lst. it should be emphasised here that mnd is not included in the four conditions stipulated in taiwan's new act. england's mca does not have any such restrictions and thus applies to both terminal and non-terminal patients. in japan, the draft ad bill does not mention specific medical conditions that would fall under the scope of the bill; presumably, however, neither the draft ad bill nor the mhlw's process guideline applies to non-terminal pvs or mnd patients. not allowing mnd patients to withdraw lst because they are not terminal seems particularly problematic in places like japan, where tracheostomy with invasive mechanical ventilation (tiv) rates for mnd patients may be the highest in the world [ ] , ranging from . to . % [ ] [ ] [ ] . even if mnd patients require artificial ventilators or anh, they can remain alive if they choose to receive these treatments. if we understand the terminal stage as an incurable and irreversible condition that, regardless of the use or withdrawal of lst, will result in death within a relatively short time [ , ] , then these mnd patients are not terminal. in japan, however, although the mnd patients' choice of withholding lst seems to be respected [ ] , detaching artificial ventilators once they have been initiated is next to impossible [ ] and physicians who detach them from mnd patients are highly likely to be prosecuted [ , ] . in such circumstances, mnd patients can choose either to receive or not to receive lst, knowing that this cannot later be withdrawn even if they desire it. in these asian countries, whether or not to allow pvs and mnd patients who are not in the terminal stage to forgo lst remains controversial. england has also struggled with the question of whether lst can be withdrawn from mcs patients. the courts initially appeared reluctant to authorise the withdrawal of anh from such patients, although they have become more willing to countenance this, at least where there is compelling evidence that the patient him-or herself would not consent to such treatment [ , ] . whether the law in this jurisdiction does enough to respect the autonomy of (now) incapacitated patients nevertheless remains a live issue [ ] . in most western countries including england, the common understanding is that there is no morally significant difference between withholding and withdrawing treatments [ , , ] . thus, withdrawing treatments is permitted in circumstances in which withholding treatments is permitted [ ] . in contrast, such a view is not necessarily the norm in asia. according to a survey of physicians who manage patients in intensive care units (icus) in countries and regions in asia, the proportion of respondents who felt that withholding and withdrawing lst were ethically dissimilar was % on average [ ] . by country or region, the largest was % in pakistan, followed by % in bangladesh, while the lowest was % in singapore, followed by % in hong kong. in japan, % responded that withholding and withdrawing were ethically dissimilar; the percentages were % in korea and % in taiwan. for example, in clinical settings in japan, the general idea is that withdrawing lst is ethically more difficult than withholding it, and that withdrawing lst is considered "unacceptable" [ , ] . legal scholars in japan are also split on the legality of withdrawing lst. for instance, according to one legal scholar, "no medical professional has been prosecuted for removing the artificial ventilator or the gastrostomy feeding tube from a terminally-ill patient" since the publication of the process guideline by the mhlw in [ , ] , implying that withdrawing lst is not illegal if one follows the protocol set by the process guideline. on the other hand, some scholars think that, whereas withholding treatment can be regarded as an instance of omission, it is difficult to view withdrawing as such, and thus claim that withdrawing and withholding treatment is not necessarily legally equivalent [ , ] . thus, while an absolute consensus exists regarding the legal status of withholding lst, differing views are held on withdrawing lst, not only among healthcare professionals but also among legal scholars. several factors may underlie the difficulty of equating withholding lst with withdrawing it. for example, a qualitative survey of emergency physicians in japan conducted between and found the following factors to be motivations for avoiding the withdrawal of artificial ventilators: ) fear of criminal prosecution and concern about unwanted media exposure; ) concern for the feelings of the patient's family; ) physicians' psychological barriers to shortening patients' lives by withdrawing lst because they regard withdrawing lst to be an act and not an omission; and ) medical factors in the acute phase of a severe condition, including the uncertainty of treatment outcome (i.e., they cannot completely deny that the patient may recover) [ ] . the fact that there is no law in japan permitting the withdrawal of lst may also be a background factor. in summary, unlike their counterparts in western countries, many healthcare professionals, as well as some legal scholars, in asian countries still find it difficult to equate withholding lst with withdrawing lst. moreover, as the above discussion suggests, it may be necessary to address the feelings of healthcare professionals adequately when considering a legal framework for endof-life care. fourth, our comparative analysis revealed that the role of the family (or, indeed, other loved ones) in england differs from those in the three asian countries. although the family plays a significant role in end-of-life decisionmaking in all three asian countries, the legal stipulations concerning their role are slightly different. in taiwan, the hospice and palliative care act stipulates the role of the family in end-of-life decision-making. the act states that patients may nominate a medical proxy in advance and that the designated family member by law may consent to forgo lst on behalf of terminally ill patients if their will cannot be expressed and no ad has been signed. the family members designated by law include (in descending order of closeness): spouses, adult children and adult grandchildren, parents, adult siblings, grandparents, great-grandparents or collateral consanguinities within the third degree, lineal relatives by affinity within the first degree. the law states that one designated family member should sign the consent form for forgoing lst. if there are disagreements concerning forgoing lst among family members, the consent of the closer family member should be prioritised following the order of closeness. in korea, the lst decision act defines two aspects of surrogate decision making, which involves family members verifying the patient's intention by witnessing (article ) and deciding for the patient (article ). the act also sets out the differing family input in each of the cases. the family members considered relevant for witnessing are: ) spouse, ) linear descendants, such as children and grandchildren, ) linear ascendants, such as parents and grandparents, and ) siblings, if there is no one corresponding to )- ). as for the witnessing, the requirement for verification is such that two family members need to witness the patient expressing his or her preferences for lst. however, the family members are different in the case of surrogate decision making, for the decision must be made by agreement of all relevant members. family members, in this case, include: ) spouses, ) lineal descendants and ascendants with one degree of kinship (i.e. parents and children,) ) lineal descendants and ascendants with two degrees of kinship (i.e. grandparents, and grandchildren) ) siblings, if there is no one corresponding to )- ). thus, the korean act and the taiwan act differ, both in how they rank family members and how the family's will is decided when there is no consensus. in japan, families have conventionally made medical decisions on behalf of patients [ , ] . the draft ad bill contains no provisions about the family's ability to forgo treatment for patients. the process guideline, however, grants this role to the family of the patient, especially when the patient is incompetent and cannot express his or her will. in that case, the family either presumes the patient's will or, when that is not possible, the healthcare team discusses with the family what will be in the patient's best interest. interestingly, the commentary of the process guideline explains that 'family' is not restricted to family members as defined by law, but can include people like trusted friends of the patient and those who have supported the patient at the end of their life. in england, the mca does not specify who may or may not be an attorney (although he or she must be an adult, i.e. of years or older). thus, unlike the three asian countries mentioned above, legally speaking, family members are not automatically authorised to make decisions about treatment on behalf of the patient who has lost his or her decision-making capacity. the legal forms of lpa list people whom the patient trusts and know well as examples of an attorney, i.e., spouses, partners, adult children, or good friends [ ] . where the patient has not made an lpa, the relevant decision-maker is still required to consult those caring for the patient or interested in his or her welfare; no limit is placed on who, precisely, should be consulted. the general rule is that, when a proxy makes decisions on behalf of the patient, it is crucial to represent the patient's prior wishes or his or her best interests. based on our analysis, we believe that the following issues regarding the role of the family need careful examination: ) whether or not family members are the best proxies or advocates for incompetent patients [ ] ; and ) whether or not there is a chance that family members may prioritise their own interests over those of the patient because they want to reduce financial or other types of burdens or even to benefit financially, for example through inheritance [ ] . in addition, there are other issues such as the definition and scope of family [ ] . , compared with england, the three asian countries traditionally place a greater emphasis on families and communities than on individuals. thus, patients will sometimes prefer to have their family and physician decide the course of their treatments rather than doing so themselves; such familybased medical decision-making is said to be an expression of filial piety [ ] [ ] [ ] [ ] [ ] . further studies will be needed to determine how a suitable framework for proxy decision-making can be established in these asian countries. decision-making in the case of a patient without family lastly, our comparative analysis found that decisionmaking with, about or for an incompetent patient who lacks family or close friends may pose a severe problem. recently, the significant increase in dementia patients has become a global issue; as of , . million people worldwide are living with dementia, and nearly million new cases emerge every year [ ] . dementia poses an enormous financial burden both on society and the individual, as well as giving rise to such issues as stigma and social isolation [ ] . in the context of endof-life decision-making, the following question is especially important: who should make, or contribute to, decisions, when patients with dementia who lack family or close friends are unable to make treatment decisions for themselves? in the three asian countries highlighted here, no specific regulatory framework covers such cases. however, japan's process guideline recommends the establishment of a committee consisting of various experts to deal with such complicated cases. in england and wales, the mca makes provision for those incapacitated adults who lack close family or friends, namely, the imca service. the purpose of the imca service is to represent and support particularly vulnerable people who have no family or friends and who lack the capacity to make important decisions about (providing, withholding, or withdrawing) serious medical treatments and changes in accommodation [ ] . clinicians must involve an imca when a decision about serious medical treatments is to be made in relation to such patients. an imca will interview or meet in private with the person who lacks mental capacity and obtain the views of professionals and anybody else who can provide information about the wishes, feelings, beliefs, or values of the person. they work to determine any alternative options, may examine relevant records, and then must write a report on their findings. the number of imca referrals regarding serious medical treatments (including withholding or withdrawing anh) was between april and march [ ] , but has been increasing every year since . among these, patients with dementia accounted for % ( referrals). the imca service of england and wales is unique among the four countries compared in this article [ ] . we suggest that when lawmakers and policymakers make regulatory frameworks concerning end-of-life care, they should discuss not only ads (including those encompassing attorneys) and proxy decision-making by the patient's family, but also make provision for cases in which people who lack ads, family, or close friends lose the capacity to make such decisions. the reason for this is that there will always be those "unbefriended patients" who cannot be helped by any of the standard legal mechanisms that protect and promote autonomy through the use of ads, family, friends, or courtappointed guardians [ ] . in such cases, establishing a service such as england's imca is one option, but the use of clinical ethics support services, such as hospitalbased committees, may be another option, as suggested by japan's process guideline. as one study has shown, ethics consultation may be useful for resolving ethical dilemmas that involve the forgoing of lst [ ] . some limitations of the present study should be noted. because our main focus was on laws and guidelines, our see (a lineal relative by affinity refers to a lineal relation of his or her spouse (such as a husband's or wife's parents or grandparents), and a spouse of his or her lineal relation (a husband or wife such as a child or a grandchild)) for different interpretations of family members in korea's and taiwan's acts. england's mca does not give such authority to family members both on decision-making and consent on behalf of individuals. findings may not fully reflect the actual practice of healthcare professionals in each of the four countries. thus, the actual practices in each country may be more similar to that in other countries, even though legal and other stipulations may suggest otherwise. furthermore, we could not compare data surrounding withdrawal of lst among these four countries due to the lack of yearround data from each [ ] [ ] [ ] [ ] . future studies should assess the actual practice(s) surrounding end-of-life care of healthcare professionals and psycho-spiritual care in end-of-life care in the four countries. additionally, in a pandemic or disaster situation such as the current covid- pandemic, ethically complex issues such as the allocation of scarce healthcare resources may adversely affect end-of-life care. future research could usefully address such situations [ ] . this study compared regulatory frameworks governing forgoing lst in japan, korea, taiwan, and england, and presented five important legal and philosophical points: ) the importance of defining the terminal stage and associated criteria for clinical judgment; ) the importance of discussions on withdrawing lst in pvs or mnd patients who are not terminal; ) the importance of (re)considering any moral and legal differences between withholding and withdrawing treatments; this is disputed particularly in asian countries; ) the importance of ascertaining the family's role in end-of-life decisionmaking; and ) the importance of devising ways to deal with incompetent patients who lack family or ads. focusing on these five points will be important for the establishment of better end-of-life regulatory frameworks for these four countries and others. abbreviations decisions on life-sustaining treatment for patients in hospice and palliative care or at the end of life right to die' program goes into effect laws & regulations database of the republic of china. patient right to autonomy act mental capacity act the hospital "the physician withdrew treatments" after switching off artificial ventilators from seven patients; they died "ethically problematic the suspicious death: the hospital in toyama prefecture "seven patients euthanized" -year-surgeon switched off artificial ventilators from patients removing artificial ventilators in the imizu municipal hospital: two physicians not prosecuted the guideline on medical decision process at the final stage of life the japanese association for acute medicine. the guideline on end-of-life care in acute care and intensive care the japan medical association. the guideline on end-of-life care the guideline on decision making process of elderly care: an adoption of anh removing an artificial ventilator: als patient requested to hospital, but the hospital director "can't answer the ripple spreads to the request for death flame of life, mind is free (living a patient - ) the law on end-of-life care: background and implication. in: presentation for international workshop: ethics at the end-of-life end-of-life care in korea: current situation biomedical ethics policy in korea: characteristics and historical development debated on end-of-life care and the legalization of "natural death" in taiwan: the practice of terminal discharge and the hospice and palliative care act the law and ethics of end-of-life care in taiwan: on the practice of terminal discharge and a criminal court case concerning the hospice and palliative care act promotion of advance care planning in taiwan palliative medicine and the hospice movement in taiwan the legislation of end-of-life care in taiwan: the passage of "patient's self-determination act london: hodder & stoughton end of life trust -v-bland [ ] wlr part death with dignity and end of life care practice direction e: applications relating to serious medical treatment withholding and withdrawing lifeprolonging medical treatment: guidance for decision making withholding and withdrawing -guidance for doctors treatment and care towards the end of life: good practice in decision making british medical association, the resuscitation council (uk) and the royal college of nursing. decisions relating to cardiopulmonary resuscitation rd edition, st revision british medical association and royal college of physicians. clinicallyassisted nutrition and hydration (canh) and adults who lack the capacity to consent: guidance for decision-making in england and wales adult: refusal of treatment re ak (medical treatment: consent) [ ] flr . . department for constitutional affairs. what is the mental capacity act ? in: mental capacity act code of practice houses of parliament, parliamentary office of science and technology. postnote no. : mental capacity and healthcare aintree university hospitals nhs foundation trust v james release: regarding the revised guideline on medical decisionmaking process at the final stage of life debates on end-of-life care and the legalization of "good death" in taiwan: the hospice and palliative care act and the patient self-determination act mental capacity act mental capacity act clarifying the best interests standard: the elaborative and enumerative strategies in public policy-making re a (male sterilisation department for constitutional affairs. what is the new independent mental capacity advocate service and how does it work? in: mental capacity act code of practice an exposition of the mental capacity act in britain: focusing on the lasting powers of attorney and the independent mental capacity advocate service - years: guidance for all doctors japan amyotrophic lateral sclerosis association "we are against the bill to refuse the treatment needed to sustain life the survey report about the end-of-life care with als patients of the als physicians in japan mechanical ventilation in amyotrophic lateral sclerosis: a cross-cultural perspective clinical features of als in japan from the registration system of intractable diseases tracheostomy with invasive ventilation for als patients: neurologists' roles in the us and japan regarding withdrawal of artificial ventilator. home health care people intractable dis some critical topics for further discussion on palliative medicine in amyotrophic lateral sclerosis (als) the yokohama district court applied inducing or aiding suicide or homicide with consent to the mother. regarding withdrawal of artificial ventilator from her child with intractable disease the place of wishes and feelings in best interests decisions: wye valley nhs trust v mr b deciding to forgo life sustaining treatment: a report on the ethical, medical, and legal issues in treatment decisions part generation of the doctrine in the united states: conditions for not receiving life sustaining treatments withdrawing and withholding life-sustaining treatments in withholding and withdrawal of lifesustaining treatments in intensive care units in asia, supplementary online content efigure attitudes of japanese and japanese-american physicians towards life-sustaining treatment end of life care and legal issues: a comparison with the united states legal issues on medical interventions in terminally ill patients the supreme court precedent: criminal case third small court precedent on kyodo hospital case: the supreme court withdrawal of mechanical ventilation from dying patients at emergency and critical care facilities in japan: a qualitative study with physicians report and recommendation of legal improvement on supporting decision making and proxy decision making in medical practice outline of a law on proxy consent regarding treatments for persons without capacity of medical consent lasting power of attorney forms the kawasaki kyodo hospital case appeal trial decision. hogaku shimpo the theory of autonomy support in the adult guardianship system in england and wales. kyoto: minerva shobo preferences for advance directives in korea cultural diversity at the end of life: issues and guidelines for family physicians family experience with difficult decisions in end of life care which factors have the greatest influence on bereaved families' willingness to execute advance directives in taiwan? cancer nurs survey of the general public's attitudes toward advance directives in japan: how to respect patients' preferences fact sheet: dementia world alzheimer report : the global impact of dementia independent mental capacity advocacy service: th annual report mental capacity act /mental capacity act code of practice unbefriended and unrepresented: better medical decision making for incapacitated patients without healthcare surrogates effect of ethics consultations on nonbeneficial life-sustaining treatments in the intensive care setting: a randomized controlled trial decision-making on withholding or withdrawing life support in the icu: a worldwide perspective end-of-life decision-making in six european countries: descriptive study euthanasie en andere medische beslissingen rond het levenseinde euthanasia laws in benelux's three countries -comparison and task the importance of addressing advance care planning and decisions about do-not-resuscitate orders during novel coronavirus (covid- ) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank proedit japan which provided professional english proofreading services. all authors contributed to the conception and design of this paper. mt wrote the draft of the paper and sk, il, yc and rh made changes to this draft, with the final draft being prepared by mt. all authors read and approved this final draft. this research was supported by the fondation brocher (swizterland), the sasakawa memorial health foundation (japan), the uehiro foundation on ethics and education (japan) and by the global research network program through the ministry of education of the republic of korea and the national research foundation of korea (nrf- s a a ). the funder did not play a role in the study design, in the collection, analysis and interpretation of research or in writing the manuscript. all materials that support our findings of this study are publicly available. professor richard huxtable is a member of the editorial board of bmc medical ethics and a member of various ethics committees, including that of the british medical association; the views stated in this article are those of the authors and should not be taken to represent those of any organisation with and for whom they work. other authors declare that they have no competing interests. key: cord- -vy r zd authors: nan title: abstracts from the (th) annual meeting of japanese association for the stusy of taste and smell (jasts ), kanazawa, japan, october - (th), (the president of the meeting was dr. takaki miwa, kanazawa medical university) date: - - journal: chem senses doi: . /chemse/bjs sha: doc_id: cord_uid: vy r zd nan since umami taste receptors t r and t r have been identified, some reports revealed their structures and functions. umami taste receptors are member of g protein-coupled receptors, belonging to family c with long n-terminal extracellular domain. t r and t r percept umami taste as a heterodimer. we focused on leucine repeats of t r and t r in their transmembrane domains. we have reported the possibility of correlation between the food preference of animals and difference in the length of leucine repeats. poly leucine (polyl) is called homo polymeric amino acid, so called hpaa. it is reported that ; % of proteins in prokaryotic and eukaryotic organisms contain hpaas. it is also suggested that polyl could interact with each other more than with the other hpaas. some inherited diseases are caused by proteins in which hpaas such as polyq, polya are expanded to excessive length. activity of the protein containing hpaas is thought to be increased by expansion of hpaas, but with expansion of more than the threshold, activity of the protein is suddenly decreased. hpaas are one of the structures to be seen frequently. it is suggested that hpaas are important structures for some proteins to function. on the other hand normal function of hpaas remains unclear. from the above point of view, polyl might affect interaction of umami taste receptor t r and t r . we also hypothesize polyl play an important role in the perception of umami. some odorant receptors also have the similar polyl structure, suggesting that under the hydrophobic condition some proteins are dimerized by interactions between leucine repeats. we named a leucine hpaas motif for protein-protein interaction as ''leucine glue.'' we are discussing the effect of polyl on protein-protein interaction by using split gfp assay. # comparative analysis of the umami receptor gene, t r from cetaceans t r and t r are known to act as umami receptor. they are member of g protein-coupled receptors (gpcr) and have seven transmembrane domains. in , it has been reported that the t r gene of giant panda has frameshift mutation, resulting in inactivation of umami receptor. originally, they are used to be a carnivorous animal. it is suggested that the mutation of the t r gene might be associated with their current food preference. we have already analyzed genomic sequences of the t r genes from two different dolphins; lagenorhynchus obliquidens and tursiops truncatus. the results strongly suggested that umami receptor genes, t r from both cetaceans are not functional, because frameshift mutations occurred in their coding sequences. nonsense codon caused by these framshift mutations appeared on their downstream region. in this study, we have also cloned two more t r genes from cetaceans, balaenoptera acutorostrata and balaenoptera physalus. both the t r genes had frameshift mutation and various mutations in their coding sequences. in addition, we have also cloned two more t r genes from cetaceans, b. acutorostrata and b. physalus. both the t r genes had frameshift mutations and various mutations in their coding sequences as well as dolphins described above. especially, in one allele from both b. acutorostrata and b. physalus, we found point mutiations at start codon atg which is a to g substitution, resulting in gtg. by this mutation, t r could not be translated. cell-based expression studies have shown that mammalian t r and t r g-protein coupled receptors combine to form a broadly tuned l-amino-acid receptor. however, contradictory data in t r knockout (ko) mouse models have been reported on taste nerve responsiveness. one study showed that taste nerve responses to amino acid stimuli in t r -ko mice were totally abolished. the other reported reduced but not abolished responses to glutamate in t r -ko mice. in the present study, we have tried to recharacterize the role of t r for the taste nerve responses to amino acids using a wide variety of taste stimuli. we have recorded from whole-nerve chorda tympani nerve responses from t r -ko mice to lingual application of various amino acid stimuli including sweet-and umami-tasting amino acids. whole-nerve recordings of chorda tympani nerve in t r -ko mice showed robust responses to all the tested amino acids. comparing to those from c bl/ wild-type mice, only small reductions of responses to glycine and l-alanine, but no reduction in responses to l-proline, l-serine, l-glutamine and l-glutamate salts have been observed. these results suggest that t r mediated receptor system may play limited role for detection of amino acids. type iii taste cells play to act to receive sour taste and also to act as ''taste integration cell'' to transmit taste information from type ii taste cells through atp. but, it is not known whether a type iii taste cell has both roles and whether two kinds of type iii taste cells share each role separately. using calcium imaging and immunocytochemistry, we revealed that atp activates type ii and iii taste cells and hcl activates type iii taste cells, and also that hcl and atp activate separate subpopulations of taste cells. we also showed that sour-responding taste cells do not have p x and p y as atp receptors in taste buds. moreover, we carried out double cell staining of mice circumvallate papillae using antibodies to atp receptor (p x , p x and p y ) and sour taste receptor (pkd l ) using an antibody to type ii taste cell marker (plcb ) and type iii taste cell marker (pgp . ). it was shown that p x and p x are expressed in taste nerves, p y is in type ii taste cells, and pkd l is in type iii taste cells. no taste cell was stained by both atp receptor and sour taste receptor. although we could not cover all the atp or sour taste receptors, these results suggest that there are the two separate subpopulations of type iii taste cells: the one sensing sour taste and the other accepting the atp released from type ii taste cells. # development of gustatory papillae in the absence of six and six six family genes encode homeobox transcription factors, and a deficiency in them leads to abnormal structures of the sensory organs. in a previous paper, six was reported to be expressed in the taste bud-bearing lingual papillae of mice, and loss of six affected the development of these gustatory papillae. we show here that embryos lacking both six and six revealed more severe abnormalities than those lacking six alone during morphogenesis of their gustatory papillae. by in situ hybridization, six was shown to be broadly distributed in the epithelium of the lateral lingual swellings at embryonic day (e) . , and in the tongue epithelium, mesenchyme, and muscles at e . . from e , six was similar in expression pattern to six , as previously reported; and in the fungiform papillae, six was expressed in the epithelium at e -e . . in the circumvallate and foliate papillae, six expression observed in the trench wall of these papillae at e . -p . although six -deficient mice had no abnormalities, six /six -deficient mice showed distinct morphological changes: fusion of the lateral lingual swellings was delayed, and the tongue was poorly developed. the primordia of fungiform papillae appeared earlier than those in the wild-type or six -deficient mice, and the papillae rapidly increased in size; thus fusion of each papilla was evident. the circumvallate papillae showed severe defects; e.g., invagination of the trenches started asymmetrically, which resulted in longer and shorter trenches. the foliate papillae elevated initially, and showed stunted trenches. therefore, six and six function synergistically to form gustatory papillae during development of the tongue. the life span of taste cells was previously estimated as - days by labeling newly proliferated taste cells with radioactive hthymidine or bromo-d-uridine (brdu) incorporated during dna synthesis. to re-evaluate this observed lifetime of taste bud cells, we repeated the analysis of lingual tissues of mice after incorporation of ethynyl-d-uridine (edu) instead of brdu, followed by detection of edu by a click reaction. we injected edu into adult mice and sacrificed them , , , , , , , , , , or days later. the vallate papillae were fixed, cryo-sectioned, immunostained for plcb (a marker for type ii cells) and examined with a confocal laser scanning microscope with optical sectioning at lm. taste buds were identified in dic images captured in parallel. numerous cells with brightly fluorescent nuclei (edu+ cells) appeared at day near and within taste buds. the number of those cells declined rapidly toward day . however, among type ii cells, very few appeared to be edu+ at day . the number of edu+ type ii cells increased at day , showed a broad peak around day , and then declined. occasional edu+ type ii cells were still detected at day . because labeling of dna with edu is much faster, brighter and more consistent than with brdu, this method will be useful to study proliferating cells in taste buds. by combining edu labeling and immunstaining for approapriate markers, the longevity of taste cell types can be examined. adiponectin is a white adipose protein that plays important roles in glucose homeostasis and lipid metabolism and is involved in cell proliferation and differentiation. the function of adiponectin is mediated by its receptors (adipor and adipor ), which have different expression patterns. in the gustatory system, adiponectin is contained in the saliva; however, the function of adiponectin signaling in gustatory tissues has not been elucidated yet. therefore, we have examined the expression patterns of adipor and adipor in mouse gustatory tissues. reverse transcription polymerase chain reaction assays have revealed that adipor and adipor mrnas were expressed in the circumvallate papillae. using immunohistochemistry, the anti-adipor antibody labeled at the taste hairs in the fungiform, foliate and circumvallate papillae. double-labeling experiments have demonstrated an expression pattern of adipor -positive cells in a subset of basal cells within the taste buds and extragemmal epithelium cells to marker proteins for taste cell types (including types i, ii, iii, and basal cells). for the marker proteins analyzed, only shh was co-expressed with adipor in a subset of basal cells within the taste buds in the circumvallate papillae. these results show that adipor at taste hairs in gustatory tissues may detect saliva adiponectin and may play a role in taste sensing in the taste buds. in addition, adipor may play a role in cell proliferation and differentiation for the shh and adipor expressing basal cells within the taste buds. mash is expressed in subsets of neuronal precursors in both the central nervous system and the peripheral nervous system. however, involvement of mash in taste bud cell differentiation remained to be demonstrated. in the present study, to begin to understand the mechanisms that regulate taste bud cell differentiation, we have investigated the role of mash in regulating taste bud cell differentiation using mash ko mice and forced expression of mash in lingual epithelial cells. in mash ko mice, aadc-ir cells are missing both in the mash mutant circumvallate papilla epithelium and in the taste buds of soft palate. in mash ko/gad -gfp mice, gfp-positive cells (gad expressing type iii cells) are also missing in the taste buds of soft palate. on the other hand, gustducin, a type ii cell marker of taste bud, is expressed in soft palate taste buds in mash mutant mice. these results suggest that mash plays an important role for expression of aadc and gad in type iii cells in taste buds. taste receptor cells are epithelial in sense that they have a limited life span and therefore must be replaced to maintain the structure of the epithelium. therefore gustatory nerves need to make synapse with appropriate taste receptor cells. however, mechanism of recognition of taste receptor cells which express appropriate taste receptor is still unknown. the cadherin superfamily of cell-cell adhesion molecules controls a series of interactions that regulates synapse formation. in this study, in order to test whether the cadherins are required for formation of synapse between gustatory nerve fibers and taste receptor cells, we have investigated expression patterns of cadherin superfamily in the taste buds. # induction of type iii-like taste cells in threedimensional co-culture and analysis of excitability of clonal cell lines derived from murine taste buds we have collected taste buds from a tongue of a p -deficient mouse and established clonal cell lines (tbd cell lines). each of tbd cell lines expresses gustducin and neural cell adhesion molecule (ncam), suggesting that tbd cell lines have characteristics of both type ii and type iii taste cells. to reveal whether tbd cell lines have excitability or not, we analyzed the expression pattern of voltage-dependent sodium channels in one of tbd cell lines, tbd-a cells and performed patch clamp recording. scn a, scn a, scn a and scn a genes coding voltage-dependent sodium channels were detected in tbd-a cells by rt-pcr. patch clamp recording revealed that tbd-a cells had tonic outward currents, but lacked transient inward currents. these results suggest that functional voltage-dependent sodium channels are not expressed in tbd-a cells. next, we tried to establish a culture model mimicking the lingual taste cells of the mouse. tbd cell lines were co-cultured with a lingual epithelial cell line ( a cell line) and a lingual mesenchymederived cell line (tmd cell line). tbd, a and tmd cell lines were maintained in a triple co-culture, in which tbd cells were preseeded as aggregates or in suspension on the collagen gel containing tmd cells and a cells were laid over the tbd cells. tbd cells in the triple co-culture expressed ncam, however gustducin was not immunohistochemically detected in tbd cells. this result suggests that tbd cells in the co-culture exhibited a characteristic of type iii taste cells. this triple co-culture model would be useful to study morphogenesis and functions of the gustatory organ. immunohistochemistry of taste-signaling molecules is useful for the distinction of taste-bud cell types. however, the optimal immunohistochemical detection depends on specimen-preparing conditions. therefore, this study aimed to examine differences in immunoreactivities under various tissue-preparing conditions in rat vallate taste buds for some typical markers of gustatory cells as follows: gustducin, type iii inositol triphosphate receptor (ip r ), synaptobrevin- (vamp ), protein gene product . (pgp . ), and neural cell adhesion molecule (ncam). staining patterns and intensities of immunoreactivities for these molecules varied according to tissue-preparing conditions, especially the fixation. confirming our previous finding, gustducin immunoreactivities were localized to the upper part of the taste bud called taste hairs in case of short fixation, but to the cell body cytoplasm excluding the taste hairs in case of long fixation. the specific immunostaining for ip r was strong in short-time fixation. on the other hand, those for vamp , pgp . , and ncam were intense in long-time fixation. the present data suggest that different experimental protocols cause the discrepancies in immunoreactivities for these markers. further investigations based on this study should provide important information on the classification of the gustatory cell type. # immunohistochemical localization of nk and vpac receptors in the frog fungiform papilla hiroshi ando , osamu tadokoro , naokazu asanuma , mihoko tomida , takami nakamura and eiji kondo department of oral physiology and department of oral anatomy, matsumoto dental university, shiojiri - , japan although the presence of substance p (sp) immunoreactive (ir) nerve fibers and vasoactive intestinal peptide (vip)-ir nerve fibers has been reported in the frog fungiform papilla, the functional roles of these fibers remain to be elucidated. to clarify whether these sp-ir and vip-ir fibers play any efferent roles in the taste disc in the fungiform papilla, we immunohistochemically examined the existence of neurokinin receptor (nk r), which is also a sp receptor, and vip and pituitary adenylate cyclase activating hormone receptor (vpac r), which is also a vip receptor, in the fungiform papilla of the frog, rana catesbeiana. many nk r-ir structures were observed in the taste disc. these were conical in shape in the middle layer of the taste disc and had a rod-like thin apical process toward the upper layer of the taste disc. double-labeling immunohistochemistry showed that only a few nk r-ir structures were located adjacent to the sp-ir nerve fibers in the taste disc. vpac r immunoreactivity was observed in the taste disc cells and the glossopharyngeal nerve bundle beneath the taste disc. vpac r-ir cells in the taste disc had either a thin or thick apical process. vip-immunonegative nerves beneath the taste disc showed vpac r-ir. vip-ir neuronal cell bodies colocalized vapc r in the grossopharyngeal nerve bundle within the tongue.the presence of the nk r and vpac r in the taste disc suggests that sp-ir and vip-ir nerve fibers play efferent roles in the taste disc in the frog fungiform papilla. # cesium-permeable potassium channel in the rod cells of frog taste disc in animal cells, the resting membrane potential is maintained by random open-shut gating of a leaky potassium channel. frog taste disc cells display the resting membrane potential of about - mv. the magnitude is not consistent with the equilibrium potential of potassium (- mv). when internal potassium was replaced with cesium in whole-cell configuration, the rod cells in frog taste disc increased a parabolic outward current gradually and displayed the resting membrane potential of - ± mv (n= ). the wing cells did not possess the outward current and displayed the resting potential of ± mv (n= ) in the condition of internal cesium solution. the replacement of external normal saline solution with cesium saline solution turned the parabolic outward current to the inward current. the inward current reversed at ± mv (n= ) which is close to the equilibrium potential of cesium ( mv). several experiments on cationic replacement showed the permeability ratio of p k :p cs :p na :p nmdg = . : . : . : . . carbenoxolone (cbx, a blocker of pannexin) inhibited the parabolic outward current moderately (ic , lm). the dialysis of lm arachidonic acid in internal cesium solution changed the parabolic outward current to the exponential one and depolarized the membrane potential by mv. the results suggest that the outward cesium current in the frog rod cells may flow through a novel channel different from mammalian cesium-permeable potassium channel. capsaicin, which is the main component of chili pepper and has many physiological functions such as increasing perspiration and energy metabolism, has negative effect of its strong pungency. capsiate was identified from Ôch- sweetÕ (capsicum annuun l.), a non-pungent cultivar of red pepper. like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system. human sensory evaluation tests demonstrated that capsiate and that analogs (capsinoids) had very weak pungency compared with capsaicin and capsaicin analogs (capsaicinoids), and the difference of threshold level between capsinoids and capsaicinoids was about -fold. however, the in vivo studies focused on the reception of them in oral cavity are few because the reception mechanism of hot taste is unclear, and the reason of weak pungency of capsinoids is not well understood. therefore, this study focuses on the reception of capsinoids and capsaicinoids in the oral cavity. firstly, we performed two-bottle preference tests using rats and confirmed that they began to avoid capsinoids at about times higher concentrations than that of capsaicinoids. in addition, we recorded the lingual trigeminal nerve responses to capsinoids and capsaicinoids and found that capsinoids induced little responses while capsaicinoids evoked apparent intensive responses. such differences were also found in immunohistochemical detection of p-erk in the trigeminal ganglion after stimulation of capsinoids and capsaicinoids. recent studies using animal models of binge eating shows that the accumbal dopaminergic neurotransmission is elevated in bingeing. it suggests that the experience of bingeing modifies the activity of midbrain dopaminergic neurons in the ventral tegmental area (vta). however, few evidences directly clarify whether the activity of the vta is elevated anticipatorily, that is, before receiving the oral (taste) and/or post-oral (viscerosensory) cues of palatable food. to address the issue, we explored possible changes in the activity of the vta neurons in mice bingeing on sugar by immunohistochemical detection of c-fos expression as a marker of neuronal activation. under food restriction, mice were subjected to a binge-type eating training for days (day - ), in which their intake of sucrose solution gradually increased day by day, resulting in its overconsumption. on day , mice were deeply anesthetized and transcardially perfused with % paraformaldehyde before receiving the sugar exposure. brain sections including the ventral midbrain were immunohistochemically processed to detect the c-fos gene expression product, fos protein. the number of fos immunopositive neurons in the vta of trained mice was larger than that of untrained naïve mice. the finding suggests that the activity of the vta is elevated anticipatorily before sugar exposure and contributes to the stimulation of the mesolimbic dopamine system, resulting in sugar bingeing. # activation of output neurons in the basolateral nucleus of the amygdala by a learned aversive taste: a manganese-enhanced mri study tadashi inui , chizuko inui-yamamoto , izumi ohzawa , yoshichika yoshioka and tsuyoshi shimura grad. sch. human sci., osaka univ., suita, - , japan, dept. oral anatomy, osaka dental univ., hirakata - , japan, grad. sch. frontier biosci., osaka univ., suita - , japan and immunology frontier research center, osaka university, suita the basolateral nucleus of the amygdala (bla) is known to play an important role in conditioned taste aversion (cta). anatomical studies have shown that the bla has reciprocal neural projections with many brain regions. it is still unclear, however, which connections are involved in the retrieval of cta. to elucidate the neural circuits for the retrieval of cta, we examined the enhancement of activity of the efferents from the bla by the presentation of learned aversive taste, using a manganese-enhanced mri technique. rats received a pairing of mm saccharin with . m lithium chloride or saline (cta or sham group, respectively). on the test day, thirty min after an injection of mm mncl into the bla, rats were intraorally infused with saccharin. sixty min after the mncl injection, we acquired triple t -weighed mr images at hourly intervals. the signal intensities of mr images, which reflect the movements of manganese by the activated bla neurons, were measured. the cta group showed significant time-dependent increase of signal intensity in the cea than did the sham group, indicating that the retrieval of cta activates efferents from the bla to the cea. furthermore, the analyses based on the injection site of mncl (rostral or caudal) demonstrated the larger movements of manganese toward the cea and cortices (insular and piriform) in the rostrally-injected cta group. therefore, the outputs of efferent neurons in the rostral part of the bla may be separated in two directions, medial (cea) and lateral (cortices). this work was supported by kakenhi ( ). the c isotope-labelled acetic acid breath test is a common clinical method to evaluate gastric emptying and is a non-invasive and repeatable method in rodents. however, the sensitivity of this test to detect changes in gastric emptying of liquid diet in mice has not been studied thoroughly. the present study aimed to validate this method by using bethanechol chloride, atropine sulfate, clonidine hydrochloride, cck- s and phenylthiocarbamide. male c bl- j mice after overnight fasting were placed in the metabolic chamber. commercial liquid diet containing c-acetic acid was used as the test meal. after intragastric administration of the test meal, the expired air was collected at -min intervals for four hours by a fully automated system. the co / co levels were measured by the mass spectrometer. the co excretion data were analyzed for calculation of gastric half excretion time (t / ) as the gastric emptying parameter. t-test was used for statistical analysis (p< . ). bethanechol chloride decreased t / of liquid diet and atropine sulfate and clonidine hydrochloride increased t / . cck- s increased t / and cck receptor antagonist, devazepide, attenuated the delay of gastric emptying by cck- s. bitter tastant, phenylthiocarbamide increased t / . our study indicated that the c-breath test was sensitive to detect difference in gastric emptying of liquid diet induced by pharmacological agents, hormones and tastants. we concluded that this technique could be used to determine the effect of physiological, pharmacological and nutritional intervention on the motility of the gastrointestinal tract. in oral surgery treatments, acrinol hydrate is often used as a disinfectant for oral membranes; however, there are few reports about the effect on taste nerve response to this disinfectant. in this study, we tested if tongue treatment with acrinol hydrate affects taste nerve responses in mice. the chorda tympani nerve responses of c bl/ mice to basic taste stimuli before and after treatment with acrinol hydrate were recorded and compared. the results of the treatment with acrinol hydrate indicated that the chorda tympani nerve response to . m sucrose was suppressed by %; however, there was no change in the responses to . m nacl, . m hcl, . m quinine hydrochloride and . m monopotassium glutamate. the suppression of the sucrose response by this treatment recovered within minute after washing. these results suggest that acrinol hydrate has an ability to suppress sweet taste. some mints, such as peppermint, are used as flavoring substances for foods and toothpaste widely. however, it is not clear whether mints affect the taste receptor mechanisms or not. in the present study, therefore, the chorda tympani nerve responses to basic tastes were compared before and after mixing with types of mints, such as . % peppermint, . % spearmint and . % menthol, in c bl/ mice. when spearmint was mixed with . m sucrose, mm denatonium benzoate, . m monopotassium glutamate (mpg), mm #-inositol monophosphate (imp) and the mixture of . m mpg and mm imp (m+i), the neural responses to these stimuli were suppressed. but mixing of spearmint did not change the responses to . m nacl, mm quinine-hydrochloride and mm hcl. mixing of peppermint or menthol did not change any responses to all tested stimuli in the present study. these results suggest that spearmint may work as a suppressant for some kind of receptors for sweet, umami and bitter. the majority of the experiments for investigating gustatory information processing in the nucleus of the solitary tract (nst) have been performed using rats as experimental animals. although the importance of mice in this kind of study is widely recognized from the view point of recent, rapid advance in gene manipulation techniques, there are very few studies using mice in this academic field. also, it is desirable for physiological studies on neural mechanisms of taste perception to use awake, or at least, weakly anesthetized animals. therefore, in the present study we tried to establish an experimental model for in vivo recording of nst neurons in slightly anesthetized or awake mice. for this, we surgically fixed a head restraining holder to the mouse scull, which made it possible to place the mouse head precisely in a stereotaxic position repeatedly without pain. after the recovery from this surgery, the gustatory area in the nst was localized electrophysiologically under anesthesia. then, single neuron activity was recorded in this area under slight anesthesia, and responsiveness to standard taste solutions (nacl, sucrose, citric acid and quinine hcl) was tested. as a result, gustatory neurons were recorded; of these, , and neurons were classified into nacl-best, sucrose-best and citric acid-best, respectively. furthermore, we found that the mouse could be easily trained to lick taste stimuli with the head restrained in the stereotaxic device. these results indicate that the present method is applicable to the investigation of gustatory information processing in the nst of mice under a physiological condition. # comparison of preference between sucrose and sugar alcohols in c bl/ mice yoshihisa katagawa , toshiaki yasuo , keika gen , takashi yamamoto and noritaka sako department of oral physiology, asahi university school of dentistry, mizuho - , japan, department of health and nutrition, faculty of health science, kio university, nara - , japan and although sorbitol tended to be less preferred compared to palatinit. the present study suggests that the sugar alcohols are less palatable than that to common sugars, such as sucrose, in mice. # novel peptides, dryamide- , dryamide- : comparison in regulatory effects on feeding behavior of the blowfly phormia regina because the drosophila orphan receptor cg is similar to the vertebrate neuropeptide y receptor, we screened its putative ligands and purified two novel peptides: dryamide- and - . to investigate their function, we injected these peptides into the thorax of the blow fly, phormia regina, and observed the subsequent changes in feeding behavior and taste sensitivity to sucrose. the blow fly was used for technical reasons, to facilitate injection. before and after peptide injection, we measured ) the sucrose intake as an indicator of appetite, ) the proboscis extension reflex (per) as an indicator of feeding motivation, and ) the electrophysiological response of the labellar contact chemosensilla to measure the magnitude of the gustatory input inducing per. there was no significant change in sucrose intake after injection. injecting both peptides depressed per and reduced the percentage of flies that performed per. the electrophysiological response of the labellar taste sensillum to sucrose seemed to decrease after injecting dryamide- , but not dryamide- . therefore, once feeding starts, these peptides might not limit the food intake of the fly. nevertheless, the results of the per test suggest that these peptides contribute to feeding initiation. together with other peptides that restrict food intake, dryamide- and - might contribute to the overall regulation of feeding behavior in phormia regina. # stand off effect between the noble bioactive peptide and appetitive odor on feeding behavior in blowfly we compared the behavioral effects of feeding suppression by the two kinds of new peptides (dryamide- , ) in blowfly. both of gustatory and olfactory systems are required to elicit appropriate motor pattern in feeding behavior. feeding behavior was also controlled by internal condition based on many bioactive peptides. proboscis extension response (per) is indicator of feeding behavior that is generally controlled by the chemical cues from the maxillary palps. the behavioral experiment showed that they detect palatable chemical signals increasing their appetite in blowfly, phormia regina. phormia usually have detected the appetite odor like mushroom ( -octen- -ol) by maxillary palps. however, some kinds of newly found peptide have an effect on feeding behavior. therefore, we compared the effect of two kinds of newly found peptides in drosophila. first, we test whether they affect feeding behavior by the amount of sucrose intake. after injection of one kind of peptide, food intake amount was obviously increased. in the next step, we investigated the odor of octen- -ol by maxillary palps did not work as appetitive odor when they are starved sufficiently in behavioral experiments. the results showed that these peptides work as the appetitesuppressant in blowfly feeding behavior, and it did worked as counteractant to odor stimulation. sodium-deficient animals ingest sodium chloride at concentrations normally rejected, and this motivated behavioral state is known as sodium appetite. for neural mechanisms of sodium appetite, there is a line of electrophysiological evidence in rats, whereas there is less evidence in mice. in order to develop a model for sodium appetite in mice, we examined the effects of repeated sodium depletion on salt intakes in c bl/ j mice. adult male c bl/ j mice (n= , weeks, - g at the beginning of the experiment) were used; each animal was housed individually in a metabolism cage during the experiment. a combination of furosemide injection with sodium-deficient diet was used to induce acute sodium deficiency. in of the mice, furosemide was injected in the st week and saline was injected in the nd week. in the remaining mice, the injection order was reversed. this pattern was repeated times over months. after hours following the injection, intakes of . m nacl and distilled water were measured. the furosemide injection shortened the latency of, and increased the amount of, . m nacl intake. furthermore, the increase in . m nacl intake was tended to be enhanced at later furosemide injections. both urine volume and -h urinary sodium excretion were greater after the furosemide injection than after the saline injection, but there were no significant differences of these measures across repeated depletions. these results show that present procedure reliably elicits sodium appetite in c bl/ j mice, suggesting that this is a suitable experimental model of sodium appetite in mice. the sensations of taste and smell are the important determinants guiding preference for foods and fluids. the dried-bonito dashi is a traditional japanese fish broth that enhances palatability of various dishes due to its specific flavor. however, the scientific evidence why dried-bonito dashi is important for japanese dishes is little known. in general, the main stimulus is considered as inosinate (an umami substance) but the dashi is a complex mixture of many taste substances such as lactate (sour), histidine (bitter), sodium (salty) and other minerals (generally bitter) as well as macromolecules (proteins and peptides) and over odorants. here we investigated mechanisms of preference for dried-bonito dashi using -h two-bottle choice tests in adult male sprague-dawley rats. we found that preference for dashi was influenced by current nutrition, i.e., ingestion of high fat or high sucrose diet suppressed preference for dashi. in addition, past experience/learning of dashi ingestion was the most important determinant that could overcome the influences of current nutrition. for example, preference of the threshold concentrations ( . %) of dashi increased gradually during repeated exposure to dashi solution. after the repeated ingestion of dashi solution, ingestion of high fat or high sucrose diet has no influence on dashi preference. in the descending concentration series, the concentration-preference function shifted -fold to the lower concentrations compared with the ascending one. these results suggested that postingestive consequences as well as oronasal sensations guide the preference for dashi solutions that are under the control of current nutrition and past experience/learning. food aversion learning is the long-term memory established after association of the properties of food, such as texture and taste (conditioned stimulus, cs), with visceral malaise (unconditioned stimulus, us). after the acquisition of the learning, the re-exposure to the cs induces the retrieval of the memory and the rejection of the cs occurs. previously, we showed that histamine release in the central nucleus of amygdala (cea) during the re-exposure to the cs was increased in soft pellets-conditioned rats, but not in sweet pellets-conditioned rats. in the present study, we studied the role of the histaminergic transmission in the cea in the retrieval of food aversion learning induced by two patterns of the cs. fos expression, a marker of neuronal activation, following the retrieval of food aversion learning induced by each cs (soft pellets or sweet pellets) and us pairing was examined. few fos-positive cells in the cea were observed by consuming both types of pellets in naïve rats. in the conditioned rats, fos expression in the cea was increased in response to the re-exposure to soft pellets while it was not elevated by the re-exposure to sweet pellets. in addition, the microinjection of h -antagonist to the bilateral cea just before the re-exposure to the cs impaired the aversive response to soft pellets but not to sweet pellets. these findings indicate that the neuronal activation through the histaminergic system via h receptor is involved in the retrieval of texture aversion learning but not in that of taste aversion learning. # food entrained feeding behavior to highly sweet food in non-food deprived mice yasunobu yasoshima, megumi tanibuchi and tsuyoshi shimura division of behavioral physiology, department of behavioral sciences, graduate school of human sciences, osaka university, suita - , japan light-regulated circadian clock in each animal species affects its original feeding rhythm. nocturnal rodents mainly consume food during the dark phase under ad libitum food condition; however, their daily feeding rhythm is entrained to mealtime when they are restricted of food. food-entrainment is suggested to be mediated by increased arousal and food-oriented motivation derived from calorie deficiency, but recent studies indicate that taste palatability seems to be important in food-entrainment. however, the role of taste palatability (hedonic aspect of taste) in food-entrainment remains not fully understood. to study the issue, we developed a new, simple mouse model. ad libitum-fed c bl/ j male mice received daily limited access to highly sweet chow (hsc) containing % sucrose for hours in the light phase ( : - : a.m.). they received the regimen for days as training. the intake of hsc gradually increased and was about -fold greater than their basal intake of normal chow at the corresponding period of time during pretraining phase. on the other hand, the intake of normal chow in another group during the corresponding timing did not increase. intake of normal chow during the dark phase in mice group receiving hsc, but not normal chow, significantly decreased during the training. the entrained increase of hsc intake was also observed in genetically hyperphagic strain, db/db (leptin receptor gene deficiency) mice. these results suggest that taste palatability and/or hedonics of sweeten food play a role in shifting of daily feeding rhythm. # sexual difference of gene expression of androgen receptor in the brain region related to extinction memory after conditioned taste aversion learning in immature mice mice acquire the conditioned taste aversion memory (ctam) by application of the conditioned stimulus (cs) such as a novel taste, sodium saccharin (sac), followed by the unconditioned stimulus (us) such as an malaise induced by an i.p. injection of licl. after conditioned taste aversion (cta) learning, the extinction of ctam is induced by repeated presentations of cs without us. recent studies have demonstrated that the extinction is a process of relearning, where mice acquire the extinction memory of cta. we have reported that the sexually mature male mice (c bl/ ) represented significantly higher retention of extinction memory (rem) than sexually immature male mice. in contrast, the significant difference was not observed in female mice. we have also reported that chronical administration of androgen into castrated mice during prematuration period results in an enhancement of rem in both sexes. here we investigated whether the sex difference of the gene expression of androgen receptor (ar) is observed in the ventral medial prefrontal cortex (vmpfc) and amygdala between sexually immature and mature mice by real-time pcr. the sexually immature male mice presented significantly higher level of ar gene in vmpfc and amygdala than the sexually mature male mice. however, there was no such difference in vmpfc and amygdala of the female mice. these results suggest that the sex difference in maturation process of rem is induced by androgen acting on vmpfc and amygdala during sexual prematuration period in mice. our previous study showed that weanling rats acquired conditioned flavor preference when the flavor was associated with low concentration of sucrose; while flavor aversion when associated with high concentration. the present study aims to confirm and extend this finding with various concentrations of sucrose. wister male -week-old rats were used. half of the rats received unsweetened grape-flavored water on odd-numbered days and sweetened (sucrose) cherry-flavored solution on even-numbered days. the remaining rats received sweetened grape-flavored solution on odd-numbered days and unsweetened cherry-flavored water on even-numbered days. during the acquisition session, the liquid was presented to each rat for min daily across consecutive days. in the following test session, each rat was presented with unsweetened cherry-and grape-flavored water simultaneously for min daily across consecutive days. the rats showed a significant preference for the flavor associated with % or % sucrose, but showed a significant aversion to the flavor associated with % sucrose, suggesting a hedonic shift from positive to negative with increasing the concentration. the association learning acquired at the age of weeks was preserved when retested at the age of weeks. short-term ( min) drinking test showed that the volume of intake was significantly smaller for % than for % and % sucrose, indicating that % sucrose is aversive because of its oropharyngeal sensation rather than its post-ingestive effects. the present study suggests that weanling experience of food is important in the formation of feeding behavior in adulthood. tastes play important roles in detecting nutrients and irritant materials in food. preference for food tastes is critical for ingestive behavior because greater palatability is to evoke greater food intake. animals generally prefer sweetness but not bitterness. such taste preferences are known to be affected by aging which causes changes in the dietary and energy requirements. however, the mechanisms of shift in taste preference by aging still remain unclear. therefore, to elucidate differences in taste preference among the life stages, we measured the amount of intake of taste solutions by the two bottle test. we used juvenile ( - weeks), young-adult ( - weeks), adult ( - weeks), and middle-aged ( - weeks) sprague-dawley male rats. all rats were fed ad libitum during all tests. we used . m or . m nacl, . m or . m hcl, . m or . m sucrose, mm saccharin-na, · - m or · - m quinine-hcl, and . m monosodium glutamate (msg). the preference ratio for . m sucrose and . m msg in middle-aged group was lower than that in juvenile and young-adult groups. on the other hand, the preference ratio for · - m quinine-hcl in middle-aged group was higher than that in juvenile and young-adult groups. there were no significant differences in the preference for hcl and nacl tastes among groups. these results suggest that aging affects taste preferences for sucrose, msg and quinine-hcl which represent sweet, umami and bitter tastes, respectively, in humans. we investigated the effects of furanones as the maillard reaction products flavors on umami and sweet taste preferences in mice. -dimethyl- -hydroxy- , -dihydrofuran- -one; sotolon and -hydroxy- , -dimethyl- ( h)-furanone; furaneol (food-grade certified products, safc supply solutions, usa) were chosen as the maillard-borne odorants. in the short time two-bottle tests, . , . , . , . lg/kg sotolon flavored umami solutions containing mm monosodium glutamate (msg) and sweet solutions containing mm saccharin were significantly consumed more than distilled water. sotolon tended to enhance umami and sweet taste preference in a dose dependant manner. whereas there were no differences of intakes between sotolon flavored water and distilled water. in addition, furaneol and citral flavor agents did not enhance umami solution preferences. these results suggested that sotolon, one of the maillard reaction products, induced enhancement of umami and sweet taste preference in mice. supported by society for research on umami taste and kakenhi ( ). zinc is one of the essential trace elements and its deficiency causes anorexia, growth retardation, and taste disorder. insulin in pancreatic beta-cells is known to be stored as microcrystals of the zinc-containing hexamer, and zinc is thought to be fundamentally important for assembly and release of insulin from beta-cells. however, the effect of dietary zinc on food intake or insulin secretion of model rats of diabetes has not been well elucidated. in this study, we followed the levels of food intake and insulin secretion in type diabetes model gk (goto-kakizaki) rats fed on diets containing different levels of dietary zinc for weeks. all the experimental rats were divided into four experimental groups of zinc-deficient (zn-def, rats fed on . mg/kg diet), zinc-sufficient (zn-suf, rats fed on . mg/kg diet), high-zinc (high-zn, rats fed on . mg/kg diet), and pair-fed (rats fed on zinc-sufficient diet with the same amount of diet consumed by zn-def one day later). food intake in zn-def gk rats followed typical cyclic-pattern seen with normal rats such as sd rats. plasma zinc concentrations and plasma insulin concentrations were found to have positive correlations in high-zn rats. orexins are neuropeptides synthesized mainly in the lateral hypothalamic area and are implicated in the regulation of food intake in addition to arousal. since swallowing is early step of feeding behavior, orexins might modify swallowing. in the present study, we examined the effects of orexins on the reflex swallowing using anaesthetized rats. orexin, which was dissolved in ringerÕs solution, was administered into the fourth ventricle. orexin- receptor antagonist (sb ), which was dissolved in dimethyl sulfoxide, was administered into the fourth ventricle. three ll of drugs were administered using a microsyringe. swallowing was induced by the electrical stimulation ( hz, sec) of the central cut end of the superior laryngeal nerve (sln) and was monitored by recording the electromyogram of the mylohyoideus muscle. the frequency of swallowing during the electrical stimulation of the sln decreased after the administration of orexin-a in a dose-dependent manner. significant reduction in number of swallowing was observed after the administration of orexin-a at the dose of or nmol. the latency of the response tended to be longer in the presence of orexin-a. the administration of orexin-b did not affect the frequency of swallowing. pre-administration of sb attenuated the magnitude of inhibition of swallowing frequency induced by the administration of orexin-a. these results suggest that orexin-a but not orexin-b inhibits reflex swallowing via type receptors situated in the dorsal medulla where swallowing center is housed. this work was supported by a grant-in-aid for scientific research from the japan society for the promotion of science (no: to mk). stimulation of water receptors in the laryngopharynx (lp) with water shortened the swallowing interval in voluntary swallowing in humans. we previously found that excitation of water receptors was due to absence or reduced concentration of clin fluids applied to the lp. unstimulated saliva contains a low concentration of cl -. thus, unstimulated saliva may excite water receptors in humans. the aim of the present study was to evaluate the role of unstimulated saliva in initiation of swallowing in humans. solutions were infused through a fine tube into the lp at a slow infusion rate ( . ml/min) that minimized the mechanical effect of infusion. facilitation of voluntary swallowing by infusion of unstimulated saliva was almost the same as that by infusion of water. topical anesthesia of the throat produced loss of facilitation of voluntary swallowing by both water infusion and unstimulated saliva infusion. the similar effects of water and unstimulated saliva on voluntary swallowing suggest that water receptors are responsible for facilitation of voluntary swallowing by unstimulated saliva. since facilitation by water infusion appeared without mechanical effect of infusion, it is likely that excitation of water receptors in the lp by a small amount of unstimulated saliva precedes excitation of mechanoreceptors caused by a certain amount of unstimulated saliva accumulated in the throat. excitation of water receptors by unstimulated saliva may initiate spontaneous swallowing and spontaneous swallowing probably plays an important role in clearance of the mouth of saliva. therefore, it appears that excitation of water receptors by unstimulated saliva may contribute to avoidance of aspiration. # peripheral chemical stimulation to trigger the swallowing reflex and its neuronal mechanisms in humans kensuke kawamoto , takuya inoue , kaya narimatsu , yuki nakamura , makoto inoue , rika yahagi and yasuyuki kitada faculty of dentistry, niigata university, - gakkocho-dori, chuo-ku, niigata - , japan, division of dysphagia rehabilitation, niigata university graduate school of medical and dental sciences, - gakkocho-dori, chuo-ku, niigata - , japan and morioka taste and swallowing research institute, - - higashi-kuroishino, morioka - , japan initiation of swallowing is mediated by the swallowing central pattern generator (cpg) in the brain stem. both peripheral and central inputs can activate the cpg. previous studies demonstrated that the ability to initiate repetitive voluntary swallowing varies even among healthy humans and proposed that appropriate chemosensory inputs, e.g., activation of water receptors in the pharynx and larynx, may compensate for difficulty in initiation of swallowing. however, whether variation also exists in reflexively evoked swallowing is unclear, and the nature of interindividual variation has not been clarified. the aim of this study was to investigate interindividual variation of swallowing initiated by central and peripheral inputs and to clarify the neuronal mechanisms that cause this variation. voluntary and reflexive swallowing activity was recorded during infusion of fluid (distilled water [dw] or . m nacl solution) into the pharynx and larynx at a very low rate in healthy volunteers. dw and nacl were used to activate and inhibit the water receptors, the activation of which facilitates initiation of swallowing. swallowing intervals (sis) were measured between the fourth and ninth swallows, and facilitatory effects (fes) were calculated by subtraction of the si of dw from that of nacl. there was a significant positive correlation between sis of voluntary and reflexive swallowing with nacl. fes of both voluntary and reflexive swallowing appeared strongly in subjects with longer sis. the present results suggest that interindividual variation in the ability to initiate swallowing depends on the potency of the swallowing cpg. the effects of taste stimuli on the change in regional prefrontal cortex blood flow (rcbf) measured by multichannel near infrared spectroscopy (nirs) were evaluated in healthy women volunteers. the test solutions were sucrose, nacl, citric acid and monosodium glutamate at three different concentrations each. six pairs of source and detector nirs probes were placed on the forehead with a cm separation which allowed the measurement of different channels every . sec. the changes in oxygenated hemoglobin (oxy-hb), which is the most sensitive indicator of changes in rcbf, were analyzed. subjects were positioned in a chair that supported their back and head while wearing a blindfold (eyemask) and headphones to minimize motion, visual and auditory disturbances. the entire oral area was exposed to ml of solution for sec applied using a syringe and rubber tube positioned above the head. another solution was applied after rinsing with deionized water, subjects were tested consecutively for different solutions in random order. each subject was tested for the same solution three times, the data were averaged and the value for the water test data subtracted in order to determine subjectÕs value for a given solution. the taste hedonic tone with a five-rating scale was also measured for the preference test. oxy-hb in all channels increased during all taste stimuli. the increases in oxy-hb and preference scale values showed significant negative correlation. these findings indicate that the prefrontal cortex activation evaluated by nirs is possibly related to pleasant or unpleasant emotions associated with taste stimuli. we receive various different stimulus modalities including tastants and other gustatory and / or somatosensory sensations in eating. sensory information from these modalities would be integrated and recognized as a ÔflavorÕ. it is useful to understand their psychophysiological properties because it may help us to develop new functional foods. we investigated the effects of vanillyl butyl ether (vbe), l-menthol (lm) and carbonated water (cw) to the arousal level of healthy subjects. these stimuli were used as hot, cold, tingling stimulus respectively. the results of contingent negative variation showed that vbe, lm and cw increased subjectÕs arousal level just after ingested by aqueous solution whereas no significant change was observed after the water ingestion. the increase of the arousal level still remained for min after ingesting vbe and lm. on the other hand, the arousal level of min after ingesting cw decreased to the same level as before ingestion. apart of the subjects were asked to rate the general stimulus intensity by point likert scale. all samples including water showed maximum intensity just after ingestion and decreased with time course. however, the significant increase of the stimulus intensity still remained for min after ingesting stimulus solutions. these discrepancies between the arousal level and subjective stimulus intensity of cw suggest that only stimulus intensity itself cannot explain the change of the arousal level. in order to understand the property of each stimulus, we need to look each stimulus into detail in the derived modality of the sensation, intensity, timing of appearance. we had reported that umami taste induced sustained saliva secretion. in this study, to investigate the effect of repetitive taste stimulation on saliva secretion, we compared the saliva weight secreted by single taste stimulation (single stimulation methods; ss) with repetitive taste stimulation (repetitive stimulation method; rs), using fourteen healthy adult subjects. in ss, the subjects sprayed the taste solution (approx. . ml) using a spray bottle into the mouth, and held for seconds and spat the whole saliva into cups at every second for minutes. in rs, the subjects sprayed the taste solution into the mouth, and held for seconds, then, spat the whole saliva into cup, and rested for minute until the next stimulation. after this procedure was repeated ten times, the subject performed the same procedure as ss. the stimuli were mm monosodium glutamate (msg) (umami), . mm citric acid (sour) and mm xylitol (sweet), which had ÔmoderateÕ taste intensity on a labeled magnitude scale. during the repetitive stimulation in the rs of msg, the salivary flow was reduced gradually according to the decrease of umami taste intensity by adaptation. however, the msg stimulation caused a sustained salivary secretion after repetitive stimulation, and there was no significant difference in the minutes saliva between ss and rs. thus, the repetitive umami stimulation did not reduce saliva secretion and could produce sustained salivary secretion. there was no adaptation in rs of citric acid and xylitol, and the saliva secretion pattern of rs was similar to that of ss, with less saliva secretion than msg. dried-bonito (db) dashi is a traditional japanese broth that enhances palatability of various cuisines due to its specific flavor. db dashi contains many components, such as histidine, lactate, inosinate, minerals, and proteins. ingestion of db dashi has been reported to have various physiological functions, e.g., effectiveness against physical and mental fatigues, enhancement of peripheral blood flow, and suppression of oxidative stress. in this study, we investigated the effects of db dashi on gastric electrical activity and hunger-satiety states in healthy japanese males. we performed a randomized crossover study for healthy males ( . ± . yr). subjects were fasted for at least h, and all measurements were performed in the morning. before and just after a single ingestion of ml of db dashi (''hondzukuri ichiban-dashi katsuo'', ajinomoto co., inc., japan) or energy adjusted water as a control, gastric myoelectrical activity was measured by electrogastrography for min. subjective motivation to eat (hunger and satiety) was rated by visual analogue scales before, immediately after, and min after the ingestion. ingestion of db dashi significantly increased both electrogastrographic normogastric power and percentage in postprandial state. in addition, db dashi produced higher satiety sensations at min after the ingestion, compared to the control diet. in conclusion, db dashi intake enhances the gastric motility and satiety in humans. we usually take sweet foods with drinking tea or coffee not only to increase nutrition but also to produce feelings of pleasantness and restfulness that belong category of emotion. when we take sweet foods, comfortable audio-visual environments, e.g. consonant interior decoration, good music and fine view may increase feelings of deliciousness. thus, it is hypothesize that environmental factors modify feelings of deliciousness. comprehensive delicious feelings during having sweet foods might be effectively produced in the comfortable environments by integration of various palatable sensory perceptions. electroencephalograms (eegs) were recorded from frontal region of the scalp of healthy participants under virtual scenes of tearoom and construction work, respectively. the participants were asked to rate deliciousness after the recordings. frequency analyses were performed from the eegs. during having the foods, occupancy rates of beta frequency band between tearoom scenes and construction work scenes were markedly different, but not in other frequency bands. during having no food, in contrast, there was no difference of occupancy rates in respective frequency bands between the two different scenes. with regard to deliciousness during having sweet foods, all participants rated high scores under the scenes of tearoom than those under the scenes of construction work. interestingly, there is a positive correlation between occupancy rates of beta frequency band and scores of deliciousness. these findings suggest that comfortable audio-visual environments play an important role for increasing delicious feelings during having sweet foods, in which beta frequency rhythms may be concerned with producing comprehensive feelings of deliciousness. though it has been reported that capsaicin possesses effects of stomach protection and appetite improvement, the influence of dietary habits of consuming pungent substance such as capsaicin on taste sensitivity has not been investigated. in order to elucidate the influence of dietary intake of capsaicin on taste and pungent sensitivities, we measured the thresholds for basic tastes and capsaicin in thai and japanese, because thai have a dietary habit of much more use of pungent substances for meal than japanese. in addition, we conducted questionnaire study about dietary habits of both groups. regarding the recognition thresholds for basic tastes, the threshold for umami tended to be higher in thai compared to japanese with no differences in other basic tastes. this result indicates that daily high intake of pungent substances hardly affect taste sensitivities. however, the threshold for capsaicin in thai was significantly higher than japanese, which was considered to result from much more use of hot pepper for meal in thai. when the thresholds for basic tastes were measured immediately after capsaicin stimulation, the thresholds for acid and bitter tastes were elevated in thai whereas they did not change in japanese. this difference may be due to the concentration of pretreatment capsaicin because suprathreshold concentration was used in the experiment, showing that high concentration of capsaicin may cause transient impairment in some taste sensitivities. in conclusion, it was suggested that daily high consumption of pungent substances hardly affects taste sensitivities but high concentration of capsaicin causes transient hyposensitivity of some tastes. this study aimed to investigate what flavors show interaction with umami taste. twenty-one female university students participated in this study. solutions of . % msg were used as taste stimuli, and the kinds of flavors were used as flavor stimuli. participants were asked to taste a pair of flavor and msg solution, and to evaluate the intensity of umami taste and the congruency of the flavor with the umami. when the participants taste msg solution with flavor of dried bonito, and of garlic, they evaluated the umami taste stronger than when they taste it without flavors (potentiation effect). when the participants taste msg solution with flavor of tomato, of garlic, and of onion, they evaluated those flavors more congruent with umami taste than when they taste it without flavors. they evaluated congruency of dried bonito and of matsutake less congruent with umami taste. there is a significant, but weak, interaction between the congruency with umami taste and the potentiation effect of umami taste (r= . , p< . ). in general, the flavors that are experienced in our daily lives are evaluated more congruent with umami taste, and have potentiation effect on umami taste perception. preceding studies also suggest that the interaction between olfaction and taste, and perception of umami taste are based on learning. this study also supports the view of ''learned synesthesia'' of olfaction and taste. acknowledgment: this study was supported by society for research on umami taste. eating with someone makes the foods more palatable, and the family ties more strong. on the other hands, eating alone (ko-sho-ku, in japanese) has many problems, such as malnutrition, unbalanced meals, not good at communication etc. ko-sho-ku is very severe problems in modern japanese society. thus, we aimed to study the effect of ko-sho-ku on palatability of the foods. in the study , ratings for taste and palatability of confectioneries were compared between when they were eaten with friends and/or family members and when eaten alone. thirty six female university students participated in this study, and were asked to eat two kinds of chocolate and two kinds of cookies in a laboratory (alone) and in their home (with someone). palatability ratings were higher when they were eaten with someone in their home. in the study , ratings for taste and palatability of jellies were compared between when they were eaten with watching a child eating same jellies in a video and without watching it. fifteen female university students participated in this study, and were asked to eat three kinds of jellies in a laboratory and to rate a questionnaire about their ability of sympathy. students with high sympathy tend to rate the jellies more palatable. these results suggested that human perceptions of taste and food palatability were affected not only by foods but also by contextual and social variables. acknowledgement: part of this study was supported by a grant titled "effects of eating with someone on palatability of foods." from asahi breweries foundation. # taste and smell of the bread made using pre-germinated brown rice sake lees kana kogiso , hiroko nakazawa , yumi yoshioka , akiko sato and mitsuo okazaki faculty of human life sciences, nagano prefectural collage, nagano, nagano - , japan and okazaki brewer co, ltd., ueda, - , japan pre-germinated brown rice is prepared by soaking brown rice in water at o c for - hours to initiate germination of sprouts not exceeding . - . mm in length. in the traditional method of brewing sake, rice bran and embryo bud are separated from brewer's brown rice during sake brewing in order to remove the rough taste. this study tried to use sake lees in fermentation with pregerminated brown rice, since the sake made from pre-germinated brown rice has hardly appeared on the market. also, the use and benefit of pre-germinated brown rice-made sake and sake lees have not been reported. in this study, we made sensory evaluation to measure the difference in taste and smell between bread made using normal sake lees and bread made using pre-germinated brown rice sake lees. the result showed that the bread made with pre-germinated brown rice sake lees had significantly better smell (p = . ) and stronger sake lees smell (p = . ) as well as a stronger sake less taste (p < . ) than the bread made with normal sake lees. the complex flavor (p = . ) and saltiness (p = . ) of the bread made with pre-germinated brown rice sake lees tended to be stronger than that of the made with normal sake lees. female college studentsÕ preference to steamed and cured slices of sweet potato was evaluated to find their sensory characteristics. evaluation was carried out with the sweet potato cultivars and lines, tamayutaka, izumi , hoshikirari, kanto , tamaotome, quick sweet, hitachi red, hamakomachi, purple sweet lord and sakukei in terms of appearance, fragrance, texture and taste by a rank-scoring method. the most preferable cultivars were hoshikirari, tamaotome, and kanto ; sweetness greatly contributed to the taste of the steamed and cured slices of these samples, while appearance and texture somehow influenced their taste scores. fish flakes are one of the traditional japanese ingredients for dashi. various methods are used to extract dashi depending on the thickness of flake. especially, the thick type flake that is often used for soba noodle sauce requires around minutes of cooking time and extracting conditions vary as well. the energy saving has become recent tendency even in culinary field, and we focused on a cooking method using remaining heat. we analyzed the free amino acid and #-imp. sensory evaluation was carried out at the same time. as a result, we can suggest that there is a method for extracting the most effective use of remaining heat for minutes after heating for minutes. we were able to explain the taste improvement from the amino acid composition determined by accq-tag tm ultra method. this condition can be considered effective for both energy saving and flavor improvement of dashi. oolong tea is usually served with oil-rich chinese food. our group has been studying on the mechanism of oil rinsing in the mouth and, in particular, on the interactions between oolong tea components and oil. our previous studies showed that oolong tea had a higher affinity for oil than deionized water, mineral water and green tea did. it may thus be more effective in washing out remaining oil from the mouth. the objective of this study is to determine the effective oolong tea components contributing to removing the residual oily sensation. for the human sensory evaluation, sweetened whipped cream was used as an aliment, and mineral water or oolong tea was used as a washer, with the result that oolong tea more effectively reduced the sense of residual oiliness. an aqueous solution of epigallocatechin gallate (egcg) and epigallocatechin (egc) as tea constituents was used to measure its interfacial tension activity in the presence of soybean oil by a face surface tensiometer. also, we emulsified soybean oil with egcg or egc and examined the resulting average particle size with a laser diffraction particle size analyzer. it was found that egcg and egc did not influence the interfacial tension and average particle size. further studies are required to find some contributory factor. commercial granulated sugars are of . % purity which is almost equivalent to the purity of a guaranteed reagent-grade sucrose. we observed that commercial sugars were different in melting point from one another and the tastes of caramel sauces prepared from these sugars were different. a white coarse bright sugar with larger grain size than that of regular granulated sugar used as an experimental sucrose crystal was milled into powder. in a differential scanning calorimetry of the crystals, a higher peak in the curve of endothermic heat flow shifted to higher temperature side after milling. the powder crystal became more stabilized on heating than the coarse crystal. using the coarse and the powder crystals, caramel sauces (''sauce c'' and ''sauce p'', respectively) were prepared. sensory tests showed that sauce c was more brownish in color and was stronger in sourness, bitterness and acridity. on the other hand, sauce p was better in aroma, stronger in sweetness and more preferable in taste and aftertaste. sauce c was lower in ph, giving stronger sourness. the use of a color difference meter showed a large difference between the two sauces (de= . ). coarse crystal was less stable on heating and sauce c was more brownish in color, having stronger bitterness. in the sensory studies for flavor development, commercial products or trial products are often used as samples, because sensory evaluation is conducted considering usual behavior of eating habit. but in the case of odor-taste interaction studies, it is desired that odorants and tastants are completely separated and presented to subjects simultaneously. in this study, we established a simple and effective way of presenting odor and taste stimuli separately under two model systems: beverage and soup. experiment. : evaluation of sweet/sour intensity in fruit flavor beverage firstly, we prepared simple equipment comprising clear plastic cup with scented papers attached on the upper inside edge. the use of non-flavored solution and flavor on the scented papers allow subjects to evaluate beverage. sugar-acid water solution was used as taste stimulus and, lemon and strawberry flavors were used as odor stimuli. untrained panel (n = ) evaluated intensity of sweet and sour characters. the result showed that lemon flavor enhanced sour character and decreased sweet character when compared with non-flavored condition. experiment. : evaluation for savory -citrus flavor interaction we prepared ordinary soup solution as taste stimulus, with two savory flavor (chicken and dried bonito) and two citrus flavor (lemon and yuzu) as odor stimuli. untrained panel (n= ) evaluated characters (saltiness, umami, sourness, mild, fatty, animalic). the results showed flavor influenced taste intensity as is the case in experiment . in conclusion, we successfully showed the important effect of odor on taste perception by using this study. # added flavorings enhance salivary hemodynamic responses to tastes detected by near-infrared spectroscopy -flavor creation using optical imaging-dried-bonito (katsuobushi) broth, an important seasoning in japanese cuisine, has long been used to reinforce the flavor of appetitive foods. not only the taste but also the aroma components of the broth contribute well to the enhancement. to elucidate the effects of aroma from dried-bonito on broth tastes caused by the central integration of flavor, we performed optical imaging of salivary hemodynamic responses using near-infrared spectroscopy (nirs). when hemodynamic responses to a reconstituted dried-bonito flavored broth were compared to those of odorless broth taste solutions, the flavored broth produced a significantly larger response than the odorless broth. this was the case for five of the ten participants, who felt that the combination of the aroma with the tastes was congruent. in the remaining five who felt the combination incongruent, the flavored broth did not cause the enhancement of responses. moreover, when (z,z)- , -tridecadienal, one of the most important key aroma components of dried-bonito extracts, was added to the flavoring, the latter five as well as the former five participants came to feel congruency of the reconstructed aroma with broth tastes. the reconstruncted flavored broth produced significantly larger hemodynamic responses than the odorless broth in both participantsÕ groups. accordingly, there is a positive correlation between the degree of congruency and increase of the salivary responses. these results indicate that nirs offers a sensitive method to detect the effect of flavoring on the taste-related salivary hemodynamic responses, dependent on the perceptual experience of the combination of aromas and tastes. minty flavors are utilized in foods, beverages, toothpastes and tobacco. minty flavors are considered to have a relaxing effect, which is necessary for us, living in modern and urban societies. the authors have reported that the participants evaluated themselves more relaxing after smelling minty odor, when they were tied out for solving hard puzzles. however, psychological and physiological effects of minty flavors are still unclear. this study was aimed to reveal brain responses to minty flavors and minty odors with nirs (near infrared spectroscopy). sixty female university students participated in this study. they received detailed explanation about the experiment and wrote consent for participating in this study. the three minty stimuli, menthol, spearmint and peppermint and the other three odor stimuli, rose, skatol and orange, were administered to the participants via the olfactometer. the participants were asked to smell the stimuli by an orthonasal or retronasal route or to taste them. brain responses were measured with nirs (bom- , omegawave, co ltd) and recorded with ad converter (powerlab) connected to the imac. the participants were also asked to evaluate the intensity of the odor continuously with the apparatus which outputs ; v being based on the participantsÕ evaluations. the results showed that the stimuli were recognized faster and stronger when the stimuli were administrated via orthonasal and retronasal routes than via oral cavity. the rose odor and orange odor were recognized faster and stronger than the menthol odor. the right forebrain showed greater responses to the stimuli via an orthonasal route than via the other routes, and greater responses to the rose odor than the menthol and the peppermint odors. currently existing odorant sensors are mainly fabricated based on metal-oxide semiconductor devices, conducting polymers, quartz crystal microbalances, or surface acoustic wave detectors. these sensors have been studied for improving on various parameters, such as sensitivity, selectivity, response speed, and portability. however, it has been difficult to develop odorant sensor elements that incorporate a combination of desirable properties. in contrast, living organisms, especially insects, use numerous olfactory sensory cells, which express odorant receptors, to sensitively detect environmental odorants in real time. so far, we have developed a compact odorant sensor that consists of a multichannel fluidic device and xenopus laevis oocytes expressing insect odorant receptors. although this odorant sensor possessed fast responsiveness and good portability, it had some technical disadvantages in terms of the stability of response measurements in the oocytes. here, we report the development of new odorant sensor elements that enable us to acquire stable odorant response measurements. we introduced insect odorant receptors and the olfactory receptor co-receptor (orco) as well as a calcium indicator protein, gcamp , into spodoptera frugiperda sf cells to construct sensor cell lines. when these cells were stimulated with a set of odorants in solution, intracellular calcium as monitored by fluorescence imaging showed sensitive responses in accordance to the ligand specificity of the expressed odorant receptors. these results show that our sensor cells can be applied as odorant sensor elements that detect various kinds of odorants with high sensitivity, selectivity, and stability. with promotion of globalization, our diet are changing in several directions (diversification, mass commercialization, or high quality). because of the change, objective evaluation techniques for taste or odor of foods and beverages are needed for effective r&d and strict quality control. in recent years, biomimetic sensors, such as taste sensor, have been more developed as practical objective evaluation tools for taste or odor. with development of these sensors, development of correlativity evaluation system between objective values (sensor outputs) and subjective values (sensory scores) is needed to realize objective quality evaluation of foods and beverages. mainly developing artificial-lipids-based taste sensors with global selectivity, our research group have studied for realization of Ôtaste-odor fusion biosensor system,Õ which estimates quality (deliciousness and safety) of foods or beverages using several sensor outputs through analysis and evaluation of subjective-objective relation. in the studies, we have estimated relations between each taste sensory and each sensor output, but did not investigate about correlativity among quality of foods or beverages and sensor outputs. hence, in this study, we investigated correlativity among sensory of deliciousness and several sensorsÕ outputs on a particular sample as the next step of realizing Ôtaste-odor fusion biosensor system.Õ as a result, we found that sensory scores of deliciousness were able to be displayed on a superspace as a hyperplane and to be estimated by the hyperplane and sensor outputs. this means that a sensory score on the particular sample can be calculated using sensor outputs and an estimation equation indicating the hyperplane. it has been reported that taste disorder caused by systemic diseases is associated with zinc deficiency. this study included patients with dysgeusia due to systemic diseases: patients with diabetes mellitus (dm), with renal failures, with digestive diseases, with digestive diseases post operation, and with liver failure. taste function was tested by electrogustometry (egm) and filter paper disk method (fpd method) in the area controlled by the chorda tympani nerve. the severe grades of taste threshold before treatment were more than % for renal and liver failure patients. all patients were treated with zinc sulfate. the rates of serum zinc deficiency (< lg/dl) were more than % for all systemic diseases. in patients with renal failure, the symptom improvement rate was the highest, and the curative period was shortest. however, in dm patients, the improvement rate was the lowest, and the curative period was longest. this study demonstrated that zinc deficiency is induced by systemic diseases, and might cause taste disorder. it is also necessary to consider the possibility of taste disorder caused by medication. this study analyzed patients with bms. the threshold of the taste function was measured by electrogustometry (egm) and the filter-paper disk (fpd) method. both basal and stimulated salivary secretions were measured. in addition, the self-rating depression scale (sds) was assessed. serum zinc and copper levels were also measured. the egm threshold was more than db in ( . %) of patients, and the taste threshold measured by the fpd method was more than points in ( . %) of patients. basal and stimulated salivary secretions were less than ml in ( . %) of patients and less than ml in ( . %) of patients, respectively. based on the sds, ( . %) and ( . %) of patients were diagnosed with neurosis and depression, respectively. blood tests showed that the serum levels of zinc were less than or that of copper were more than lg/dl, respectively, in ( . %) of patients. the clinical outcome was cure in . %, improvement in . %, recurrence in . %, unchanged in . %, and dropped out in . %. in this study, basal salivary secretions lower than the normal range compared with stimulated salivary secretions. this suggests that, even if the salivary secretory function is maintained to some extent, the sympathetic nerve activity is predominant due to tension. it is necessary to accumulate more clinical data, such as trigeminal sensitivity of the tongue in comparison with the electric taste threshold. postoperative oral tumor patients show a variety of conditions and often display taste dysfunction resulting from anti-cancer drugs, irradiation and defects of oral tissue. prosthetic rehabilitation should be required for the recovery of oral functional disorders. however, no reports have investigated taste disorder for patients wearing maxillofacial prostheses. the aim of this study was to clarify taste sensation in postoperative oral tumor patients using the filter-paper disc method, salivary secretions measured by gum chewing, serum zinc concentrations by blood tests. we investigated patients who had undergone prosthodontic treatment in the department of prothodontics and oral rehabilitation at iwate medical university dental center. results were as follows: ) taste disorder was seen in % of cases; ) within years after radiation exposure, the degree of taste disorder was severe; ) disease severity did not necessarily accord with awareness of taste disorder, and some patients with serious taste decline were unaware of their taste disorder; and ) patients who were aware of their taste decline also showed declines in chewing score. objective inspection and liaison with other departments to include an otolaryngologist, doctor of psychosomatic medicine, and a dietitian is important. it is known that more than % of patients with taste dysfunction have zinc deficiency. carbonic anhydrase (ca) vi, also known as gustin, is a , -da zinc metallo-protein found in human saliva and is involved in taste dysfunction. it can be detected by enzymelinked immunosorbent assay, but the test takes time. therefore, in this study, we developed a new quick and simple method of diagnosis using an immunochromatography assay to be used as an on-site test. first, reactivity with a serum antibody (prepared using a synthetic peptide designed from a - amino acid chain of human ca vi as a hapten conjugated to keyhole limpet hemocyanin and injected into two new zealand white rabbits) was evaluated and compared to a control (swine antirabbit igg) using a hi-flow plus membrane and detected with colloidal gold. a difference in color development was observed between samples of pbs (-) and the positive control (synthetic peptide). furthermore, it was density dependent, and in serially diluted parotid gland saliva, the color development was seen to be times dilution density from times dilution density. the results suggest that an immunochromatography assay using this novel antibody can be used to quantitatively measure ca vi, which may be useful in the diagnosis of taste dysfunction caused by zinc deficiency. # the epithelium of recessus olfactorius is a part of the accessory olfactory system in the japanese toad (bufo japonicus) hideo nakazawa , kimiko hagino-yamagishi , atsushi c. suzuki and takatoshi nagai the recessus olfactorius (ro) is a small pouch observed in the antero-lateral olfactory epithelium of anurans. ro is mostly covered with the epithelial cells with cilia, which is reminiscent of sensory function. however, if ro is a part of the main olfactory system has not been studied. we studied the epithelium of ro (ro epithelium) in japanese toads regarding central projection, ultrastructure and expression of proteins involved in olfactory transduction. fluorescent carbocyanine dye (dii) was placed on the ro epithelium to show a neuronal projection from ro. fluorescent signal was not detected in the main olfactory bulb, but in the accessory olfactory bulb. in contrast to the olfactory epithelium which does not contain microvillous receptor cells, the ro epithelium contained both ciliated cells and micro-villous cells. the expression of a-subunit of g-proteins (g aolf and g ao ) in the ro epithelium was examined by immunohistochemistry and in situ hybridization. regionally different expression of g aolf and g ao was detected in the ro epithelium, namely expression of g aolf in the apical cell layer and g ao in the basal cell layer. such a pattern of g-protein expression in the ro epithelium was similar to that in the vomeronasal epithelium. these results suggest that the ro epithelium belongs to the accessory olfactory system. however, the ro epithelium contained secretory cells with granules that were not contained in the vomeronasal epithelium. physiological experiments will further clarify if the ro epithelium is functionally similar to the vomeronasal epithelium. in mice, the female memorizes some urinary pheromones of the mating male, thereby preventing the pheromones from inducing pregnancy block. the memory depends on the neural changes in the accessory olfactory bulb (aob), the first relay in the vomeronasal system. microcircuits in the aob include the prominent reciprocal dendrodendritic synapse between mitral cell projection neurons and granule cell interneurons. a previous study has shown that in vivo infusion of a protein synthesis inhibitor, anisomycin, in the aob is able to prevent the pheromonal memory formation. other reports have indicated that antidromical stimulation of mitral cell axons induces long-term potentiation (ltp) at the mitral-to-granule cell synapse in slice preparations of the aob. using aob slices, we measured field epsps derived from granule cells to examine the effects of protein synthesis inhibition on ltp at the mitral-to-granule cell synapse. high frequency stimulation, consisting of a hz, pulse train applied four times at min intervals, induced ltp lasting for min. under bath application of anisomycin ( lm), high frequency stimulation induced short-term potentiation and early-phase ltp, but failed to induce late-phase ltp. the results suggest that protein synthesis underlies late-phase ltp at the mitral-to-granule cell synapse in the aob. the goal of our research is to understand the mechanism of synaptic transmission in the aob that has been demonstrated to be critical to memory formation for male mouse pheromones. by measuring the reciprocal synaptic currents from mitral cells in the aob, we have demonstrated that an agonist for group ii metabotropic glutamate receptors (mglur /mglur ), dcg-iv, suppressed dendrodendritic inhibition (ddi) in a reversible manner while the mglur /mglur antagonist ly enhanced it. the effects of these drugs were markedly impaired by genetic ablation of mglur , indicating that dcg-iv-mediated suppression of ddi is mediated by mglur . in these studies, glutamate release was triggered by an application of a voltage step from - to mv. in the present study, to see whether mglur has similar effects on the ddi elicited by more physiological stimuli, ipsps triggered by spike trains in mitral cells in slice preparations were recorded using the patch-clamp technique in whole-cell configuration. aob slices were prepared from -to -day-old balb/c mice. ly enhanced it. together with previous results, the present result suggests that mglur /mglur can be activated by endogenous glutamate release from mitral cells, which results in the suppression of the synaptic transmission from mitral to granule cells in the aob. phenols are predominant in the total flavor compounds of dried bonito (katsuobushi). the influence of odors of , , -trimethoxybenzene (tm; m= ), , -dimethoxyphenol (dt; m= ), and guaiacol (gu; m= ), which were the chemicals responsible for the katsuobushi aroma, on the production of b-endorphin (be) in the hypothalamus were examined in f male rats. rats ( weeks of age) were exposed to . % of odors dissolved in either ethyl alcohol (etoh: m= ) or triethyl citrate (tec: m= ) for h/ day for one or two weeks. be levels in water soluble extracts of hypothalamus were examined by elisa kits. among three phenols, tm is the most potent odor to increase be levels in rat hypothalamus, followed by dt and gu: the increasing rate as compared with the control was %, % and %, respectively. these phenols may activate glomeruli clusterd in the lateral domain of the dorsal surface of the mammalian olfactory bulb and probably suppress the activities of sympathetic nervous system. these results suggest that the long-term exposure to bonito bouillon-flavored diet enhanced ratÕs preference for bonito bouillon. neurogenesis occurs in the forebrain subventricular zone (svz) throughout life. these neurons migrate to the main olfactory bulb (mob) via the rostral migratory stream (rms), and upon reaching the mob they differentiate into granule cells and periglomerular cells. the information broadcast by general odorants is received by the olfactory sensory neurons and transmitted to the mob. newly generated neurons at the svz play important roles in odor discrimination and odor memory. recent studies have shown that reduction of mastication impairs neurogenesis at the hippocampus and brain functions. in the present study, bromodeoxyuridine-immunoreactive (brdu-ir) structures in the sagittal section of the svz, rms, mob and accessory olfactory bulb (aob) of female adult mice fed a soft diet were studied to explore the effects of reduction of mastication on newly generated neurons at the svz and mob. the numbers of brdu-ir cells in the svz of adult mice fed a soft diet for , , or months were lower than those of mice fed a hard diet. the odor preferences of individual female mice to butyric acid were tested in a plexiglas y-maze preference apparatus. feeding the soft-diet affected the adverse responses of mice to butyric acid. the results suggest that feeding with a soft-diet reduces neurogenesis at the svz, which in turn reduces olfactory function at the mob. epigenetic mechanisms play an important role in memory formation and synaptic plasticity. specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. before eye opening young rats depend on somatosensory and olfactory function for survival, as they can learn their damÕs odor and approach her without visual information. in order to establish olfactory learning, the pairing of odor and somatosensory stimulation is crucial. we have shown that synaptic plasticity in the ob underlies aversive olfactory learning. our behavioral pharmacological experiments have shown that long-term olfactory memory requires activation of creb. western blot analyses have revealed that expression of p-mapk/erk is increased for hour after odor-shock training, followed by increase of p-creb lasting for hours. therefore, we examined whether intrabulbar infusion of trichostatin a (tsa), a histone deacetylase (hdac) inhibitor, facilitates olfactory learning in young rats. tsa infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner. we further tested whether odor-shock training leads to histone acetylation in the ob and defined the time course of the activation. the acetylation of histone h was significantly increased for hour after odor-shock pairing compared with odor only or no stimulation. tsa infusion significantly increased histone acetylation levels as well. these results show that hdac inhibition is associated with aversive olfactory learning in young rats. the endopiriform nucleus (epn) is a large group of multipolar cells located in the depth of the pirifprm cortex (pc). although many studies have suggested that the epn plays a role in temporal lobe epilepsy, the normal function of the epn remains to be elucidated. by using optical imaging with voltage-sensitive dye in slice preparations, we previously found that paired-pulse stimulation to pc and/or gustatory cortex (gc) evoked the excitation propagation to the epn, agranular division (ai) of the insular cortex and further to the claustrum (paired-pulse facilitation). in the present study, we investigated the electrophysiological properties of neurons in the epn, claustrum and ai in anaesthetized rats. in response to electrical stimulation of the olfactory bulb, the evoked potentials with a latency of about ms were elicited in these regions. paired-pulse facilitation of the evoked potential was also observed. of the neurons in these regions tested with odor and gustatory stimulation, ( %) showed excitatory responses to both odor and taste stimulation, ( %) had excitatory responses to odor but not to taste stimulation, ( %) had excitatory responses to taste but not to odor stimulation, and the remaining ( %) had no response. further, we previously found significant increases in the numbers of c-fospositive cells in the pc, gc, epn, ai and claustrum following natural stimuli, such as feeding an apple. these results, together with previous results, suggest that the epn, claustrum and ai are the multimodal-chemosensory regions, where olfactory and gustatory information is centrally integrated. department of science education, faculty of education, chiba university, chiba - , japan we found that a simple and typical association learning of a flower odor with proboscis extension reflex to nectar in honeybees was inhibited by exposure to extremely low frequency (elf) alternating magnetic fields. we postulated that the elf magnetic fields could be detected by magneto-receptors. honeybees have two types of putative magneto-receptors which either contain magnetite crystals, abdominal dark short hairs or abdominal trophocystes. we previously reported that a cutting of the abdomen longitudinal connective nerve between rd ganglion (first ganglion in abdomen) and th ganglion had no effect on the inhibitory action of alternating magnetic fields. and we concluded that the trophocystes had no role in magnetoreception. in this work, we confirmed that the dark hairs had a role in magnetoreception and inhibitory action of olfactory learning by magnetic fields. an exposure to hz alternating magnetic fields of approximate lt inhibited the performance of conditioning learning in intact bees. when all dark hairs were pull out, an exposure to alternating magnetic fields had no inhibitory effects on the conditioning. so, it was shown that the dark hairs had a role in magnetoreception which impaired olfactory learning. # prediction of glomerular activity patterns using the graph structure of odorant molecules zu soh , toshio tsuji , yuichi kurita , noboru takiguchi and hisao ohtake grad. school of engineering, osaka univ., suita - , grad. school of engineering, hiroshima univ., higashihiroshima - , grad. school of natural science and technology, kanazawa univ., quantitative evaluation of odor qualities is an important process especially in the fragrance, food and beverage industries. however, most of the conventional odor sensing systems are unable to assess odor qualities perceived by humans. this study therefore aims to develop an artificial sensory evaluation system that can be used to evaluate odors and support sensory testing. as the first step in the development of such a system, we proposed a neural network model for prediction of olfactory glomerular activity based on the results of previous studies, which reported that odors evoking similar glomerular activity patterns have similar odorant qualities. the proposed model consists of marginalized graph kernels and a multilayer neural network. the former is used to evaluate distances between odorant structures, and the latter converts these distances into glomerular activity patterns. however, a number of parameters included in the model required further discussion. we therefore explored the two parameters that most significantly influenced the prediction accuracy of the model. these were the number of the gaussian units and the number of neuron units in the neural network. the results indicated that optimal prediction ability was achieved when the number of the gaussian units was approximately sixty and the number of neuron units in the hidden layer was approximately double than in the input layer. based on these outcomes, future plans for further improvement of prediction ability were discussed. in food and beverage industries, quantitative assessment of odors is an important process in developing new products. however, conventional odor sensing systems focus on identifying or detecting specific odors, and are unable to assess odor qualities perceived by humans. against this background, our study aims to develop an odor assessment system that can be used to evaluate odors and support sensory testing. toward the development of a model for the evaluation of odor qualities, this study focuses on neural activity evoked by odorant stimuli in the olfactory system. this is because the results of previous studies suggested that odorants prompting common neural activity patterns in the olfactory system had similar odorant qualities. as a first step, we conducted a series of human sensory tests to investigate perceptual similarities between odorants, and then compared the results with activity patterns evoked on the glomerular layer of the olfactory bulb in rats. these animals were chosen because the structure of their olfactory system is similar to that of humans, and measurement data relating to their glomerular activity is available online. we defined three indices (correlation, overlap rate of activation, and sum of squares of difference between histograms) to describe similarities and differences between activity patterns, and also conducted sensory testing to determine similarities to human perception. comparison of the results revealed that each index showed a weak correlation to perceptual similarities. we therefore examined whether similarities between odorants could be predicted by combining the three indices using svm (support vector machine), and the results indicated a certain level of prediction ability. novel esters, alcohols, and ethers containing a tetrahydropyran ring were derived from ( , , -trimethyltetrahydropyran- -yl)acetic acid ( ) or -methyl-( , , -trimethyltetra-hydropyran- -yl)propanoic acid ( ), and their odor properties were investigated. the relationships between the structures of the derived compounds and their odor properties were also examined. the methyl ester of carboxylic acid ( ) had a refreshing, floral, and herbaceous odor, and the methyl ester of carboxylic acid ( ) had a refreshing, woody odor. the propyl ester of carboxylic acid ( ) had an adzuki-bean odor, and the propyl ester of carboxylic acid ( ) had a vegetable and spicy odor. thus, the odor of methyl ester of carboxylic acid ( ) was thus similar to that of methyl ester of carboxylic acid ( ), but considerably different from that of the propyl ester. moreover, the odor of propyl ester of carboxylic acid ( ) was considerably different from that of propyl ester of carboxylic acid ( ). to determine the most stable structures of these esters, the molecular orbital calculation was carried out for each ester using mopac pm . the bulkiness of the side chain of a tetrahydropyran ring of methyl ester of carboxylic acid ( ) was comparable to that of methyl ester of carboxylic acid ( ). however, the side chain of propyl ester of carboxylic acid ( ) was bulkier than that of its methyl ester, and this increased bulkiness prevents the free rotation of the side chain of propyl ester. similarly, the bulkiness of the side chain of propyl ester of carboxylic acid ( ) makes its free rotation more difficult than that of propyl ester of carboxylic acid ( ). thus, the bulkiness and the degree of free rotation of the side chain of a tetrahydropyran ring influence the odor of compounds containing a tetrahydropyran ring. measured odor thresholds depend on both subject sensitivity and method. yet, threshold-methods have received relatively little systematic attention in olfaction. we measured psychometric, i.e., proportion correct detection vs. concentration, functions for acetic acid. a -outof- , forced-choice procedure was used. stimuli were precisely controlled using an air-dilution olfactometer. the design had four factors, all randomized or counter-balanced: ) practiced subjects vs. unpracticed subjects (between subjects); ) -second inter-trial interval (iti) vs. -second iti (within subjects); ) re-sampling allowed (i.e., subjects could smell each of the stimuli presented during a trial as many times as they wished) vs. not allowed (within subjects); ) concentrations presented in ascending order (lowest concentration first, moving up to the highest concentration, then starting again at the lowest concentration after a break) vs. random order (within subjects). a four-way anova (the above four factors) revealed a significant main effect of re-sample condition. these results highlight the importance of methods for measured thresholds, and have implications for laboratory practice. many researches have reported the influence of information to identify an odor. we tried to measure the prefrontal cortex activity during the smelling an odor using functional near-infrared spectroscopy (fnirs) to find out the effects of positive and negative information with a same odor. we used three kinds of odors which have been evaluated as moderately hedonic values by a preliminary experiment. eighteen subjects who had normal sense of odor were presented these odors with visual stimulation on a display. the visual stimulation showed the explanations of each odor with positive or negative information. during the experiment, subjects were measured for their prefrontal cortex activity with channels ( x distributions) by fnirs (foire- , shimadzu corp). these odors were presented by odor-stimulation system through tubes and a mask. the experiment was conducted by the following protocol; pre-resting period ( s), task period ( s), evaluation period ( s) and post-resting period ( s). we had trials ( odors x positive/negative information x trials) and control tasks with odorless. during the evaluation period, subjects evaluated the intensity, pleasantness/unpleasantness and familiarity of each odor. the stimuli were presented by random order. as a result, we showed the different evaluation value on the same odor but different information condition. the positive information condition showed significantly higher pleasant value than negative information condition (p<. ). fnirs data also showed the significantly different prefrontal cortex activation between positive and negative information conditions. we showed the effects of positive and negative information on a same odor. first impression of a stranger is created by several perceptions, such as face, tone of voice, speed of words etc. there are many researches on development of first impressions, but there are few researches that study a role of olfaction on development of first impressions. we wear perfume not only because we like it, but also because we want someone to have good impressions. this study aimed to investigate whether the perfume that we wear affects othersÕ first impression for us. sixty female university students participated in this study, and all of them were not acquaintance of the experimenters. ck one, citrus-floral perfume, and coco chanel, fresh oriental note perfume, were used as olfactory stimuli. the method to investigate for participantsÕ impression for the female model (experimenter) was teg ii (tokyo university egogram ver. ii, published by kaneko shobo). when the model wore ck one, she was evaluated as having stronger sense of responsibility and the makings of leader. on the other hand, when the model wore coco, she was evaluated as having unique personality and strong self-assertion. these results show the perfume that we wear changes othersÕ first impressions for us. the authors are continuing to study about this phenomenon, and confirm it with another models and with another perfumes. # a study of the image congruency between bottle appearance and fragrance of perfume appearance of bottles might influence on the evaluation of perfumes as visual information before sniffing fragrance. we investigated such congruencies/ incongruencies between visual and odor information in the market products of perfume through the two experiments. in the experiment , we investigated broad constructs of image aroused from appearance of bottles and flagrance of perfumes, and reviewed congruencies between bottles and fragrances. we selected products as samples, and used ''napping'' for experiment . in napping, subject sort the samples on the space reflecting sensory similarities. our subjects carried out napping under the two conditions. subjects were asked to sort samples according to similarities of scents. these scents were sniffed actually in the sniffing condition, but in the viewing condition, scents were imagined from appearance of bottles. distances matrixes between samples were measured from results of napping, and were used for multi-dimensional scaling (mds). the result of mds showed that dimension could represent major image of perfumes (for men or female) in the both conditions, and that diman-sion in the viewing condition could represent lightness of scents related to transparencies of bottles. however, we could not find systematic feature change of scents on dimension in sniffing condition. it may suggest complexity of scents sniffed actually. we then examined the qualitative aspects in congruencies/ incongruencies between bottles and flagrancies in the experiment . we selected among perfumes used in experiment , and used descriptive terms to evaluate each of the samples. in the results of experiment , we could not find any relations between terms with perfumes. changes of the physiological parameters (saliva amylase, heart rate, and brain waves) of subjects during the smelling of the bark oil of lindera umbellata were measured. the value of saliva amylase did not change during the smelling of the bark oil. the proportion of the parasympathetic nervous system increased significantly during the smelling of the oil, while that of the sympathetic nervous system decreased. on the other hand, the proportion of b-wave decreased significantly during the smelling of the oil. thus, the bark oil exhibited the relaxation effects for the subjects. the bark oils included monoterpene alcohols ( . %) and ketones ( . %), monoterpene oxides ( . %), monoterpene hydrocarbons ( . %), and others. among of these, linalool ( . %) was the most abundant component. here we investigated relationship between the number of constituent odors and odor pleasantness. we hypothesized that the stability of the olfactory perception depends on the number of odor components and their molecular variability. we especially focused on odor pleasantness, which best explains molecular variability of odorants in the natural world. three different categories of odors, aliphatics, aromatics and terpenoids, were used in this study. we used or odors for preparing multi component odors. odor pleasantness for the multi component odors tended to be more stable against concentration changes than that of the single component odors. notably, multi component odors containing odorants of all categories showed higher stability than odors containing single odor category. future study for large number of components with different molecular variability will reveal rules for the prediction of the odor perception for odor mixtures. # fmri of brain responses to complex odor and visual stimuli one of the most important features of odor perception is the hedonic or emotional component. the purpose of this study was to measure the brain activity associated with human olfaction using functional magnetic resonance imaging (fmri) and to evaluate the influence of the pleasantness of odors by complex odor and visual stimuli. seventeen healthy right-handed subjects ( men; women) with normal olfaction participated in the study. an event-related mri method was used to analyze olfaction-induced responses on fmri. a puff of odorant was delivered to the nose of each subject through a nose mask with electrically controlled pneumatic valves. for the event-related task in the fmri experiment, two odorants (pleasant odor: amyl-acetate, unpleasant odor: iso-valeric acid) and two types of visual stimuli (pleasant image, unpleasant image) were randomly presented. two cross-modal sensual interactions were tested using spm-software for both matching/mismatching conditions and pleasant/unpleasant conditions, respectively. results revealed specific brain areas that were activated by cross-modal interactions with odor and visual stimuli. in advance of the fmri experiment, psychological odor experiments (cross-modal, complex stimuli with a visual and odor evaluation test) were conducted using a subjective judgment method or pleasantness/unpleasantness, respectively. based on the fmri and psychological experiments, we estimated the most suitable conditions for evaluating the pleasantness of odors. from analysis of the fmri experiments, two responses ( : emotional response to pleasantness/unpleasantness, and : response to matching/ mismatching) were obtained with the cross-modal, complex stimuli using odors and images. from the analysis of our experiments, activity associated with the response to pleasantness/unpleasantness was seen mainly in the right orbital frontal cerebrum and the supramarginal gyrus, middle frontal gyrus and amygdala. on the other hand, activity associated with the response to matching/mismatching was seen in areas of the brain corresponding to memory and cognition. thus, cross-modal, complex effects were suggested to be an emotional response to pleasantness/unpleasantness and cognitive and memory responses to matching/mismatching. as otorhinolaryngologists, it is important to grasp the grade of subjective symptoms and quality of life (qol) when we examine outpatients with olfactory dysfunction. however, the influence that the sense of smell exerts on the qol related to health is not clear. therefore, we investigated outpatientsÕ qol using the ''health-related qol measure (sf- ),'' which is widely used around the world. concurrently, we applied a ) visual analog scale (vas) to grade subjective symptoms (the smell of food and drink, perfumes, excreta and gases) and the deliciousness of meals (qol), ) smell questionnaire for daily life, ) t&t olfactometry, ) intravenous olfaction test (alinamin test) and ) card-type smell identification test (open essence). we then analyzed those tests and sf- for correlations. as the results, ''vitality (vt),'' which is a subscale of sf- , correlated weakly with the subjective symptom vas for ''smell of perfume'' (r s = . ) and the t&t recognition threshold (r s = . ). ''role functioning emotional (re),'' which is another subscale of sf , correlated weakly with the subjective symptom vas for each of ''smell of food and drink'' (r s = . ), ''smell of perfume'' (r s = . ) and ''smell of gas'' (r s = . ). no other correlation was found for any of the olfactory tests. these results showed that, although olfactory dysfunction affected the vt and re of the patientsÕ health-related qol, it was very slight. even if olfactory dysfunction develops, the effect on the overall qol of the patient is slight compared with other diseases. this likely explains why these patients are slow to consult a doctor. ½introduction olfactory dysfunction is most commonly caused by chronic rhinosinusitis (crs) in japan. within crs, eosinophilic chronic rhinosinusitis (ecrs) is said to cause severe olfactory dysfunction. however, there have been few reports that have compared ecrs with non eosinophilic chronic rhinosinusitis (necrs). ½patients and methods a prospective study was carried out at three institutions. a total of patients underwent endoscopic sinus surgery between april and march and were pathologically diagnosed with crs. of those, patients ( males, females; mean age: . years) were included in this study because they had undergone olfactory evaluation preoperatively. olfactory dysfunction was assessed using the standard olfactory threshold test (t&t) and subjective symptom score ( ; ). data were analyzed using spss software ver. . ½results the patients consisted of necrs patients, ecrs patients, patients with allergic fungal rhinosinusitis (afrs), patients with odontogenic maxillary sinusitis and patients with fungal rhinosinusitis. t&t found that . % of the total patients had olfactory dysfunction with a mean recognition threshold of ‡ . ( of necrs patients; . %, of ecrs patients; . %), while . % of the total patients had olfactory dysfunction with a subjective symptom score of ‡ ( of necrs patients; . %, of ecrs patients; . %). both the t&t result (or: . ) and the subjective symptom score (or: . ) were significantly worse in the ecrs patients than in the necrs patients. [introduction] we investigated the hypothesis that the prognosis of olfactory dysfunction (od) due to chronic rhinosinusitis (crs) becomes worse as the duration of od becomes longer. [patients and methods] patients ( males, females; age range: - years; mean age: . ± . years) with od due to crs underwent appropriate surgery and postoperative treatment, and were followed up for at least months. a visual analog scale (vas) was used to evaluate their pre-and post-treatment symptoms of od. [results and discussion] patients with an od duration of ‡ years showed significantly less improvement compared with an od duration of < years. the od duration and the post treatment vas showed a significant negative correlation (r s =À. , n= ). it was particularly strong in male patients aged ‡ years (r s =À. , n= ), and in the aspirin-intolerant asthma (r s =À. , n= ), peripheral eosinophil count of ‡ /microl (r s =À. , n= ) and current smoking (r s =À. , n= ) subgroups. multiple logistic regression analysis of patients with severe od even after appropriate treatment identified the following as poor prognostic factors: ) male gender (or= . ), ) od duration ‡ years ( . ), ) age ‡ years ( . ) and ) peripheral eosinophil count of ‡ microl ( . ) (predictive rate: . %). these results indicate that the prognosis of od due to crs becomes poorer as the duration of the disease becomes longer. in particular, patients who have these risk factors should be started on treatment as soon as possible and should be advised to quit smoking. motohiko suzuki, yoshihisa nakamura, hiroshi kawaguchi and shingo murakami causative viruses of postviral olfactory dysfunction have not yet been identified. we investigated causative viruses in patients with postviral olfactory dysfunction. we examined the presence of viruses in nasal discharge and examined the time course, with regard to changes in olfactory dysfunction and nasal obstruction in patients with postviral olfactory dysfunction, using questionnaires, acoustic rhinometry, and olfactory tests. nasal discharge was collected from patients with postviral olfactory dysfunction. rhinoviruses were detected in patients. viral serotypes were identified by nucleotide sequences to be human rhinovirus- , human rhinovirus- , human rhinovirus- , and human rhinovirus- . one of the four patients complained of dysosmia, whereas another complained of anosmia. results of acoustic rhinometry significantly improved in the four patients, although olfactory testing did not show significant improvement. two of the four patients complained of olfactory dysfunction even months after the first visit. coronavirus and parainfluenza virus were detected in one patient each, and epstein-barr viruses were detected in three patients. the present study detected rhinovirus, coronavirus, parainfluenza virus, and epstein-barr virus in nasal discharge of patients with postviral olfactory dysfunction. furthermore, this study suggests that rhinoviruses cause olfactory dysfunction through mechanisms other than nasal obstruction. in this study, we evaluated patients with olfactory dysfunction induced by central nervous damage. six of patients developed hyposmia after head trauma, patients by brain tumor, one patient after subarachnoid bleeding and one patient by encephalitis. five of patients did not sense any smell by venous injection of prosultiamine. psychophysical evaluation of olfactory function was performed by t&t olfactometry, and the average threshold score of recognition was . (severe hyposmia). eight patients got mri brain scans and each of them had one or more intracranial lesions. frontal lobe abnormalities were seen in of patients. intracranial lesions were also observed in temporal lobes and one parietal lobe and one occipital lobe. these mri findings suggested most of the olfactory disorders were come from damage of orbitofrontal cortex in frontal lobe, which is thought to be the olfactory center. on the other hand, out of patients developed taste disorder, despite no pathogenic lesion was found in medullary, thalamus and insula, which are thought to be the central pathway of taste and taste cortex. currently, we have yet to establish effective treatment of olfactory loss caused by central pathway damage. in clinical practice, some of these patients show improvements in olfactory function with several years, thus long-term follow-up of patients is needed to develop new clinical studies. the clinical study of patients with drug-induced olfactory disturbances cases ( . %) of drug-induced olfactory disturbance were investigated. anti-cancer drugs occupied % in the frequency of causative drugs. tegafur was most common in the causes of olfactory disturbance by anti-cancer drugs. we showed the characteristics of drug-induced olfactory disturbance by tegafur. a total of patients were identified. the averaged recognition threshold was . and the averaged detection threshold was . in t&t olfactometry. after treatment, re-examinations were performed in patients. the averaged recognition key: cord- - zd hpj authors: soma, takehisa; ohinata, tsuyoshi; ishii, hiroshi; takahashi, toshikazu; taharaguchi, satoshi; hara, motonobu title: detection and genotyping of canine coronavirus rna in diarrheic dogs in japan date: - - journal: res vet sci doi: . /j.rvsc. . . sha: doc_id: cord_uid: zd hpj to clarify the prevalence of canine coronavirus (ccov) infection in japan, faecal samples from dogs with diarrhoea were examined for ccov rna together with canine parvovirus type (cpv- ) dna. the detection rates of ccov and cpv- for dogs aged less than year were . % and . %, while those for dogs aged year or older were . % and . %, respectively, which were significantly different (p < . and p = . , respectively), indicating not cpv- but ccov is an important diarrhoea-causing organism in juvenile dogs. among the ccov-positive dogs, . % and . % showed to be positive for ccov types i and ii, respectively, and simultaneous detection rate of both types was high at . %. furthermore, transmissible gastroenteritis virus (tgev)-like ccov rna was detected from dogs. these findings indicate that ccov type i and tgev-like ccov are already circulating in japan, though no reports have been presented to date. canine coronavirus (ccov) is a positive stranded rna virus, and has been classified as a group coronavirus along with feline coronavirus (fcov), transmissible gastroenteritis virus (tgev), and human coronavirus e (pratelli, ) . ccov infection occurs mainly by oral ingestion of the virus in excreted faeces, and the major symptoms are digestive conditions such as diarrhoea and vomiting. juvenile dogs and dogs under stress tend to be affected more frequently. furthermore, the symptoms become more severe in dogs co-infected with canine parvovirus type (cpv- ) (appel, ) . ccov was initially isolated from military dogs with diarrhoea in (pratelli, ) . the virus has spread extensively throughout the world and in japan it was first isolated in (yasoshima et al., ) . thereafter, evidence of ccov infections in japan has been confirmed by serological and genetic studies (bandai et al., ; mochizuki et al., mochizuki et al., , soma et al., ) . ccov has long been recognized as a single genotype. however, pratelli et al. ( b pratelli et al. ( , reported two genotypes of ccov; atypical ccov, designated as ccov type i with a spike-protein gene similar to that of fcov, and the well-known typical ccov, designated as ccov type ii. it has been confirmed that ccov type i is in circulation in europe and china (benetka et al., ; decaro et al., ; pratelli et al., ; stavisky et al., ; yesilbag et al., ; wang et al., ) . furthermore, wesley ( ) reported a novel ccov type ii named tgev-like ccov, which possesses an s gene similar to that of tgev, and this virus also appears to be circulating in dogs in europe and the usa (decaro et al., elia et al., ; erles and brownlie, ). it is highly likely that ccov type i and tgev-like ccov are already in circulation in japan, though no reports have been presented to date. in the present study, for the purpose of clarifying the present state of ccov infection in japan, faecal samples of dogs with diarrhoea were examined for ccov rna and detected ccov strains were examined for genotype. in addition, cpv- dna was investigated and its detection rate was compared with that of ccov rna. rectal swab samples were collected from a total of privately owned (not kennelled) family pet dogs with diarrhoea which visited veterinary clinics in japan in the period from september to december . none had received ccov live vaccine and cpv- vaccine derived from cpv- new type, the antigenic variants referred to as types a, b and c, though some dogs had received the vaccine from the original cpv- (old type). the swab samples were soaked in a small amount of sterile saline and immediately preserved in a frozen state. viral rna was extracted from the swab samples using qiaamp viral rna mini kit (qiagen, tokyo, japan) according to the manufacturer's instruction. rt-pcr for detecting ccov rna was performed using qiagen onestep rt-pcr kit (qiagen, tokyo, japan). about . ll of the extracted rna was added to the ll reaction mixtures containing ll of  - /$ -see front matter Ó elsevier ltd. all rights reserved. doi: . /j.rvsc. . . buffer (finally . mm mgcl ), . mm of each deoxynucleozide triphosphate (dntp), u rnase inhibitor (qiagen, tokyo, japan), . lm of the ccv -ccv primer pair providing a bp amplicon of ccov-m region (pratelli et al., ) and . ll enzyme mix. the rna was reverse transcribed at °c for min, followed by inactivation of reverse transcriptase and denaturation of cdna template at °c for min. the cdna was amplified in sequential cycles of denaturation at °c for min, annealing at °c for min and extension at °c for min, followed by a final extension of °c for min. the pcr products were electrophoresed at v for min in a % agarose gel. then, they were stained with ethididium bromide, and were visualized using a uv transilluminator at nm. the rt-pcrs for ccov genotyping were performed using el f-el r, s -s and cepol- -tgsp- primer pairs, which amplify ccov type i, ccov type ii and tgev-like ccov genes, respectively (erles and brownlie, ) , in a manner similar to the ccv -ccv primer pair except for annealing temperature of cepol- -tgsp- primer pair ( °c). the el f-el r, s -s and cepol- -tgsp- primer pairs provided , and bp amplicons, respectively. in addition, cpv- pcr was performed with fy -ry primer pair, which can detect cpv- new type (cpvs- a, b and c) specifically (senda et al., ) . in brief, viral dna extracted by qiaamp dna mini kit (qiagen, tokyo, japan) was amplified in cycles at °c for s, at °c for min and at °c for min, followed by the agarose gel-electrophoresis and the ethididium bromide-staining. ccov rna (m gene) and cpv- dna were detected in ( . %) and ( . %), respectively, of dogs, while both were detected in dogs ( . %). the detection rates of ccov-m and cpv- in dogs aged less than year were . % ( / ) and . % ( / ), respectively, while those in dogs aged year or older were . % ( / ) and . % ( / ), respectively, which were significantly different (p < . , v = . and p = . , v = . , respectively) ( table ) . no significant differences were observed for ccov-m detection rates in regard to gender, breed, or season (p > . ) (data not shown). the genotypes of ccov-m positive samples were also examined, as shown in table . ccov type i was detected in samples from dogs ( . %), ccov type ii in ( . %), and both in ( . %). of the dogs positive for ccov type i, ( . %) were negative for ccov type ii and cpv- . tgev-like ccov was detected in dogs ( . %), and dogs of which were positive for ccov type ii alone and both types i and ii, respectively. ccov rna detection rates determined by rt-pcr in previous reports ranged from . % to . % (bandai et al., ; decaro et al., decaro et al., , mochizuki et al., ; wang et al., ; yesilbag et al., ) and the present results ( . %) were within that range. in previous serological examinations, the seropositivity of juvenile dogs was found to be higher than that of adult dogs, however the difference between those age groups was not as remarkable as that observed in the present study (priestnall et al., ; soma et al., ) . as for the causal factors, it is considered that the antibody is not detected immediately after infection, whereas it is sometimes detected for a prolonged period even after the infection has been terminated. on the other hand, it is considered that detection of virus genes is a more accurate method of determining infection because it reflects viral replication, though ccov infection have persisted for weeks to months after disappearance of clinical signs in some cases (pratelli et al., a; tennant et al., ) . in the present study, ccov infection was quite frequent in dogs aged less than year, while it was extremely rare in dogs aged year or older, indicating that ccov is an important causative organism of diarrhoea in juvenile dogs. furthermore, simultaneous infections with ccov and cpv- were observed in quite a few dogs ( . %). on the basis of these findings, it is necessary to use vaccines to prevent not only cpv- , but also ccov, especially in juvenile dogs. currently, ccov type i has been reported to be circulating in europe and china (benetka et al., ; decaro et al., ; pratelli et al., ; stavisky et al., ; wang et al., ; yesilbag et al., ) , while the present results indicate that it is also effectively circulating in japan. the pathogenicity of ccov type i in dogs has not been clarified, because of failure to isolate the organism (pratelli, ) . however, it has been observed that diarrhoea tends to be more serious in dogs with simultaneous infection with ccov types i and ii as compared to in those infected with only ccov type ii (decaro et al., ) . the simultaneous infections have been observed in . - . % of ccov rna-positive dogs decaro et al., decaro et al., , . the present result ( . %) was in the range of those previous reports. on the other hand, of positives for ccov type i ( . %) were negative for ccov type ii and cpv- , indicating that ccov type i infection may be a potential cause of diarrhoea, though some other factors play roles in diarrhoea. thus far, tgev-like ccov has been detected in dogs from europe and the usa (decaro et al., elia et al., ; erles and brownlie, ). in the present study, this viral gene was detected in . % ( / ) of the dogs with diarrhoea, indicating that this virus is also circulating in japan, and the prevalence was similar to that reported previously ( . %) . tgevlike ccov has poorly serological reactivity with classical ccov type ii, from which ccov vaccines available currently are derived (decaro et al., ) , therefore, the effect of the vaccination may be re-evaluated in vivo. however, erles and brownlie ( ) detected the viral gene in . % ( / ) of asymptomatic dogs by rt-pcr using the same primer pair as in the present study, indicating that the pathogenicity of tgev-like ccov may be low, though it has been observed that this virus induced mild diarrhoea in an experimental infection . few reports have been published regarding infections with ccov type i and tgev-like 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dogs and cats recent epidemiological status of canine viral enteric infectious and giardia infection in japan genetic evolution of canine coronavirus and recent advances in prophylaxis development of a nested pcr assay for the detection of canine coronavirus m gene evolution of canine coronavirus in naturally infected dogs pcr assay for the detection and the identification of atypical canine coronavirus in dogs genetic diversity a canine coronavirus detected in pups with diarrhoea in italy two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus detection by pcr of wild-type canine parvovirus which contaminates dog vaccines antibody testing against canine coronavirus by immunoperoxidase plaque staining prevalence of canine enteric coronavirus in a crosssectional survey of dogs presenting at veterinary practices studies on the epizootiology of canine coronavirus detection of canine coronaviruses genotype i and ii in raised canidae animals in china the s gene canine coronavirus, strain ucd- , is more closely related to the s gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus case report on mixed infection of canine parvovirus and canine coronavirus-electron microscopy and recovery of canine coronavirus canine coronavirus infection in turkish dog population the authors thank veterinarians of animal clinics for providing faecal samples. key: cord- -q lpwjt authors: ujike, makoto; shimabukuro, kozue; mochizuki, kiku; obuchi, masatsugu; kageyama, tsutomu; shirakura, masayuki; kishida, noriko; yamashita, kazuyo; horikawa, hiroshi; kato, yumiko; fujita, nobuyuki; tashiro, masato; odagiri, takato title: oseltamivir-resistant influenza viruses a (h n ) during – influenza seasons, japan date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: q lpwjt to monitor oseltamivir-resistant influenza viruses a (h n ) (orvs) with h y in neuraminidase (na) in japan during influenza seasons, we analyzed , clinical samples by na sequencing and/or na inhibition assay. the total frequency of orvs was . % ( / , ) during the – season and . % ( , / , ) during the – season, indicating a marked increase in orvs in japan during influenza season. the na gene of orvs in the – season fell into distinct lineages by d g substitution, whereas that of orvs in the – season fell into lineage. na inhibition assay and m sequencing showed that almost all the orvs were sensitive to zanamivir and amantadine. the hemagglutination inhibition test showed that orvs were antigenetically similar to the – vaccine strain a/brisbane/ / . our data indicate that the current vaccine or zanamivir and amantadine are effective against recent orvs, but continuous surveillance remains necessary. i nfl uenza a and b viruses are major pathogens that represent a threat to public health with subsequent economic losses worldwide ( ) . vaccination is the primary method for prevention; antiviral drugs are used mainly for prophylaxis and therapy. currently, classes of drugs, matrix (m ) blockers and neuraminidase inhibitors (nais) are available, but m blockers such as amantadine and rimantadine are not commonly used because of the rapid generation of resistance and lack of effi cacy against infl uenza b virus ( ) ( ) ( ) . the nais zanamivir and oseltamivir are widely used because of effects against infl uenza a and b viruses and a low frequency of resistance. nai virus surveillance studies by several groups have demonstrated that < % of viruses tested show naturally occurring resistance to oseltamivir as of ( ) ( ) ( ) ( ) ( ) ( ) , indicating limited human-to-human transmission of these viruses. at the beginning of the - infl uenza season, however, detection of a substantially increased number of oseltamivir-resistant infl uenza viruses a (h n ) (orvs) was reported, mainly in countries in europe where the prevalence varies, with the highest levels in norway ( %) and france ( %) ( ) ( ) ( ) ( ) . these viruses showed a specifi c na mutation with a histidine-to-tyrosine substitution at the aa position (n numbering, h y), conferring highlevel resistance to oseltamivir. most of these orvs were isolated from nai-untreated patients and retained similar ability of human-to-human transmission to oseltamivirsensitive infl uenza viruses a (h n ) (osvs) ( , ) . in response to public health concerns about orvs, the world health organization (who) directed global infl uenza surveillance network laboratories to intensify nai surveillance and announced regularly updated summaries of orv data collected from each laboratory on its website ( ) . this site reported that the global frequency increased from % (october -march ) to % (april -september ) to % (october -january ), indicating that orvs have spread rapidly around the world. japan has the highest annual level of oseltamivir usage per capita in the world, comprising > % of world consumption ( ) . such high use of oseltamivir has raised concerns about emergence of osvs with increased resistance to this drug. moreover, in japan, recent infl uenza seasons were dominated by infl uenza viruses a (h n ) (figure ). if a high prevalence of orvs is observed, primary selection of oseltamivir treatment for infl uenza patients should be reconsidered. thus, monitoring orvs is a serious public health issue. to estimate the frequency of orvs and characterize these viruses, we analyzed , clinical samples isolated from the - season and , isolates from the - season by na sequencing and/or nai inhibition assay. the total frequencies were . % in the - season and . % in the - season, indicating that orvs increased dramatically in japan. the phylogenetic tree of na and ha genes was constructed by neighbor-joining methods. the phylogenetic tree was described by representative orvs and osvs isolated from several prefectures in japan. sequence information for isolates from other countries was obtained from the global initiative on sharing avian infl uenza data and the los alamos national laboratory database. all amino acid positions in the phylogenetic tree were described by n numbering. the chemiluminescent na inhibition assay was per-emerging infectious diseases • www.cdc.gov/eid • vol. formed by using the na star kit (applied biosystems, tokyo, japan) with slight modifi cations of the instructions provided by the manufacturer. the fi nal drug concentration ranged from . nmol/l to , nmol/l for oseltamivir and from . nmol/l to , nmol/l for zanamivir. chemiluminescent light emission was measured by using an lb plate reader (berthhold technologies, bad wildbad, germany). drug concentrations required to inhibit na activity by % (ic ) were calculated by a -parameter method using mikrowin version software (mikrotek laborsysteme gmbh, overath, germany). the hi test was performed to evaluate the reactivity of ferret antiserum against - vaccine strain a/ brisbane/ / , as described by the who manual ( ) . antiserum was treated by receptor-destroying enzyme ii (denka seiken, tokyo, japan) and adsorbed with packed turkey erythrocytes before testing to prevent nonspecifi c reaction. a . % suspension of turkey erythrocytes was used for the hi test. viruses with > -fold reduced hi titer to the homologous titer of a/brisbane/ / antiserum were regarded as antigenic variants. to determine the cutoff value between nai-resistant (outlier) and -sensitive viruses, box-and-whisker plots were used. the cutoff value was defi ned as upper quartile + . ×interquartile range from the th to th percentile. in this study, orvs with h y were excluded from the overall population for statistical analysis. outliers were excluded from the calculation of mean values and standard deviations for ic . to estimate the frequency of infl uenza a (h n ) orvs in each prefecture of japan, , isolates during the - season and , isolates during the - season were collected from all prefectures and examined by na sequencing to detect the h y mutation in na protein. in the - season, viruses possessing h y mutation (total frequency of orvs . %; figure , panel a) were observed in prefectures, indicating that the frequency of orvs was signifi cantly lower than that in countries in europe and the united states ( , ( ) ( ) ( ) ( ) . in tottori prefecture, however, of infl uenza viruses a (h n ) tested possessed h y, showing a markedly higher frequency ( . %) than that in other prefectures. in the - season, however, orvs were observed nationwide. of , infl uenza viruses a (h n ), , viruses possessed a h y mutation, for a total frequency of . % ( figure , , and the other orvs are also genetically close to osvs from japan (online appendix figure) . these ha genes were also genetically identical or close together (online appendix figure) , suggesting that almost all orvs from japan with the hawaii lineage are derived from osvs from japan. on the other hand, in the northern-eu lineage, osv counterparts were not observed, but foreign orvs genetically close to orvs from japan were observed. during the - season, the na gene of orvs from japan was close to that of orvs isolated from countries in europe (i.e., a/paris/ / and a/ england/ / ). during the - season, the orvs from japan, which shared a t on ha protein, were further divided into subclades (c- to c- ) by common amino acid changes on ha and/or na (online appendix figure) . orvs from japan in c- and c- were genetically close to the orvs isolated from north america or hawaii (e.g., a/memphis/ / and a/hawaii/ / ), whereas orvs in c- , representing most infl uenza a (h n ) viruses from the - season in japan, and orvs in c- were close to orvs isolated from south africa and australia in the southern hemisphere (e.g., a/ kenya/ / and a/victoria/ / ). all orvs except c- were isolated before the emergence of orvs from japan in each subclade. these fi ndings suggest that orvs from japan within a northern-eu lineage would not have emerged domestically but instead may have been introduced from various countries. of the viruses ( isolates in the - season and isolates in the - season) tested by na inhibition assay, possessed a h y substitution. with the na inhibition assay, although precise ic values were calculated from a normal sigmoid curve (figure , panels a and b), some viruses generated types of unusual sigmoid curves (figure , panels c and d) resulting from the mixed population of nai-resistant and -sensitive viruses, as previously reported ( ) . tentative ic values were calculated from type a curves (figure , panel c) and included in overall statistical analysis, but values could not be calculated from type b curves (figure , panel d) . later viruses were regarded as resistant candidates. in the na inhibition assay for oseltamivir, osvs showed a mean ic ± sd of . ± . nmol/l (range . - . nmol/l), and orvs had a mean ± sd ic of . ± . nmol/l (range . - . nmol/l), showing a reduction of > -fold in susceptibility to oseltamivir. one osv identifi ed as a statistical outlier (cutoff ic , > . nmol/l; upper quartile + . × interquartile range) showed a d e substitution on the na protein (table ). in the na inhibition assay for zanamivir, statistical analysis showed that viruses were regarded as the zanamivir-sensitive viruses, with a mean ± sd ic of . ± . nmol/l (range . - . nmol/l), and viruses ( orvs and osvs) were identifi ed as outliers (cutoff ic , > . nmol/l) ( table ) (table ) . these data suggest that d changes have a substantial effect on sensitivity to zanamivir (and oseltamivir). moreover, a/tottori/ / with h y and d d/g substitutions conferred high-level resistance to both nais ( figure , panels a and b) . however, a recent study reported that a d e change was detected only after virus propagation in cell culture, but not in the original clinical specimen ( ) tottori prefecture is located in the western part of the main island of japan. comprising cities and geographically divided into areas, this prefecture has the lowest population in japan (figure , panel b) . despite a low frequency of only . % in japan during - season, an unexpectedly high frequency ( . %) of orvs was observed in tottori prefecture (figure , panel a) . orvs from tottori were collected from cities in areas with no systematic bias apparent in the sampling process ( figure , panel b ). phylogenetic analysis of na genes showed that these orvs formed subclades (figure to orvs were observed in t- , suggesting that orvs in t- would be derived from osvs in tottori prefecture. a mapping study for orvs showed that all orvs in the hawaii lineage were collected from tottori city only, primarily at the end of january, whereas orvs with the northern-eu lineage were collected from cities, including tottori city, during february and march. genetically diverse orvs belonging to t -t were cocirculating only in tottori city in the eastern area (figure , panel b) . the tottori case raised concern about the possibility that these tottori orvs could survive to become an origin orv for the - season in japan. however, phylogenetic analysis showed that all orvs isolated during the - season were not genetically close to orvs from tottori (online appendix figure) . as a result, all orvs from tottori seemed to have been eliminated in the - season, and orvs that may have been introduced from other counties were circulating during - in japan. our study demonstrated that orvs dramatically increased in japan from the - season ( . %) to the - season ( . %). all tested orvs showed a reduction of > -fold in susceptibility to oseltamivir by na inhibition assay. on the other hand, almost all orvs remained sensitive to the other antiviral-drugs, e.g., zanamivir, and m inhibitors. hi testing suggested that the current vaccine, a/brisbane/ / , would be effective against recent orvs. in addition, recent studies have reported that symptoms and hospitalization rates of patients infected with orvs are no different from those seen with osvs ( , ) . figure , panels c (type a) and d (type b). although the viruses with d d/g tend to generate both patterns from repeat testing for the same samples, type b was selected in this case. §although a/tottori/ / showed mixed population of d d/g, it tended to show a normal curve fit (figure , panel b) . ¶the ic values of most viruses with d d/e tend to be higher than mean ic values but do not exceed the cutoff value. japan has the largest per capita use of oseltamivir (> %) in the world ( ) . because this use could cause effi cient selection of orvs in individual patients, japan might be the initial site of worldwide spread of orvs. however, long-term nai surveillance in japan during - and recent surveillance showed a low frequency of nai-resistant viruses for any strains and subtypes ( , , ) , suggesting that transmissibility of orvs selected by drug pressure was remarkably decreased. in addition, previous nai surveillance ( ) ( ) ( ) ( ) ( ) ( ) and several animal studies ( ) ( ) ( ) ( ) also suggested that nai-resistant viruses would become defective viruses with attenuated infectivity and transmissibility to human. in contrast, despite little nai use, a high emergence of orvs has been detected in several countries in europe since november . these orvs had as effi cient transmissibility as osvs in humanto-human transmission, resulting in worldwide spread in a short period of time. although whether the initial orv detected in norway in the - season appeared because of nai drug pressure is unknown, those orvs may have obtained amino acid changes on na and/or other proteins to compensate for the defect, in addition to the h y substitution on the na protein. most orvs belong genetically to the northern-eu lineage in clade b, suggesting that the gene constellation may contain a big advantage to retain infectivity and transmissibility. an interesting question arose as to where the orvs in japan originated. in the hawaii lineage, almost all orvs in japan would be derived from osvs in japan because the na gene of orvs was similar to osv counterparts isolated at similar times or from similar regions (online appendix figure) . on the other hand, in the northern-eu lineage, orvs in japan would have been introduced from other countries. in - , almost all orvs would be imported from countries in europe. in - , the orvs in c- , which comprised most isolates in - , and orvs in c- were genetically similar to orvs isolated from the southern hemisphere. because infl uenza activity in the southern hemisphere occurs half a year earlier than that in the northern hemisphere, most orvs in japan conceivably could have been imported from the southern hemisphere. orvs in c- and c- were genetically similar to orvs isolated in north america and hawaii, but the collection month of orvs in c- were similar to each other, suggesting that orvs in c- might be derived from an unknown common origin orv. the orvs obtained during - were not genetically similar to any orvs isolated in tot-tori during - , indicating that orvs from tottori had been eliminated and had not formed the origin orvs for the - season in japan. as for a/yokohama/ / belonging to clade c, the patient from which this virus was isolated was known to have taken oseltamivir before sampling ( ) , indicating that selective drug pressure in this person might have selected for this orv. in the na inhibition assay for zanamivir, some viruses, including orvs and osvs, showed reduced sensitivity to zanamivir. na sequencing of these viruses showed types of amino acid changes. one virus, a/tottori/ / (osv), possessed a q k substitution, which reportedly confers resistance to zanamivir ( , ) . conversely, most of the other viruses possessed d g/v/n. the amino acid changes d to n or e among subtype h n viruses and to a, g, e, n, or v among h n have been reported ( , , ) , and viruses with d substitutions often exhibit reduced sensitivity to nais ( , , ) . however, a recent study reported a possible role for cell culture in selecting these d variant viruses ( ) . in the present study, d variations (d g/e/n) also were not detected from available original clinical specimens (table ) , supporting the previous fi nding. we thus concluded that viruses with d variations would not have emerged naturally, and all orvs would remain sensitive to zanamivir. by sequencing of m gene, we confi rmed that almost all japanese orvs belonging to clade b retained sensitive genotype to m inhibitors, consistent with previously reports that recent clade b viruses are sensitive to m inhibitors, but clade c viruses are resistant ( ) . during the - seasons, we also addressed nai surveillance for a/h n and type b circulating in japan and identifi ed no viruses resistant to both nais. conversely, in march and early april , a new swine-origin infl uenza virus a (h n ) (now known as pandemic [h n ] virus) emerged in mexico and the united states and spread rapidly to many countries, including japan ( ) ( ) ( ) ( ) . in june , detection of pandemic (h n ) virus with h y on the na protein was reported from denmark, hong kong special administrative region, people's republic of china, and japan, but all orvs of pandemic (h n ) virus emerged as sporadic cases with no evidence of effi cient human-to-human transmission ( ) . although oseltamivir remains a valuable drug for treatment of pandemic (h n ) , many orvs were isolated after prophylaxis with a half dose of the drug. therefore, prophylaxis with oseltamivir may not be recommended as asumi hirata(tochigi prefectural institute of public health and environmental science), hiroyuki tsukagoshi (gunma prefectural institute of public health and environmental sciences), yuka uno (saitama city institute of yumiko kawakami (tokushima prefectural centre for public health and environmental sciences), ootsuka yuka (ehime prefecture institute of public health and environmental science), daisuke kawamoto (fukuoka city institute for hygiene and the environment), takayuki hirano and hisato masumoto (saga prefectural institute of public health and pharmaceutical research) estimating the impact of school closure on infl uenza transmission from sentinel data managing infl uenza: amantadine, rimantadine and beyond amantadine-resistant infl uenza a (h n ) virus in japan increased incidence of adamantane-resistant infl uenza a(h n ) and a(h n ) viruses during the - infl uenza season in japan monitoring of neuraminidase inhibitor resistance among clinical infl uenza virus isolates in japan during - infl uenza seasons detection of infl uenza viruses resistant to neuraminidase inhibitors in global surveillance during the fi rst years of their use neuraminidase sequence analysis and susceptibilities of infl uenza virus clinical isolates to zanamivir and oseltamivir surveillance for neuraminidase inhibitor resistance among human infl uenza a and b viruses circulating worldwide from use of oseltamivir in european countries between and -lack of association with the appearance of oseltamivir-resistant infl uenza a(h n ) viruses surveillance for neuraminidase-inhibitor-resistant infl uenza viruses in japan enzymatic properties of the neuraminidase of seasonal h n infl uenza viruses provide insights for the emergence of natural resistance to oseltamivir european infl uenza surveillance scheme. oseltamivir-resistant infl uenza virus a (h n ), europe, / season emergence of resistance to oseltamivir among infl uenza a(h n ) viruses in europe oseltamivir-resistant infl uenza viruses a (h n ), norway, / world health organization. who/ecdc frequently asked questions for oseltamivir resistance infl uenza a(h n ) virus resistance to oseltamivir manual on animal infl uenza diagnosis and surveillance evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of infl uenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal infl uenza a viruses infections with oseltamivir-resistant infl uenza a(h n ) virus in the united states oseltamivir-resistant infl uenza a viruses circulating in japan isolation of oseltamivir-resistant infl uenza a/h n virus of different origins in yokohama city, japan, during the - infl uenza season the h y mutation in the infl uenza a/h n neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo infl uenza virus carrying neuraminidase with reduced sensitivity to oseltamivir carboxylate has altered properties in vitro and is compromised for infectivity and replicative ability in vivo infl uenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets infl uenza virus carrying an r k mutation in the neuraminidase gene is not transmitted in ferrets adamantane resistance in infl uenza a(h ) viruses increased in in south east asia but decreased in australia and some other countries zanamivir-resistant infl uenza viruses with a novel neuraminidase mutation importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of infl uenza viruses antigenic and genetic characteristics of swine-origin a(h n ) infl uenza viruses circulating in humans emergence of a novel swine-origin infl uenza a (h n ) virus in humans triple-reassortant swine infl uenza a (h ) in humans in the united states origins and evolutionary genomics of the swineorigin h n infl uenza a epidemic world health organization. viruses resistant to oseltamivir (tamifl u) identifi ed world health organization. antiviral use and the risk of drug resistance. pandemic (h n ) briefi ng note we thank miho ejima and mariko tokunaga for the production of several fi gures. key: cord- -d h w authors: kashiwagi, hirokazu; kuwana, masataka; hato, takaaki; takafuta, toshiro; fujimura, kingo; kurata, yoshiyuki; murata, mitsuru; tomiyama, yoshiaki title: reference guide for management of adult immune thrombocytopenia in japan: revision date: - - journal: int j hematol doi: . /s - - -z sha: doc_id: cord_uid: d h w nan in , itp group of blood coagulation abnormalities research team with funding from the ministry of health, labour and welfare research grant for overcoming intractable diseases in japan published a reference guide for management of adult immune thrombocytopenia (itp) [ ] . the reference guide was based on the existing global standards on itp treatment, as well as research outcomes and expert opinions, and reflected the experience in japan at that time. a feature of the reference guide was clarification of h. pylori-associated itp, which is a unique characteristic of itp cases in japan. the reference guide also clarified the criteria for starting treatment for itp, as well as the treatment target and presented a flowchart of treatment, with corticosteroids as first-line treatment, splenectomy as second-line and other treatments as third-line, together with the respective recommendation levels. the basic concepts of management of adult itp have not changed significantly since that time, and the reference guide remains adequately effective. however, in the reference guide, thrombopoietin receptor agonists (tpo-ras) were positioned as third-line treatment, as these products had only recently been approved in japan. since then, we have accumulated experience in the use of these drugs, and their long-term efficacy and safety continue to become clear. furthermore, in , the indications for rituximab, which has been positioned as second-line treatment in europe and the us, were extended to adult itp in japan. conversely, the number of patients undergoing splenectomy is declining, and there is an increasingly large divergence between decisions made in clinical practice and the reference guide regarding the selection of second-line treatment for patients resistant to corticosteroids. there is also an accumulation of new evidence on itp treatment, including administration methods for corticosteroids used as first-line treatment, namely high-dose dexamethasone (hd-dex). for these reasons, this study group decided to examine the latest reports on adult itp treatment and present a revised version of the reference guide for the management of adult itp in japan. the most significant revision is the new recommendation of tpo-ras, rituximab,b and splenectomy as second-line treatment, in addition to recommending that each treatment is selected on a case-by-case basis, in line with the patient's conditions and lifestyle. these revisions were drafted and discussed by the "reference guide for management of adult itp" preparatory committee, and were created based on the opinions of the researchers and research collaborators on blood coagulation abnormalities research team, research on rare and intractable disease. the main aim of this reference guide is for it to be effectively used in clinical practice, so we also created some practical questions together with recommendation based on literature searches and opinions of the committee. the recommendation levels for each treatment were determined using the grade system (grading of recommendations, assessment, development and evaluation), as used in the previous reference guide (table ) [ ] . please refer to previously published our consensus report in the case of management of itp with pregnancy [ ] . we hope that this revised reference guide will be utilized effectively by physicians who manage itp patients every day. primary immune thrombocytopenia (itp) is an acquired disease that causes thrombocytopenia through an immunological mechanism [ , ] . based on the number of designated intractable disease medical care certificates issued, approximately , people are affected by this condition in japan. the number of new cases each year is estimated to be . people per , , and the disease commonly occurs in children aged years or younger, women aged - , and the elderly [ ] . primary itp is classified as newly diagnosed itp (within months after diagnosis), persistent itp ( - months) and chronic itp ( months or longer) based on the onset timing [ ] . the pathological mechanism of itp is thrombocytopenia caused by accelerated destruction of platelets opsonized by anti-platelet autoantibodies that mainly target platelet membrane glycoproteins [gpiib/iiia(cd /cd ), gpib/ ix(cd ), etc.] by reticuloendothelial systems in the spleen and other organs. the anti-platelet autoantibodies also induce maturation failure and apoptosis of megakaryocytes, leading to impairment of platelet production. in addition to anti-platelet autoantibodies, immune complexes, complement and cytotoxic t cells may also play a role in thrombocytopenia [ , [ ] [ ] [ ] [ ] [ ] . thus, itp is regarded as a syndrome that involves various immunological abnormalities, and itp treatment mainly aims to (i) inhibit production of anti-platelet autoantibodies, (ii) inhibit platelet destruction and phagocytosis, and (iii) recover platelet production. however, at present, the inability to accurately ascertain the mechanism of thrombocytopenia of individual patients is an impediment for treatment selection. no disease-specific tests for itp have been established, so itp is diagnosed basically by exclusion. that is, itp is diagnosed if a patient has thrombocytopenia (< , / μl), but erythrocytes (excluding anemia caused by bleeding or chronic iron deficiency) and leukocytes are normal, and other possible diseases that cause thrombocytopenia are excluded. clinical findings and tests that may help for diagnosis are described below. medical interview and examination findings the medical interview should confirm the clinical course of thrombocytopenia and bleeding symptoms, whether the patient had an antecedent infection, the status of complications and concomitant medication, and family history. the medical interview should also take note of the presence/absence of bleeding symptoms and the characteristics of these symptoms. the purpura seen in itp often ranges from petechiae to ecchymosis. mucosal bleeding (epistaxis, gastrointestinal bleeding, hematuria, etc.) is often seen in serious cases of thrombocytopenia, where the platelet count has fallen to ≤ , /μl, and fatal cerebral hemorrhage occurs in around % of adult cases and around . % of pediatric cases [ ] . deep bleeding, such as intraarticular bleeding and intramuscular bleeding, is rare. many adult cases with chronic itp lack bleeding symptoms. laboratory tests i. peripheral blood: it is important to carefully monitor peripheral blood smears together with the blood count. platelet aggregates are seen in pseudothrombocytopenia. the size of the platelets often increases in itp, but when there is a notable increase in giant platelets, the condition may be caused by bernard-soulier syndrome or myh -related disorders (may-hegglin anomaly, etc.). the presence of schistocytes is suggestive of thrombotic thrombocytopenic purpura (ttp) or disseminated intravascular coagulation (dic). ii. bone marrow examination: bone marrow examination is important to rule out leukemia and myelodysplastic syndrome (mds), as well as thrombocytopenia caused by bone marrow hypoplasia, but there are no findings specific to itp and it is not considered to be an essential for the diagnosis of itp [ ] . however, the examination of bone marrow should be performed to rule out other diseases when abnormalities are found in erythrocytes or leukocytes, or in patients resistant to itp treatments. iii. percentage of reticulated platelets (rp%) and plasma (serum) thrombopoietin (tpo) concentration: the platelet lifespan is shortened in itp, so the rp%, which shows the percentage of immature young platelets, is often high in itp patients [ , ] . the percentage of immature platelet fraction (ipf%), measured with an automated blood cell counter, is equally useful as rp% [ ] . the plasma (serum) tpo concentration stays within the normal range to slightly elevated in itp, but it is markedly high in thrombocytopenia caused by bone marrow hypoplasia. these tests may be useful for differentiating itp from hypoplastic thrombocytopenia such as aplastic anemia [ , , ] , but they are not covered by japanese health insurance. iv. autoantibodies against gpiib/iiia (or gpib/ix): currently, paigg (platelet-associated igg) test is covered by japanese health insurance, but it has low specificity for itp and the diagnostic value is poor [ ] . on the other hand, detection of autoantibodies against gpiib/ iiia or gpib/ix, or detection of gpiib/iiia-reactive b cells (by the elispot method) may be useful for itp diagnosis [ ] , but only a limited number of laboratories can perform these tests, and they are not covered by japanese health insurance. the therapeutic target for chronic itp is not to return the platelet count to normal, but rather to maintain a platelet count sufficient to prevent serious bleeding (usually ≥ , /μl). the adverse reactions associated with long-term excessive administration of drugs to normalize the platelet count often reduce the patient's quality of life (qol), so it should be avoided. therapeutic effect is determined in accordance with the following criteria. it is essential that platelet count measurements, acquired using two or more measurements taken at least week apart, satisfy the criteria. complete response (cr): platelet count is ≥ , /μl with no bleeding symptoms. partial response (pr): platelet count is ≥ , /μl and two or more times higher than the pre-treatment level, with no bleeding symptoms. no response (nr): applicable to at least one of the following: the platelet count is < , /μl, or less than two or more times higher than the pre-treatment level, with bleeding symptoms. when diagnosed with itp, it must first be determined whether the patient has a helicobacter pylori (h. pylori) infection. eradication therapy in patients with h. pylori infection results in an increased platelet count in - % of patients for whom eradication is successful. however, emergency treatment is prioritized in patients with severe bleeding symptoms, and in patients who are at risk of fatal bleeding. in patients who test negative for h. pylori infection or whose platelet count does not increase with eradication therapy, indication for treatment is decided based on the bleeding symptoms and platelet count. when the platelet count is ≥ , /μl and the patient has no or mild bleeding symptoms, their condition could be monitored without treatment. in patients with a platelet count of ≥ , /μl and < , /μl with no or mild bleeding symptoms, it is recommended that their condition is carefully monitored, and indications for treatment should be determined considering the individual patient's bleeding risk, including their age and comorbidities. in patients with a platelet count of < , /μl or with serious bleeding symptoms (cerebral hemorrhage, melena, hematemesis, hematuria, excessive genital bleeding, severe epistaxis or oral hemorrhage, injury where hemostasis is difficult, etc.), multiple purpura, petechiae or mucosal bleeding, immediate start of treatment is recommended. aggressive therapy is particularly important in severe cases when the platelet count is < , /μl and when there is gastrointestinal bleeding or intracranial bleeding. the first-line treatment is corticosteroids. patients who do not achieve the therapeutic target with corticosteroids or require long-term administration of high-dose corticosteroids, or patients unable to tolerate corticosteroids due to complications or adverse drug reactions, are transitioned to second-line treatment. tpo-ras, rituximab and splenectomy are recommended as second-line treatments. selection of the second-line treatment should be based on consideration of the advantages and disadvantages of each treatment, and to suit the situation of each individual patient. thirdline treatment should be considered for refractory itp cases where the second-line treatment is ineffective or is difficult to implement due to complications or other factors, after fully considering the necessity of the treatment. high-dose intravenous immunoglobulin, methylprednisolone pulse therapy, or platelet transfusion should be considered for patients with severe bleeding symptoms, marked thrombocytopenia or who urgently require an increase in their platelet count because of surgery or for some other reason. since the report of gasbarrini et al. [ ] which demonstrated that platelet count increases after eradication of h. pylori in adult itp patients, the effect of this treatment for itp has been demonstrated in a large number of retrospective and prospective observational studies. meta-analysis has also been implemented, and it has been proven that the platelet count increases with successful eradication in adult itp cases infected with h. pylori [ ] [ ] [ ] . studies conducted in japan have reported that platelet response is obtained in about - % of patients in cases treated with eradication therapy. in cases where the platelet count recovers to ≥ , /μl, few recurrences occur in the long-term follow-up, not only in mild cases but also in refractory cases not responsive to splenectomy [ ] . patients responsive to h. pylori eradication therapy have reduced production of anti-platelet antibodies, suggesting that their autoimmune pathophysiology is suppressed by eradication [ ] . therefore, within itp, there is a unique subtype in which h. pylori infection is significantly involved in the pathology. this type is called h. pylori-associated itp and is classified as secondary itp in europe and the us. h. pylori eradication therapy is more effective than corticosteroids, at least in japan, and has fewer adverse reactions, so the reference guide recommends testing for h. pylori infection in all patients diagnosed with itp and, if the patient is confirmed to be a carrier, that eradication therapy should be prioritized irrespective of the platelet count or bleeding symptoms [ ] . in japan, itp was added to the indications of h. pylori eradication therapy in . there are geographic differences in the efficacy rate of h. pylori eradication therapy for itp, with an efficacy rate of % or more in east asia, including japan, central and south america and italy, but an efficacy rate of less than % in reports from european countries other than italy and from north america [ ] . the efficacy rate is also low in children, and the treatment is normally ineffective in secondary itp with underlying diseases, such as systemic lupus erythematosus [ ] . this treatment is indicated for all cases in which the patient is infected with h. pylori. at first, all patients, including in cases where response was achieved with emergency treatment, should receive evaluation of h. pylori infection. preferential tests are (i) a urea breath test, (ii) a h. pylori stool antigen test, and (iii) a serum or urine anti-h. pylori igg antibody test. invasive methods that use an endoscope (histological examination, rapid urease test, cultivation) should be avoided. the test may produce a false-negative result if the patient is taking antibiotics, proton pump inhibitors (ppi), or potassium-competitive acid blockers (p-cab), or if the patient is being treated with immunosuppressant therapy, so it is recommended that two or more tests be conducted. treatment should be in accordance with the revision of the guidelines for diagnosis and treatment of h. pylori infection issued by the japanese society for helicobacter research [ ] . primary eradication involves a three-drug combination of ppi or p-cab with clarithromycin and amoxicillin twice a day (after breakfast and dinner), taken together for days. p-cab, vonoprazan, containing regimen has a higher eradication success rate than the conventional regimen using ppi [ ] . -ppi or p-cab (omeprazole, lansoprazole, rabeprazole, esomeprazole, or vonoprazan), twice daily for days -amoxicillin mg, twice daily for days -clarithromycin mg or mg, twice daily for days -from to weeks after completing eradication regimen, implement the urea breath test or h. pylori stool antigen test to determine if eradication was successful. it is necessary to wait at least months before conducting the serum anti-h. pylori igg antibody test. -metronidazole can be used instead of clarithromycin for secondary eradication, but the eradication success rate is lower than primary eradication, so it is preferable for these cases to be treated by a gastroenterologist or a doctor accredited by the japanese society for helicobacter research. -convenient -drug pack preparations are widely used (rabecure, lansap, vonosap, rabefine, lampion, vonopion). -cases with a temporary reduction in platelet counts and worsening of bleeding symptoms after eradication therapy have been reported [ ] , so it is recommended that eradication therapy be implemented after increasing the platelet count with corticosteroids or high-dose immunoglobulin in patients with a platelet count of ≤ , /μl or clinically significant bleeding symptoms. a national survey conducted in japan found that platelet counts increased by % of patients with successful eradication [ ] . a systematic review of cases, including data from europe and the us, where the efficacy of h. pylori eradication therapy is low, reported that the platelet count increased to ≥ , /μl in . % of patients, while the platelet count increased to ≥ , /μl and double the previous level in . % of patients [ ] . adverse drug reactions to eradication therapy that occurred in ≥ % of cases include gastrointestinal symptoms, such as soft stool, diarrhea and abdominal distension, as well as taste disorders, but the reactions were mild and disappeared after completion of treatment. rashes are seen in ≥ % of cases, and some patients have an allergic reaction not only to the drugs used for treatment but also to extracellular vesicles released from dead h. pylori [ ] . rarely this treatment causes toxic epidermal necrosis and stevens-johnson syndrome. it can also cause temporary thrombocytopenia, so treatment should be avoided in patients with significant bleeding symptoms. it is preferable to continue taking the drugs for week even if adverse drug reactions develop, provided the reactions are mild, to increase the eradication success rate and prevent the occurrence of antibiotic-resistant bacteria. corticosteroids mainly demonstrate an effect on itp by (i) inhibiting destruction of antibody-bound platelets by the reticuloendothelial system, (ii) inhibiting production of anti-platelet autoantibodies, and (iii) inhibiting destruction of megakaryocytes by macrophages in the reticuloendothelial system, such as spleen, or cytotoxic t cells in the bone marrow [ , , , , ] . corticosteroids have been key drugs for itp treatment since the s. in , george et al. reviewed reports on adult itp treatment and found that of itp cases were treated with corticosteroids, of which cases (approximately %) achieved a complete response [ ] . when using this treatment, it is essential to be fully cognizant of the adverse drug reactions associated with corticosteroid treatment. corticosteroids are selected as first-line treatment for patients who test negative for h. pylori at initial itp diagnosis or patients who test positive but are unresponsive to eradication, who have a platelet count of < , /μl or critical bleeding symptoms, multiple purpura, petechiae and/or mucosal bleeding. it is important to consider the risk factors for bleeding, including the age of the individual patient and comorbidities, to determine indications for treatment in patients with a platelet count of , - , /μl. in patients with underlying diseases (hypertension,diabetes,active infections, chronic infections,impaired immune status,osteoporosis,dys lipidemia,peptic ulcer, etc.) with a high probability of problematic adverse drug reactions associated with corticosteroid treatment, it is recommended that corticosteroids be started while controlling complications. as initial treatment, prednisolone (psl) . - mg/kg/day is given orally for - weeks. then, irrespective of whether the platelet count has increased, the dose is tapered over - weeks until it is reduced to mg/day or less [ , , , , , ] . consider starting the initial corticosteroid dose at . mg/kg/day for patients with the aforementioned underlying diseases and elderly patients aged years and older. reduce the corticosteroid dose, and if possible, stop administration in patients who achieve cr with corticosteroid therapy. for patients who do not reach cr, reduce the corticosteroid dose in which the platelet count can be maintained at ≥ , /μl, a level which does not affect survival prognosis. namely, monitor their progress at a maintenance dose of psl ≤ mg/day and aim to maintain good qol, including reducing drug-induced adverse drug reactions. if these therapeutic effects are not seen, the patient's condition is deteriorating, or this treatment cannot be used because of adverse drug reactions and/or complications, then select second-line treatment to avoid bleeding risks and improve qol. the platelet count increases to ≥ , /μl in approximately % of patients, and the platelet count increases to ≥ , /μl in approximately % or more of those patients. however, the platelet count declines as the corticosteroid dose is tapered, and reports indicate that only - % of patients are able to cease corticosteroids with remission [ , ] . there are various adverse drug reactions associated with corticosteroids, and the incidence of adverse drug reactions is also affected by individual differences, including the patient's age, and whether they have an underlying disease, so it is often necessary to adjust the dose or stop administration. immunodeficiency patients' immune status becomes impaired and they become immunocompromised, particularly with long-term administration of corticosteroids. particular attention should be paid to cases of opportunistic infection, such as fungal infections, including pneumocystis pneumonia, and herpes virus infections, etc. hepatitis b virus (hbv) can be reactivated when using a moderate or higher dose of corticosteroids [ ] . referencing the "guideline for treating hepatitis b caused by immunosuppression or chemotherapy" issued by the japan society of hepatology, hb antigens, and hbc and hbs antibodies should be measured before starting corticosteroids. if the patient tests positive for any of these, it is essential to determine whether the patient currently has a hbv infection or has a history of infection by measuring hbv-dna. diabetes mellitus it is important that patients who have been diagnosed with diabetes continue to strictly control their condition. elevated blood sugar can become problematic after starting corticosteroids in patients with borderline diabetes, obese patients, and elderly patients; thus, caution is needed. elevated blood sugar can also become problematic even in patients without a relevant medical history or underlying disease, so the blood sugar levels of all patients should be regularly monitored after starting treatment. peptic ulcer corticosteroids can induce the onset of peptic ulcers such as gastric ulcers. a hemorrhagic ulcer occurring while the platelet count is notably reduced can cause anemia and can also trigger critical conditions such as hypotension, so corticosteroids should be administered with ppi as appropriate. it is recommended to proactively perform endoscopy for patients who may have a peptic ulcer. other it is vital to take sufficient precautions for general adverse drug reactions to corticosteroids, including hypertension, dyslipidemia, and osteoporosis. refer to the guidelines for the respective disease regarding the treatment of each disease. the results of a meta-analysis on treatment using hd-dex and psl [ ] . according to that meta-analysis, the overall platelet count response in five randomized control trials (rcts) (n = ) was significantly more effective in the hd-dex group than in the psl group after days (hd-dex: % vs psl: %), but there was no significant difference between the two groups after months ( % vs %). these findings suggest that hd-dex may be more effective than psl at promoting early recovery of the platelet count, but there is little evidence that its efficacy exceeds that of psl in the long term. therefore, at the current time, conventional psl is recommended as first-line therapy for newly-diagnosed itp. the results of the metaanalysis found that hd-dex had a lower incidence of adverse drug reactions, but it can induce notable immunodeficiency in elderly patients and other adverse effects, so careful administration is essential. note that hd-dex is not covered by japanese health insurance. tpo-ras, rituximab, and splenectomy are recommended as second-line treatment. it has been years since the oral tpo-ra agent eltrombopag and the subcutaneous injection romiplostim received marketing authorization in japan. the efficacy and safety of long-term use of both medications have been reported [ , ] . the efficacy rate is high, but continuous treatment is required except a few cases, and caution is needed regarding adverse drug reactions, such as thrombosis. the efficacy of rituximab is inferior to splenectomy and tpo-ras, but treatment is completed in a short timeframe, and the treatment enables early recovery of the platelet count and remission in the long term in certain cases. splenectomy has the highest efficacy rate and can be expected to be curative in approximately two-thirds of cases. however, this procedure results in life-long reduced immunity against streptococcus pneumoniae, neisseria meningitidis and haemophilus influenzae, and also increases the risk of thrombosis. no rcts have directly compared these treatments. there are advantages and disadvantages to each treatment, which makes it impossible to uniformly determine the priorities of each treatment ( table ). the condition and situation of each individual patient, including their complications, age and lifestyle, must be ascertained, and the treatment should be selected with consideration for the patient's own wishes. patients who are non-responsive to corticosteroid therapy or who are unable to take sufficient doses of corticosteroids due to complications (corticosteroid intolerance) are indicated for this treatment. many patients who respond to corticosteroids have a reduction in their platelet count as the dose of the corticosteroids is reduced, and quite often these cases have been treated by increasing the corticosteroid dose again. however, long-term administration of corticosteroids increases the risk of adverse drug reactions, so now that multiple effective therapeutic options are available, early transition to second-line treatment should be considered. however, it is preferable to consider splenectomy - months after onset. tpo-ras are drugs that bind to the tpo receptors (c-mpl) expressed on megakaryocytes and hematopoietic stem cells, which enhance platelet production by promoting differentiation and maturation of megakaryocytes. the platelet count starts to increase about - days after starting administration and peaks at day - . continuous use can maintain the effect of increased platelet count. the currently available drugs are romiplostim, eltrombopag and lusutrombopag, but only romiplostim and eltrombopag are approved for the treatment of itp in japan. • romiplostim: - μg/kg, once a week, via subcutaneous injection, starting from μg/kg at the initial dose, with the dose increased or reduced as appropriate depending on platelet count and symptoms. • eltrombopag: . - mg/day, once a day, taken on an empty stomach h before or after meals, starting from a . mg oral dose, with the dose increasing every weeks up to mg until sufficient effect is achieved. efficacy a number of rcts have been conducted, including studies conducted in japan, for both romiplostim and eltrombopag on corticosteroid-resistant refractory itp cases [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . meta-analyses that compiled the results of these rcts found that both drugs significantly improved overall and continuous platelet response compared to the control group [ , ] . a comparison of a group treated with splenectomy and a group not treated with splenectomy found that both drugs had slightly reduced reactivity in the splenectomy group, but they were still effective in ≥ % of cases (romiplostim: splenectomy group = %, non-splenectomy group = %, eltrombopag: splenectomy group = %, nonsplenectomy group = . %) [ , ] . the incidence of all bleeding events and critical bleeding events, and events that required emergency treatment were significantly lower in the tpo-ras group, and there were also significantly more patients in the tpo-ras group who were able to reduce the dose or stop concomitant medication altogether (mainly corticosteroids) [ , ] . these results were also confirmed in large-scale observational studies in europe [ , ] . it was also reported that long-term remission was achieved even after stopping tpo-ras for both romiplostim and eltrombopag in around - % of patients in the clinical trials and around - % of patients in subsequent retrospective reports [ ] [ ] [ ] [ ] . the mechanism of inducing remission with tpo-ras is unknown, but possible mechanisms are as follows: (i) immunotolerance to platelet autoantigens may have been induced as a result of the increased platelet number by tpo-ras or (ii) tpo-ras may have improved the function of regulatory t cells (cd +, cd hi) [ , ] . thus, when the platelet count is maintained at ≥ , /µl, reduction of the tpo-ras dose may be considered, while monitoring fluctuations in platelet count. there are concerns about adverse drug reactions such as those listed below, given the pharmacological action of tpo-ras. thrombosis tpo may lead to pre-activated state of platelets [ ] , so an increase in thrombosis is the most concerning the potential adverse event. however, large-scale cohort studies have shown that itp itself increases arterial and venous thrombosis [ ] [ ] [ ] , which makes it difficult to determine the effect of the tpo-ra itself. there was no significant increase in the incidence of thrombosis in the clinical trials on romiplostim and eltrombopag [ , ] . however, nearly all cases of thrombosis in users of eltrombopag, both arterial and venous thrombosis, occur within year after starting treatment, and reports indicate that there are almost no new cases from years post-treatment onwards [ ] , thus, it is essential to be mindful of the onset of thrombosis, particularly in the early stages of tpo-ras treatment. use of tpo-ras should be carefully considered in patients with congenital or acquired thrombophilia, namely, patients with a history of thrombosis, hypertension, smoking, obesity, diabetes, dyslipidemia, antiphospholipid antibodies, and in patients with cancer. myelofibrosis a report on long-term administration of eltrombopag found that / ( . %) patients developed myelofibrosis requiring clinical attention, but there were no symptoms suggestive of an abnormal blood count or morphology, or bone marrow failure [ ] . it was also reported that increase of reticuline fibers improved on stopping of tpo-ras treatment [ ] . on the other hand, another report from a single institution found that clinically significant fibrosis (mf- or higher) may occur in one-fifth of patients administered tpo-ras long term [ ] . while using tpo-ras, it is essential to pay attention to peripheral blood findings, including smears, and if findings suggestive of myelofibrosis are found, then a bone marrow biopsy should be performed in advance and discontinuation of tpo-ras must be considered. increase in blast cells tpo receptors are expressed not only on megakaryocytes but on hematopoietic stem cells as well, so an increase in blast cells is a concern when using tpo-ras in patients with leukemia. there have been no reports of acute leukemia in clinical trials on eltrombopag and romiplostim for itp, and although malignant tumors, including lymphoma, have been reported, it is unlikely that these events were directly related to tpo-ras [ ] . progression of acute myeloid leukemia (aml) was a concern in an interim analysis of a clinical trial investigating the effect of romiplostim on mds [ ] , but the results of the subsequent final analysis demonstrated that use of romiplostim was unlikely to increase the incidence of aml [ ] . however, since there is also a report suggesting a correlation between tpo-ras use and leukemia [ ] , it is preferable to rule out the possibility of mds with bone marrow examination before starting tpo-ras. other an adverse drug reaction associated with eltrombopag that requires attention is liver dysfunction, and it has been seen in approximately % of treated cases [ , ] . cataracts were indicated in toxicity tests of eltrombopag using rodents [ ] . this may reflect the effect of concomitant drugs, such as corticosteroids, but regular ophthalmological examinations are recommended. there have been reports of anti-drug antibody production associated with romiplostim, but there have been no reports to date of production of antibodies that cross-react with endogenous tpo [ ] . answer. there is no difference in efficacy or safety, so drug selection should be based on consideration for the method of administration, complications, and the there is no rct that directly compares eltrombopag and romiplostim, but based on the results of a meta-analysis of rcts comparing each drug with placebo, there is no significant difference in efficacy or safety between the two drugs [ ] . therefore, determining which drug to use should be rituximab is a human-mouse chimeric monoclonal antibody that recognizes cd antigens expressed on b cells, and it works to reduce anti-platelet antibodies by reducing b cells. the indication of rituximab was expanded to include itp in japan in march . efficacy multiple rcts have been conducted on rituximab for itp patients and a meta-analysis on those rcts has also been reported [ ] . in five rcts on adult itp patients [ ] [ ] [ ] [ ] [ ] . % of patients in the rituximab treatment group achieved a complete response with a platelet count of ≥ , /μl, while this figure was . % in the standard treatment group, suggesting that the rituximab group was superior, with a relative risk of . ( % ci: . - . , p = . ). however, the percentage of patients who had a partial response with a platelet count of , - , /μl was . % in the rituximab group and . % in the standard treatment group, with a relative risk of . ( % ci: . - . , p = . ), indicating that there was no significant difference in the bleeding inhibition effect between the two groups. there was also no difference between the two groups in terms of onset of infection. in three of these five rcts, rituximab was combined with dexamethasone and was compared with a dexamethasone monotherapy group, so the results reflect the additional effects of rituximab combined with dexamethasone [ , , ] . in the remain-ing two rcts, rituximab monotherapy was compared with saline infusion in a blinded manner, although both groups were permitted concomitant use of low-dose corticosteroids. these two studies address the clinical question as to whether rituximab is effective in corticosteroid-resistant or -dependent patients [ , ] . there was no significant difference in any of the aforementioned indicators, including complete response rate, in the two rcts. however, the time to relapse was significantly longer in the rituximab group compared to the placebo group ( weeks vs weeks, p = . ), so there are benefits for patients who respond to rituximab [ ] . an investigator-initiated clinical trial in japan found that of patients ( . %) administered rituximab had a complete response, and . % ( % ci: . - . %) had a partial response with a platelet count of ≥ , /μl [ ] . this study was conducted in itp patients resistant to other treatments and found that rituximab had a certain level of clinical efficacy. an increase in the platelet count is seen approximately - months after starting administration of rituximab [ ] [ ] [ ] . the median duration of the platelet response to rituximab is year, and relapse occurs over time even if remission is achieved, approximately - % of patients maintain a platelet count of ≥ , /μl over or more years after rituximab administration [ ] [ ] [ ] . re-administration of rituximab after relapse produces a similar response as the initial administration in % of patients [ ] . some patients achieve remission again with rituximab after each subsequent relapse [ ] . the long-term prognosis of the effect and safety in itp patients repeatedly administered rituximab is unknown. there are retrospective reports that rituximab is more effective in women, patients younger than years of age, and patients treated within year of itp diagnosis [ , , , ] . however, there are also reports indicating no correlation with these factors [ , , ] as well as a report stating that the treatment tends to be more effective in men [ ] , so these are not established predictors of the effects of rituximab. the relationship between the presence and/or elimination of autoantibodies against platelet glycoproteins and the response to rituximab has also been investigated, but at present there is no definitive opinion on this matter [ ] [ ] [ ] . the effect of rituximab is unrelated to whether or not the patient has had a splenectomy [ , ] . safety acute infusion reactions generally, these reactions are observed between . and h after starting administration, and often occur with the first administration. the symptoms include fever, chills, headache, rash, angioedema, and cough. rarely it can cause anaphylactic-like symptoms, lung disorders (bronchospasm, infiltration of the lungs, acute respiratory distress syndrome) and cardiac disorders (myocardial infarction, ventricular fibrillation, cardiogenic shock). administration of antihistamines, antipyretic analgesics or corticosteroid min before administering rituximab and maintenance of a slow infusion rate at the start of administration are effective in mitigating these symptoms. reactivation of hbv administering rituximab to hbv carriers and hbv infected patients can reactivate hbv and increases the risk of de novo hepatitis. it is essential to follow the "guideline for treating hepatitis b caused by immunosuppression or chemotherapy" published by the japan society of hepatology when treating these patients. infection there are concerns of inducing a compromised state based on the mechanism of action of rituximab, but no difference in the incidence of infection between the rituximab group and the control group was detected in a metaanalysis of rcts on itp patients [ . % vs . %, relative risk . ( % ci: . - . , p = . )] [ ] . the results of a large-scale, long-term prospective study (median observation period was years, itp patients n = ) revealed that the infection incidence rate was . / human-years, and three of patients with infection died [ ] . only one case of opportunistic infection was observed, in the form of sinus aspergillosis. this treatment is also known to cause progressive multifocal leukoencephalopathy (pml) through reactivation of jc virus, and there have been two cases reported after rituximab was administered to itp patients [ , ] . other rituximab is known to cause neutropenia, and this has been observed in itp patients following administration of rituximab [ , ] . peripheral b lymphocytes disappeared after rituximab administration, but plasma cells were not damaged, and there was no significant change in blood immunoglobulin levels in all three itp clinical trials that measured immunoglobulin over time [ , , ] . answer. at the moment, the standard dose of mg/m /week doses is recommended. the rituximab dose most commonly used in studies to date is the standard dose which is used to treat lymphoma, mg/m /week doses, while some studies have used a lowdose regimen of mg/m /week doses [ ] [ ] [ ] . no rcts have directly compared these two dose regimens. although the lowdose regimen was effective in the two observational studies, it had a shorter duration of effect and a lower response rate than the standard-dose regimen [ , ] . there is a report that the incidence of adverse events is lower with the low-dose regimen [ ] , but overall the standard dose is recommended because it has virtually the same profile in the examination of a large number of cases [ , ] . an rct was conducted to examine other doses, comparing three groups: mg/m /week , mg/m /week treatment groups and the standard treatment group, but there was no difference in response rate [ ] . there is also a report on a prospective observational study with mg/ weeks , but there was no difference to the standard dose [ ] . in the pathogenesis of itp, the spleen is the site where macrophages phagocytose platelets with anti-platelet autoantibodies, and also functions as the site of anti-platelet autoantibody production by b cells [ ] [ ] [ ] . therefore, particularly in refractory itp cases, the platelet count is expected to recover with splenectomy, and often this procedure enables the patient to stop pharmacotherapy, including corticosteroids. however, there are cases of spontaneous remission in itp and in some cases, it takes time for the therapeutic effect to manifest, so it is preferable to consider splenectomy - months after diagnosis at the earliest [ , , , ] . a number of factors with potential predictive value have been investigated, but no factors that enable prediction of the effect of splenectomy are known. therefore, when selecting the option of splenectomy, various factors must be carefully considered, including the risks associated with surgery caused by underlying diseases and other factors, and it is important to fully explain the related factors, including the necessity of splenectomy, preoperative approaches, the surgical procedure, prognosis, efficacy rate, duration of surgery, and postoperative complications, and to obtain informed consent. in terms of splenectomy for elderly patients with itp, reports have shown that while elderly patients have a higher risk of perioperative bleeding and efficacy is inferior than in younger patients, at least % achieve long-term remission [ ] . considering that this procedure enables patients to stop pharmacotherapy such as corticosteroids, it can improve long-term qol in elderly patients, thus splenectomy may also be considered for elderly patients. preoperative management and surgical procedure ( ) preoperative management. it is preferable to maintain a platelet count of ≥ , /μl when performing splenectomy. implement methods to systematically increase the platelet count, such as high-dose intravenous immunoglobulin (ivig), and platelet transfusion, based on consideration of the surgery date. recently, there has been a report on a splenectomy implemented with the platelet count maintained with tpo-ras, and the efficacy of this treatment was demonstrated, but caution is essential due to the increased risk of postoperative thrombosis [ ] . ( ) surgical procedure. laparoscopic splenectomy is recommended over open splenectomy (explained in the question section). however, the decision about the procedure is left to the surgeon. efforts should be made to remove the accessory spleen, which may cause recurrence. efficacy a platelet increase response is seen in % of cases, but relapse occurs in approximately % of cases, and a permanent effect is said to occur in approximately % of cases [ ] . in a previous report by this research team, it was found that . % of cases maintained remission at the final observation point, or more years after splenectomy [ ] . safety general postoperative complications associated with the surgical procedure such as ileus due to gastrointestinal adhesions, and intraabdominal bleeding, can occur after splenectomy. other complications that require attention include infection and thrombosis (portal vein thrombosis, etc.) [ ] . infection the natural igm antibodies required to eliminate encapsulated bacteria such as s. pneumoniae, n. meningitidis and h. influenzae are produced by b cells in the spleen. therefore, the ability to defend against these bacteria is impaired after splenectomy, and there is an increased risk of overwhelming post-splenectomy infection (opsi); thus, it is essential to prevent infection with appropriate vaccinations [ ] . post-splenectomy infections can become more serious than normal infections, so early treatment is essential. when the infection is suspected due to fever or other symptoms, promptly administer penicillin-based or new quinolone-based antibiotics and carefully monitor the patient for signs of the infection worsening. many patients who undergo splenectomy are refractory cases who have had long-term treatment with immunosuppressants such as corticosteroids, so it is vital to be aware that some patients may have immunodeficiency associated with their previous treatments. thrombosis there have been reports of patients who develop abdominal venous thromboembolism (abvte), including portal vein and splenic vein thrombosis, or other venous thromboembolisms (vte), such as lower limb venous thromboembolism, after splenectomy. boyle et al. collected data on itp patients, including splenectomy cases, and investigated complications associated with splenectomy [ ] , and reported that the incidence of abvte in non-splenectomy cases was %, while that figure increased to . % soon after surgery, within less than days, in splenectomy cases. there was no such increase from days onwards, so the increase in abvte may be affected by the splenectomy procedure itself. in a number of reports on vte, splenectomy cases showed an associated, but slight, increase in the incidence of vte compared to non-splenectomy cases [ ] . the risk of opsi increases post-splenectomy and the mortality rate of opsi can reach %. thus, it is strongly recommended to vaccinate the patient with a -valent vaccine (pneumovax) to prevent s. pneumoniae infection, which is the most common pneumonia-causative bacteria in japan [ , , ] . it is preferable to vaccinate the patient up to weeks before splenectomy to inhibit the onset of perioperative opsi, but when preoperative vaccination is difficult, the patient should be vaccinated soon after surgery [ ] . n. meningitidis and h. influenzae can also cause opsi, but these bacteria are less common in japan, and neither of the vaccines has been approved. vaccinations should be considered based on the circumstances of individual patients, such as their intent to travel overseas. conventionally, around - % of itp cases do not respond to corticosteroids and splenectomy. many different types of treatments have been attempted for such "refractory" itp cases [ , ] . with the repositioning of tpo-ras and rituximab to second-line treatment in this revised guide, the number of multi-refractory itp cases that do not respond to these treatments is expected to be considerably limited, and there are no reports that have summarized how effective drugs used as third-line treatment are in such cases. therefore, the efficacy of drugs shown in this section is mainly for patients unresponsive to corticosteroids, including patients with initial onset of itp. furthermore, these reports are all from small cohorts of patients, and there are no drugs for which large-scale studies or rcts have been implemented since the previous reference guide, so it is essential to note that the evidence levels of these treatments are low. these limitations noted, the drugs listed below show potential efficacy for patients with multi-refractory itp, or patients who have no indication for second-line treatment due to complications or other factors. these drugs may be effective as monotherapies, but they may be more effective if used in combination with other drugs, including tpo-ras [ , ] . each type of second-line treatment is for patients who did not achieve pr or better, or for patients for whom secondline treatment is contraindicated due to complications or other factors. however, before transitioning to third-line treatment, it is preferable to reconfirm the itp diagnosis, for example by repeated bone marrow examination. one report has indicated that multi-refractory itp cases are often secondary itp with chronic lymphocytic leukemia or sle [ ] . note that in patients with mild bleeding symptoms, it is preferable to base treatment decisions on the bleeding symptoms, rather than strictly referring to the platelet count, while fully considering the adverse drug reactions associated with each drug. drugs with treatment regimen, efficacy and safety . azathioprine (recommendation level: c, not covered by japanese health insurance). azathioprine is reported to have a - % efficacy rate with oral administration of - mg/kg/day (maximum dose mg/day) [ , ] . adverse drug reactions include myelosuppression, liver dysfunction, and pancreatitis. . cyclosporine (recommendation level: c, not covered by japanese health insurance). cyclosporine is reported to have a - % efficacy rate by maintaining a blood concentration trough level of - ng/ml with two doses of - mg/kg/day [ ] . there are also reports of small-scale studies that achieved a high degree of efficacy with -drug combination therapy with azathioprine and mycophenolate mofetil (mmf), or by combining cyclosporine with high-dose dexamethasone and lowdose rituximab [ , ] . problematic adverse drug reactions include kidney dysfunction and hypertension. . cyclophosphamide (recommendation level: c, not covered by japanese health insurance). cyclophosphamide has been reported to have a - % efficacy rate with - mg/m intravenous infusion repeated every - weeks, or - mg/kg/day oral administration [ , ] . adverse drug reactions include alopecia, myelosuppression, hemorrhagic cystitis, infertility, and secondary primary cancer. it is preferable to avoid administration in young patients who wish to become pregnant. . diaminodiphenyl sulfone (dapsone, , -diaminodiphenylsulphone, dds. recommendation level: c, not covered by japanese health insurance). diaminodiphenyl sulfone is known as a drug used to treat leprosy. the efficacy of this drug in treating itp has been reported mainly from europe since the s, but the mechanism of action is unknown. according to a review published in , which summarized eight small-scale prospective and retrospective studies, the efficacy rate is - % and serious adverse drug reactions are reported to occur in ≤ % patients [ ] . however, there are also reports that this drug has a lower efficacy rate ( - %) [ , ] . known adverse drug reactions include hemolysis (dds syndrome), hematopoietic disorders, and mucocutaneous disorders. . danazol (recommendation level: c, not covered by japanese health insurance). reports on the efficacy of the testosterone derivative danazol for treating itp appeared in the literature for more than years [ ] . the normal dose is - mg/day, but there are also reports of low-dose treatments ( - mg/day). efficacy varies significantly, from to % depending upon the report [ , ] . a recently reported meta-analysis, which summarized the results of reports and cases, reported the efficacy as % ( % ci: - %), and the complete response rate as % ( - %), but many patients were administered - mg/day [ ] . adverse drug reactions include liver dysfunction, amenorrhea, thrombosis, hoarseness, hirsutism, and androgenization. . vinca alkaloid (recommendation level: c, not covered by japanese health insurance). the efficacy of vinca alkaloid has been reported since the s. there are various reports on the specific vinca alkaloid used for treatment (vincristine or vinblastine), the administration method (slow venous infusion or bolus), as well as the dose and number of doses. the efficacy varies significantly from to % depending on the report [ , ] . normally, this treatment is temporarily effective but does not produce long-term remission. administration may be considered when attempting to increase the platelet count temporarily in patients non-responsive to ivig [ ] . adverse drug reactions include constipation and peripheral neuropathy. . mycophenolate mofetil (mmf) (recommendation level: c, not covered by japanese health insurance). use of mmf for itp is mainly reported from europe, and an efficacy rate of - % has been reported with oral administration of - mg × times/day [ , [ ] [ ] [ ] . the adverse drug reactions are relatively mild, but gastrointestinal disorders are common. in patients with life-threatening bleeding associated with profound thrombocytopenia, such as gastrointestinal bleeding or cerebral hemorrhage, and patients who require surgery, it is essential to rapidly increase platelet count to achieve hemostasis. recommended treatments for such patients are ivig, methylprednisolone pulse therapy, and platelet transfusion. emergency treatment is indicated for patients with a platelet count of ≤ , /μl causing bleeding into a major organ (brain, lung, gastrointestinal tract, urinary system, intraabdominal bleeding), or patients at high risk of such an event. table shows platelet counts necessary for patients who require surgery or invasive treatment. treatment regimen administer mg/kg/day of human immunoglobulin intravenously for consecutive days. the platelet count starts to increase about days after starting treatment and tends to peak at an average of days after starting treatment. it steadily declines thereafter, with the platelet count remaining higher than the pretreatment level for - weeks. in europe and the us, typically mg/kg/ day is administered for - days. efficacy a report on the effect of ivig in itp patients found that % of patients reached a platelet count of ≥ , /μl and % increased their platelet count to ≥ , /μl [ ] . a small-scale (n = ) rct was conducted that compared the effect of different ivig doses, mg/kg/day × days and mg/kg/day × days, and found that although the latter dose increased the platelet count more rapidly by about day, the duration of platelet count elevation was shorter [ ] . there are no reliable indicators for predicting ivig effect. safety this treatment can cause anaphylactic shock, so the patient should be carefully monitored at the initial administration. it rarely causes acute kidney dysfunction or thromboembolism. headache is relatively common and tends to occur with rapid infusion. anti-inflammatory analgesics are effective. methylprednisolone pulse therapy (recommendation level: b, not covered by japanese health insurance) treatment regimen administer g/day methylprednisolone intravenously for consecutive days. the platelet count starts to increase from about day , and an increase in the platelet count is seen in approximately % of cases, but in many cases, the increase is temporary, so after pulse therapy, oral prednisolone should be used as maintenance therapy, and the dose tapered thereafter. this treatment can be combined with ivig and/or platelet transfusion during critical bleeding episodes. efficacy a report on using methylprednisolone pulse therapy for patients with a platelet count of < , /μl and mucosal bleeding found that % of patients were responsive, with their platelet count increasing to ≥ , /μl, taking an average of . days to reach that level [ ] . a study on patients with a platelet count of ≤ , /μl found that the treatment was effective in % of patients and % of patients reached a platelet count of ≥ , /μl within days [ ] . an rct that compared pulse therapy and ivig therapy found that ivig was superior in terms of the time required to increase the platelet count to , /μl and the duration of the effect [ ] . safety adverse events seen at early stages of treatment include arrhythmia and hyperglycemia, so ecg monitoring is recommended, and blood sugar measurements should be taken, giving note to these issues from the start of administration. also be aware of other general corticosteroid adverse drug reactions. question. is methylprednisolone pulse therapy more effective than conventional oral psl therapy in an emergency? answer. methylprednisolone treatment increases the platelet count more rapidly than conventional psl, and the response rate is also higher, so methylprednisolone pulse therapy is recommended when urgent treatment is required. a study (non-rct) comparing methylprednisolone pulse therapy (maintained with oral psl after pulse therapy) and conventional oral psl ( mg/kg/day) therapy found that pulse therapy increased the platelet count earlier ( . days vs . days, p< . ), and the initial response rate was also better ( % vs . %, p< . ). however, the longterm response rate was the same ( % vs %, p=ns) [ , ] . pulse therapy was also effective for some patients who were nonresponsive to conventional oral psl therapy [ ] . an rct was conducted comparing a group administered oral psl mg/kg/day for days after methylprednisolone pulse therapy (dose was tapered thereafter) with a group administered a placebo instead of oral psl, and the study found that the oral psl treatment group had a significantly better rate of platelet count increase and duration of effect. therefore, using oral psl maintenance therapy after pulse therapy appears to be a superior option [ ] . treatment regimen ~ × platelets ( - units of platelet concentrates) are administered as a single dose. this treatment has the fastest hemostatic effect, but the presence of anti-platelet antibodies leads to shorter survival of the transfused platelets, and an increase in the platelet count occurs is only temporary. combining platelet transfusion with ivig is more effective than either alone, so these treatments are often combined in emergencies. in theory, the platelets transfused into itp patients are rapidly destroyed by anti-platelet antibodies, so it is not guaranteed that the transfusion can contribute to hemostasis of the bleeding site. however, there are a number of reports of cases in which platelet transfusion alone or in combination with ivig was clinically effective [ ] [ ] [ ] [ ] . safety take note of adverse reactions associated with transfusion, including fever, urticaria, and anaphylaxis. platelet transfusion for itp does not increase the risk of thrombosis [ ] . tpo-ras and rituximab can now be used for refractory itp, which makes the selection of treatment methods more difficult. this revision of the reference guide for the management of adult itp contains detailed information particularly on the efficacy and adverse drug reactions/complications associated with second-line treatment for corticosteroid resistance. we endeavored to provide a more detailed explanation on individual matters by adding q&a items to address clinical questions. we hope this new reference guide will be widely utilized to assist in management of adult itp patients in the medical setting. reference guide for management of adult idiopathic thrombocytopenic purpura (itp) version the 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thrombocytopenic patients neuraxial anesthesia in parturients with thrombocytopenia: a multisite retrospective cohort study key: cord- -ozyx u authors: takahari, daisuke; shinozaki, eiji; wakatsuki, takeru; ooki, akira; ozaka, masato; suzuki, takeshi; nakayama, izuma; osumi, hiroki; kamiimabeppu, daisaku; sato, taro; ogura, mariko; suenaga, mitsukuni; chin, keisho; yamaguchi, kensei title: managing a gastrointestinal oncology practice in japan during the covid- pandemic: single institutional experience in the cancer institute hospital of japanese foundation for cancer research date: - - journal: int j clin oncol doi: . /s - - - sha: doc_id: cord_uid: ozyx u coronavirus disease (covid- ) was declared to be a global pandemic by the world health organization on march , . on april , , a state of emergency was declared in japan, as had been by other nations worldwide. this unprecedented crisis has profound implications for patients undergoing chemotherapy and for practicing healthcare professionals. various reports have shown data indicating that cancer patients with covid- have high morbidity and mortality rates. in order to reduce the use of medical resources to avoid the risk of covid- infections in both cancer patients and health care providers, oncologists now have to draw the line for cancer treatments by maintaining their efficacy while avoiding severe adverse events. in this article, we outlined the decisions made regarding the practice of gastrointestinal oncology in our institution during the covid pandemic. a case of coronavirus disease (covid- ) , which is caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), was first reported in wuhan city, china in december . the world health organization (who) declared a global pandemic on march , [ ] . as of may , , there were , , confirmed cases and , confirmed deaths worldwide [ ] . the pandemic continues to spread over the globe. in japan, as of may, , there were , cases of covid- infection and deaths [ ] . on april , , the government of japan declared a state of emergency under the act on special measures for pandemic influenza and new infectious diseases preparedness and response. the areas where emergency measures were first implemented consisted of seven prefectures with the highest numbers of covid- cases. however, on april , the emergency measures were expanded to include the entire country. the initial duration for the state of emergency was from april to may , , but then extended until may , . fortunately, it was lifted by may , but there is still a lot of caution to be taken. even with the state of emergency, the government of japan has worked to minimize its impact on social and economic functioning, and has not taken compulsory measures such as implementation of a "lockdown", which has been and is being implemented in other countries [ , ] . for cancer patients, of course, hospital visits for treatment and assessments are not subject to restriction, but the visits themselves can be a risk of infection, especially for individuals who have risk factors of covid- infection. the basic principle of preventing infection with sars-cov- is to reduce close contact with other people. although evidence remains insufficient, several reports have indicated that cancer patients have significantly increased severity and complications associated with covid- infection [ ] [ ] [ ] . a meta-analysis found that the odds ratio of severe complications was . [ % confidence interval (ci) . - . ] for patients with cancer [ ] . the data suggest that patients with cancer more likely have severe disease leading to the need for intensive care and mechanical ventilation. and also, cancer patients with covid- have high mortality rates [ ] [ ] [ ] [ ] [ ] [ ] . the odds ratio of covid- for death in patients with cancer and covid- was reported to be . ( % ci . - . , p = . ) [ ] . for this reason, cancer societies around the world have issued guidelines for cancer patients and cancer treatment during the covid- pandemic. the american society of clinical oncology (asco) has issued a statement on general care for clinicians, the cancer care delivery team, and patients with cancer during the covid- pandemic [ ] ; and the european society of medical oncology (esmo) also has issued a statement of encouragement for medical oncologists as follows: ( ) ensure the continuum of care, ( ) be prepared for new routines, ( ) protect yourself to protect your patients, and ( ) reinforce support to patients [ ] . in addition, several recommendations, including united states (us), french, and italian guidelines were published [ ] [ ] [ ] . in japan, three societies [the japanese cancer society, the japanese society of clinical oncology, and the japanese society of medical oncology (jsmo)] recently established a joint working group that issued the publication "new coronavirus infection and cancer care (q&a for patients)" [ ] . however, few specific recommendations on chemotherapy have been issued for patients with gastrointestinal (gi) cancers. lou et al. recently proposed recommendations for minimizing risks to patients with gi cancers in the us [ ] . the cancer institute hospital of japanese foundation for cancer research is located in the center of tokyo and is one of the largest cancer centers in japan where provides approximately , gi cancer chemotherapy annually. in this article, we present our clinical recommendations on how to modify the treatment but maintain effectiveness as much as possible for gi cancer patients receiving chemotherapies during the covid- pandemic. several guidelines from the us and europe on chemotherapy during the covid- pandemic have been published [ , ] . the common strategies in these guidelines consist of the following: ( ) balancing the benefits of treatment with risk of covid- infection and exacerbation of cancer due to lack of treatment, and ( ) reducing visits to the cancer center to reduce the risk of infection with sars-cov- . the risks of chemotherapy during the covid- pandemic combined with the lack of guidance that takes into consideration the local circumstances in japan have motivated us to develop a guidance for the use of chemotherapy in patients with gi cancers. the guidance was developed with the consensus of medical oncologists involved in gi cancer care in our institution (including jsmocertified oncology practitioners), who carefully considered whether the benefits of chemotherapy outweighed the risk of covid- infection. we examined the following questions: it was then discussed and finalized at each cancer multidisciplinary team conference with surgical and radiation oncologists, nurses, and pharmacists. table shows the guidance on changes in treatments such as reducing the intensity of chemotherapy, delaying treatment, skipping cycles, or stopping according to the degree of severity of covid- epidemic. risk factors associated with covid- are as follows: age (≥ years), performance status ≥ , comorbidity (hypertension, diabetes mellitus, liver or renal or lung dysfunction). low risk is defined as the presence of no risk factor. high risk is defined as the presence of one or more risk factors. based on this concept, changes in treatments are considered for each type of gi cancer. oncologists now have to draw the line for cancer treatments by maintaining their efficacy while avoiding severe adverse events and reducing the use of medical resources during the covid- pandemic. in accordance with asco recommendations [ ] , access to the institution is restricted to one entrance: all patients and visitors are screened there for fever, whether they have had contact with a patient with covid- , and screened for signs/symptoms (cough, dyspnea, and alterations in sense of smell or taste) of covid- . if the patient has had a fever greater than . °c during the previous days or on the day of visit or any relevant signs/symptoms within weeks, the patient will be transferred to a separate outpatient facility and undergo chest computed tomography (ct). if pneumonia is found or the attending physician deems it necessary after interviewing the patient, a polymerase chain reaction (pcr)-based sars-cov- assay of a patient specimen to detect the virus will be performed, and chemotherapy will not be administered on the same day, regardless of the patient's signs/symptoms. unlike influenza, the severity of covid- is associated with acute respiratory distress syndrome (ards) rather than secondary bacterial pneumonia [ ] . therefore, the neutropenia resulting from chemotherapy might not pose a risk of mortality in covid- ; however, in general, immunosuppression should be avoided. except for the regimens of docetaxel, cisplatin, -fluorouracil (dcf) used in esophageal squamous cell carcinoma (escc), -fluorouracil, leucovorin, irinotecan, oxaliplatin (folfoxiri) used in colorectal cancer (crc), and fol-firinox in pancreatic cancer (pc), severe immunosuppression is not commonly associated with the chemotherapy regimens used in gi oncology. under low risk of covid- infection in the community, our routine clinical practice can usually follow established chemotherapy protocols. by contrast, for treating patients with high risk and/or during the pandemic, we must choose between reducing the intensity of chemotherapy (e.g., omission of the -fu bolus for crc, replacement of infusional -fu by capecitabine or s- for escc, gastric cancer (gc), and crc), delaying treatment, skipping cycles, or stopping (i.e., treatment maintenance phase and salvage chemotherapy with relatively few benefits). in addition, the empiric use of the prophylactic granulocyte colony-stimulating factor or antibiotics should be considered. we recommend the use of telemedicine for monitoring the side effects when treatment is delayed or chemotherapy cycles are skipped. with regard to immunotherapy for gi cancers, immunecheckpoint inhibitor (ici)s (nivolumab and pembrolizumab) can cause immune-related adverse events, including pneumonitis. we should accordingly weigh the potential clinical harm of immunotherapy in relation to its benefits, when considering its administration to a patient. furthermore, with regard to the use of anti-angiogenic agents (e.g., bevacizumab, ramucirumab, and aflibercept) for the treatment of gc and crc, these agents are associated with a risk of arterial and venous thromboembolism, which have also been reported to occur at high rates in patients with covid- [ , ] . although no evidence to avoid these anti-angiogenic agents during the pandemic is available, it is incumbent upon us to provide this information on covid- to patients and healthcare providers. moreover, hypertension is a frequent ae of antiangiogenic therapy, and antihypertensive drugs such as angiotensin ii type receptor blockers arbs) or angiotensin-converting enzyme (ace) inhibitors may affect expression of the ace , the receptor of sars-cov- . so far, whether ace inhibitors and arbs worsen covid- -induced lung injury or not has been controversial [ , ] . thus, there is no need to discontinue it at this time, but caution should be exercised in the future. the standard neoadjuvant chemotherapy (nac) used in japan for clinical stage ii/iii escc is the combination of cisplatin plus -fluorouracil (cf) [ , ] . a randomized controlled trial (jcog ) revealed its clinical benefit for curative resection and survival [ ] . we should follow this principle of achieving curative resection and longterm survival, despite the covid- pandemic. several phase ii studies [ , ] found a clinical benefit for dcf in locally advanced escc. however, dcf, which is now being tested in the phase iii trial jcog , should not be recommended for nac in clinical practice because of its hematological toxicity. in situations where surgery must be postponed or aborted because of various social situations, adjuvant cf is indicated for patients in japan with clinical stage ii/iii escc and with pathological nodal metastatic disease who received surgery without nac [ ] . however, the survival benefit of adjuvant cf was not definitive in the jcog trial [ ] . accordingly, adjuvant cf should be omitted during the covid- pandemic. we also avoid dcf in the palliative setting because of the reasons listed in the previous section on 'neoadjuvant chemotherapy'. with regard to palliative chemotherapy in the patients with high risk of covid- infection, reduction in treatment intensity should be considered. best supportive care can be an option, depending on the situation. for first-line systemic chemotherapy, we might consider changing from cf to oral s- monotherapy. for second-line chemotherapy, nivolumab, which showed survival benefit in the att rac tion- trial [ ] , should be considered for administration with longer intervals between each administration. in addition, for patients receiving nivolumab for longer than months, pausing its administration could be considered [ , ] . taxanes such as paclitaxel or docetaxel can sometimes be candidates for third-line chemotherapy. intervals between administration of these agents should also be longer, as the situation demands, because they sometimes cause severe neutropenia. prophylactic granulocyte-colony stimulating factor should also be considered. a total radiation dose of gy is often used for chemoradiotherapy in stage i or advanced escc [ , ] . however, according to the rtog regimen, . gy of rt is generally used worldwide [ ] . according to the rads (remote, avoid, defer, shorten) principle [ ] , we have adopted gy/ fractions (fr) instead of gy/ fr to reduce the immunosuppressive effect of rt during the covid- pandemic. table lists the principles of chemotherapy for escc according to each risk group in the covid- pandemic. nac is not the standard treatment for resectable gc in japan [ ] . thus, based on the results of the jcog trial [ ] , we only recommend nac for resectable gc with bulky and/ or para-aortic lymph node metastases for patients with low risk of covid- infection. gastrectomy followed by adjuvant chemotherapy is the standard of care for patients with resectable gc in japan [ ] . the adjuvant chemotherapy trial of s- for gastric cancer (acts-gc) trial verified the efficacy of s- monotherapy as adjuvant chemotherapy for stage ii or iii gc [ , ] . the japan clinical cancer research organization (jaccro)-gc trial recently demonstrated that compared to s- monotherapy, the addition of docetaxel to s- (ds) significantly prolonged recurrence-free survival (rfs) for stage iii gc [ ] . based on the results of these trials, s- monotherapy for stage ii and ds for stage iii gc patients is the current standard adjuvant chemotherapy [ ] . although the interim analysis of the jaccro-gc trial showed that the superiority of ds compared with s- monotherapy in terms of -year rfs, the improvement in overall survival (os) remains unclear. in addition, severe (grade / ) neutropenia was observed twice as often in the ds ( . %) than in the s- ( . %) patients [ ] . furthermore, the acts-gc trial did not prove that s- was effective as adjuvant chemotherapy for patients aged older than years [ ] . taken together, regarding indications of adjuvant chemotherapy, s- alone for stage iii gc patients with high risk of covid- infection is recommended and adjuvant chemotherapy for elderly patients (> years) is not recommended. patients receiving ongoing adjuvant chemotherapy and without severe hematologic toxicity before the onset of the covid- pandemic should continue receiving that same treatment during the pandemic; however, dose reduction or stopping should be considered for patients with high risk of covid- infection. in principle, we recommend continuing of the administration of the same standard chemotherapy to patients with low risk of covid- infection, even during the covid- pandemic. however, reducing the frequency of hospital visits by modifying treatment schedules (such as reducing the intensity of chemotherapy, delaying treatment or skipping cycles) for patients with high risk of covid- infection may be considered. as first-line chemotherapy, for patients with high tumor burden or tumor-related signs/symptoms or low risk of covid- infection, combination of fluoropyrimidine and oxaliplatin (if her is positive, plus trastuzumab) is recommended as a standard regimen [ ] . for patients with cytology-positive (cy ) tumors who were considered for conversion surgery, the treatment intensity should be maintained. on the other hand, for patients with low tumor burden or high risk of covid- infection, non-intensive monotherapy [s- plus oxaliplatin (sox)] with reduced dose of oxaliplatin, s- monotherapy, or -fu/leucovorin) is an alternative regimen. prescription for two courses at once will be allowed for patients who received s- monotherapy as maintenance therapy to reduce the frequency of their hospital visits. as second line therapy, ramucirumab plus paclitaxel / nanoparticle albumin-bound (nab)-paclitaxel is the standard of care in japan [ , , ] . we propose skipping the administration of paclitaxel /nab-paclitaxel at day to avoid neutropenia and to reduce the frequency of hospital visits. a previously published study has indicated that biweekly administration of ramucirumab plus paclitaxel did not compromise efficacy [ ] . as third-line therapy, the subgroup analysis of japanese patients in the att rac tion- trial reported a very low frequency of immune-related pneumonitis (n = , . % of patients) [ ] . therefore, nivolumab is recommended in third or later line, but also consider prolonging the interval between administration ( - weeks) for responders. irinotecan and trifluridine/tipiracil are also third-line or later treatment options for gc [ , ] . however, these two agents show a relatively high risk of severe hematologic toxicity (> %) [ , ] . thus, the treatment modification (reducing the intensity of chemotherapy, delaying treatment, skipping cycles, or stopping) should be considered, especially for patients with high-risk factors of covid- infection. because it is associated with less hematologic toxicity than irinotecan and trifluridine/tipiracil, ramucirumab monotherapy could also be a treatment option for patients with high risk of covid- infection [ , ] . table lists the principles of chemotherapy for gc according to each risk group in the covid- pandemic. for patients with low risk of covid- infection, we should start and continue standard neoadjuvant chemotherapy (chemoradiotherapy), if possible. however, shortcourse radiotherapy ( × gy) followed by delayed surgery may be the best option, except for t rectal cancer. it has been demonstrated that the pathological complete response rate was significantly higher in patients whose surgery was delayed than in patients who underwent immediate surgery, although there were no differences in rates of preservation of sphincters and negative margins of resected tumors between two groups [ ] . neoadjuvant chemotherapy is not recommended for patients with high risk of covid- infection. for patients with low risk of covid- infection, we should start and continue standard adjuvant chemotherapy if possible (t and n : capecitabine plus oxaliplatin (capeox) - months, t and/or n : capeox months) [ ] . however, adjuvant chemotherapy that reduces the intensity and shortens the duration of chemotherapy is an alternative option. treatment should be performed at a local hospital near where a patient resides if traveling to the cancer center is too difficult. for patients with high risk of covid- infection, adjuvant chemotherapy is not recommended. fluoropyrimidine monotherapy is the alternative option of the adjuvant chemotherapy. for patients with microsatellite instability high (msi-h)/deficient mismatch repair (dmmr), patients receiving adjuvant chemotherapy for stages ii and iii msi-h/dmmr crc have a lower recurrence rate than mss/pmmr crc patients, and also a favorable prognosis [ ] . we should start and continue standard chemotherapy according to the national comprehensive cancer network (nccn)/esmo/japanese society for cancer of the colon and rectum (jsccr) guidelines for patients with metastatic crc and high tumor burden or who expect to undergo conversion surgery, [ ] [ ] [ ] if possible. furthermore, nonintensive chemotherapies such as fluoropyrimidines ( -fu, capecitabine and s- ) and bevacizumab, fluoropyrimidine monotherapy, or a stop-and-go strategy are alternative options. for patients with a low tumor burden or high-risk factors, non-intensive chemotherapy may be desirable. furthermore, delaying treatment and treating at the patient's local hospital should be considered if traveling to the cancer center is too difficult. table principles of chemotherapy for colorectal cancer according to each risk group in the covid- pandemic risk factors associated with covid- are: age (≥ years), performance status ≥ , comorbidity (hypertension, diabetes mellitus, liver or renal or lung dysfunction). these recommendations are based on single institutional experience msi-h microsatellite instability high, dmmr deficient mismatch repair, rt radiotherapy, fu montherapy: capecitabine, s- , tegafur-uracil/leucovorin for months, non-intensive chemotherapy: fluoropyrimidine and bevacizumab, fluoropyrimidine monotherapy or a stop-and-go strategy a low risk is defined as the presence of no risk factor b high risk is defined as the presence of one or more risk factors we consider using pembrolizumab for patients with msi-h/dmmr regardless of risk factors associated with severe covid- [ ] , although there are several controversial issues, such as the differential diagnosis between covid- -related lung injury and ici-associated immune-related adverse events and the risk of worsening lung injury by activating the immune response. table lists the principles of chemotherapy for crc according to each risk group in the covid- pandemic. the survival benefit of s- monotherapy as adjuvant chemotherapy for pc has been shown by the jaspac- study [ ] . the recent results of the prep- /jsap trial have revealed the survival benefit of gemcitabine plus s- (gs) as neoadjuvant chemotherapy for resectable pc [ ] . based on these results, nac with gs and postoperative adjuvant chemotherapy with s- have become the standard of care for patients with resectable pc in japan. considering the poor outcome of treating pc with surgery alone and the significant survival-prolonging effect of adjuvant (neoadjuvant) chemotherapy, nac with gs and postoperative adjuvant chemotherapy with s- are recommended even during the covid- pandemic. the initiation of adjuvant chemotherapy can be delayed until weeks after surgery if the patient does not recover sufficiently after surgery. folfirinox is not recommended as postoperative adjuvant chemotherapy, because severe neutropenia has been reported, and it is not covered by insurance in japan. there is no indication for discontinuing or changing the current chemotherapy in patients with low risk of covid- infection. however, if a new treatment is planned, gemcitabine plus nab-paclitaxel, which has a relatively low incidence of hematologic toxicity, is recommended [ ] . folfox may be considered in cases in which platinum is preferred, such as in patients with breast cancer gene (brca) mutations [ ] . for elderly patients or those with high risk of covid- infection, changes in treatment intervals or a change to s- monotherapy [ , ] may be considered. for patients undergoing gemcitabine plus nab-paclitaxel, the deletion of day of the standard regimen, namely, providing infusions of both drugs on days and of each -day cycle, should be considered to avoid neutropenia and to reduce the frequency of hospital visits. some studies have reported that this approach improves patient tolerance without significantly compromising efficacy [ , ] . concerning second-line treatment, we need to evaluate the indications for the treatment carefully for not only new patients but also patients currently undergoing treatment. best supportive care can be an option as the situation demands. in japan, there is no evidence of improved survival for biliary tract cancer patients treated with adjuvant or neoadjuvant chemotherapy. adjuvant/neoadjuvant chemotherapy should not be administered to biliary tract cancer patients during the covid- pandemic. there is no indication for discontinuing or changing current chemotherapy in patients with low risk of covid- infections. gemcitabine plus cisplatin is the standard of care for patients with unresectable biliary tract cancer [ ] . intervals between chemotherapy cycles, such as a biweekly regimen, should be lengthened as the situation demands because gemcitabine plus cisplatin sometimes causes severe neutropenia. table lists the principles of chemotherapy used for biliary-pancreatic cancer according to each risk group in the covid- pandemic. in this article, we outlined the recommendations for making decisions on chemotherapies for gi cancers during the covid pandemic. there are several recommendations and guidelines already published worldwide ( ) ( ) ( ) ( ) ( ) ( ) . however, few specific recommendations on chemotherapy have been issued for patients with gi cancers. moreover, practices differ in many aspects in gi cancers between us/europe and japan (e.g., oral agent such as s- are widely used). and the guidelines from japan are general and do not specifically address how to modify the treatment but maintain effectiveness as this article described. in this respect, we believe that our guidance is based on actual clinical practice of gi oncology in japan and would be useful not only to cancer center institutions but also to general hospitals. now, providing normal medical care is also being somewhat limited because of covid- . the standard approach for gi cancer, early detection and early treatment, such as that of endoscopic screening and endoscopic submucosal dissection (esd), respectively, which have been very prevalent in japanese society, is in a crisis. in addition, compared to other countries, a higher number of japanese patients have received subsequent chemotherapy after disease progression [ ] . this strategy of early detection and treatment and higher rates of subsequent implementation of chemotherapy might have contributed to longer survival in japanese patients than the survival of other populations. however, retaining a similar treatment strategy after the onset of the covid- pandemic might be difficult. despite these difficulties, we must continue to administer chemotherapy to the patients who need it. the most important goal of treatment was to complete the protocol without reducing the intensity of treatment, especially if cure is desired. in the palliative setting, however (in fact, the majority of cases are in the palliative setting), minimizing the risk of developing severe covid- that is associated with chemotherapy and maximizing survival and symptom relief are important. in order to achieve these important goals, the duration of treatments should be modified; and dose reduction, treatments with reduced toxicity, change to oral agents such as s- that require reduced visits to the cancer center, and empirical use of granulocyte-stimulating factor should be considered. along with protecting the patient from infection, infection of healthcare providers must be avoided. if healthcare providers are infected, and the medical supply system is not maintained, anticancer treatments must be reduced. for example, our department has created a business continuity plan that limits the number of treatments based on proportion of staff that is absent because of covid- infection. such preparations are important for any medical institution. with regard to ongoing clinical trials, with the considerations for protecting patients and the institutional standards for good clinical practice (gcp) during the covid- pandemic state, recruitment of new patients was avoided as own consensus in our institution, and the safety for the current participants should be prioritized. guidance is also provided by the food and drug administration [ ] and referring to it is recommended. the limitation of this guidance is that our recommendations are not comprehensively evidence-based and are largely based on expert opinion. we have just started using it in our institution and its feasibility should be confirmed. currently, several issues on covid- and cancer chemotherapy have been published in addition to ours, but all of them remain controversial. the accumulation of evidencebased data should lead to the establishment of optimal treatment policies. as effective treatments and vaccines for covid- are developed, this guidance may need updating, and there will be additional new issues to consider, such as the risks associated with concomitant use of vaccine against covid- and ici therapy. staying updated with new information for the treatments of gi cancers is important. in summary, we in the field of gi oncology recommend considering modifications to conventional chemotherapy that are based on the prevalence of covid- , treatment goals and patients' vulnerabilities. even if the pandemic is successfully suppressed, subsequent pandemics will inevitably occur. these recommendations should, therefore, be useful in the post-corona era as well. conflict of interest daisuke takahari has received lecture fees from taiho pharmaceutical co.,ono pharmaceutical co., eli lilly japan, and bristol-myers squibb. eiji shinozaki has received lecture fee from eli lilly japan. masato ozaka has received lecture fees from yakult honsha co., ltd. and taiho pharmaceutical co. kensei yamaguchi has received research grants and lecture fees from taiho pharmaceutical co., eli lilly japan, ono pharmaceutical co., bristol-myers squibb, yakult honsha co., ltd., sanofi and chugai pharmaceutical co., ltd., and lecture fees from takeda pharmaceutical co. ltd, bayer, daiichi-sankyo, dainippon-sumitomo pharma, merck sharp and dohme, and gilead sciences, inc. takeru wakatsuki, akira ooki, takeshi suzuki, izuma nakayama, hiroki osumi, daisaku kamiimabeppu, taro sato, mariko ogura, mitsukuni suenaga and keisho chin have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons 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version pan-asian adapted esmo consensus guidelines for the management of patients with metastatic colorectal cancer: a jsmo-esmo initiative endorsed by csco japanese society for cancer of the colon and rectum (jsccr) guidelines for the treatment of colorectal cancer nivolumab in patients with metastatic dna mismatch repair-deficient or microsatellite instability-high colorectal cancer (checkmate ): an open-label, multicentre, phase study adjuvant chemotherapy of s- versus gemcitabine for resected pancreatic cancer: a phase , open-label, randomised, non-inferiority trial (jaspac ) randomized phase ii/ iii trial of neoadjuvantchemotherapy with gemcitabine and s- versus upfront surgery for resectable pancreatic cancer( prep- / jsap- ) increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine family history as a marker of platinum sensitivity in pancreatic adenocarcinoma s- monotherapy as second-line treatment for advanced pancreatic cancer after gemcitabine failure phase ii study of s- in patients with gemcitabine-resistant advanced pancreatic cancer a modified regimen of biweekly gemcitabine and nab-paclitaxel in patients with metastatic pancreatic cancer is both tolerable and effective: a retrospective analysis biweekly gemcitabine/nab-paclitaxel as first-line treatment for advanced pancreatic cancer cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer regional differences in advanced gastric cancer: exploratory analyses of the avagast placebo arm the food and drug administration ( ) fda guidance on conduct of key: cord- -fucyt zc authors: yoshikawa, toru; chua, gavin title: covid- and japanese shareholder activism: brief respite for japan’s self-healing concrete date: - - journal: asian bus manage doi: . /s - - - sha: doc_id: cord_uid: fucyt zc extrapolating from modern international understanding of corporate japan’s distinct form of managerial capitalism, we elaborate on the growing momentum of shareholder activism in japan leading up to the covid- health crisis, so as to inform the subsequent discussion on the relevant primary considerations that belie the future direction of shareholder activism in japan post-covid- . on an initial logical extrapolation, it appears probable that covid- could mark the peak of japanese activism. however, it is crucial to acknowledge that the success of japan’s managerial capitalism have also declined, which poses a question on to which direction japanese corporate governance may be shifting. finally, we point out how corporate japan’s impeding market developments will provide a useful case-study for global financial discourse. the covid- health crisis has catalyzed a demand-side, real economy-led economic downturn, throwing financial markets around the world into a period of considerable uncertainty and volatility. equity capital markets are not exempt, and shareholder activists' ability to shrewdly navigate the turbulent storm will be tested. we canvas the corporate terrain leading up to the covid- health crisis, and then present some considerations from both the outside investor's perspective and the corporate insider's perspective. finally, we discuss a possible direction of shareholder activism in japan and the future japanese market-sensitivity to international corporate governance norms. prior to covid- , there was an almost unanimous acknowledgement of the tremendous momentum behind shareholder activism (both confrontational and quiet), as an expression of shareholder primacy, in japan (buchanan et al. ) . this is unsurprising given the abundance of public companies with solid performance, undervalued stock prices, excess cash reserves, vulnerable capital policies and often insulated management. in , japan was the most-targeted non-us jurisdiction, with local records of activist campaigns, $ . billion in capital deployed, public companies targeted, shareholder demands for board representation and proxy fights (activist insight ). as observed by many researchers (e.g., ahmadjian and robins ; yoshikawa and mcguire ) , incremental convergence between japan's insider or stakeholder-oriented corporate governance system and anglo-american shareholder-centric model had been occurring slowly but surely, partially catalyzed by the bubble economy collapse and the globalization of capital markets. per the reflexive nature of market development, the growing success of early shareholder activists, coupled with attendant legal and regulatory developments, laid the groundwork for the current boom in japanese shareholder activism, prominently led by paul singer's elliott management, daniel loeb's third point management and yoshiaki murakamirelated parties. the movement was further buttressed by prime minister shinzo abe's corporate governance reform initiatives through the stewardship code and corporate governance code. nonetheless, certain crucial structural impediments remained, pushing back against complete convergence. first, the complex corporate governance situation stemming from japanese firms' unique capital structures must be appreciated. a byproduct of its war-era, bankbased economy, cross-shareholdings among affiliated firms meant that a portion of a public company's shares would be held by the company's banks, customers, suppliers, and other friendly partners. this leads to a structural conflict of interest, since such stable shareholders wearing two hats would often prioritize ongoing business relationships and stable business ties over high investment returns, decreasing pressure on firm's profit performance and cash returns (via dividends or share buybacks) and reinforcing managerial independence (yoshikawa and mcguire ) . second, as discussed by many observers (e.g., ahmadjian and robins ) , group-focused loyalties and identities persist in corporate japan's mental universe, leading to a stakeholder-oriented model. though cracks had begun to emerge with the increasing acceptance of shareholder value maximization rhetoric, certain setbacks to the activist momentum (e.g., murakami's arrest for insider trading) potentially offered a rare glimpse into japan, inc.'s closing ranks. this cultural bias has translated into a practical constrain on the usual activist strategies seen in more divisive equity capital markets like the us. covid- and japanese shareholder activism: brief respite… thus, pre-covid- , corporate japan was undergoing a transitionary period that was potentially a crucial determinant of japan's future corporate governance norms. the covid- health crisis could not have come at a worse time. deep-rooted cultural change requires time for truly lasting cracks to cement, and this pandemic-catalyzed economic downturn looks to stop the winds in the activists' sails. momentum, once lost, can be hard to regain. no doubt, for certain ongoing high-stake corporate tussles (e.g., softbank and elliott management), the concurrent decline in real economy performance and liquidity will bring tensions to a head. but for the vast majority of activist actions, the heightened volatility and economic uncertainty will pose drastic challenges (gottfried and donahue ), notwithstanding increased opportunities from lowered valuations (topix plunged % from -week highs, similar to s&p 's % plunge from -week highs) and potential target firm vulnerability due to relatively weaker shareholder rights plans (koh et al. ) . first, the inherent nature of this real economy-led economic downturn means that firm preservation, not shareholder value maximization (since cash distributions adversely impact a firm's liquidity), will be at the forefront of companies' peripheries. japan's unique capital structure of cross-shareholdings and stable shareholdings will only exacerbate such concerns, since a proportion of the listed company's shareholders will have the exact same skin in the game. therefore, such conflicted shareholders may lend immediate managerial support now, in hopes that it may lead to reciprocal support. as such, shareholder activists may soon find that the support they can count on (e.g., domestic institutions) in proxy fights are now diminished during this period, as management distraction concerns and further instability exacerbation outweigh any potential benefits. second, on the ideological front, usual activists' inefficiency arguments against cash-hoarders during prosperous times will now be poignantly countered with the raw experience of resiliency during these turbulent times (lewis ). on a micro-scale, there is no doubt that covid- has the real potential to represent the inflection point for shareholder activism in japan, as market mood turns against corporate outsiders. but whether this subsequently manifests in reality depends much on the surrounding circumstances that prevail long past covid- . first, given the fact that the japanese market has long matured, and growth opportunities are increasingly scarce, the viability and sustainability of japan's managerial capitalism may simply no longer hold true. capital inefficiency due to managerial preference to lock in capital within the firm will generate ever-increasing agency costs. second, the post-war-era agency cost hedges no longer operate to the same extent. for example, the economic incentive systems (lifetime employment, seniority-based wages) designed to align managerial interests within the company have eroded over time (yoshikawa and mcguire ) , coupled with ever-increasing reliance on temporary workers, as a creative adaptation of japan's existing employment institutions, to cope with greater market pressures. thus, the short-term inefficiencies of managerial capitalism may no longer produce long-term economic growth (to the same extent) given the modern circumstances of japan's economy. on a macro-scale, the corporate discussions to follow provide an interesting casestudy for global discourse. the current hot potato in the financial realm surrounds the proper primary purpose of a company: should it be a purely profit-seeking private entity or should it resemble quasi-public entities focused on broader stakeholder considerations and thus indirectly, economic growth? the covid- aftermath will produce unique invaluable research domain to better discern the resiliency of japan's stakeholder-model economy (e.g., the number of japanese corporate insolvencies vis-àvis the u.s.) and thus contribute to discussions on comparative corporate governance. on a glass half-full approach, one could even posit that the timing of this black swan event could not be better, as the arguments for both potential points on the pareto frontier can now be discussed adequately and frankly before significant transitionary costs are incurred as japan (and many other economies) may be further shifting to a marketbased, shareholder-oriented model. after all, there is no universal method for economic efficiency, and much stems from strategies tailored to a countries' current comparative institutional advantages. activist insight. . the activist investing annual review a clash of capitalism: foreign shareholders and corporate restructuring in s japan hedge fund activism in japan: the limits of shareholder primacy harvard law school forum on corporate governance land of the falling 'poison' pill: understanding defensive measures in japan on their own terms japan's corporate cash-hoarders have moment in the sun. financial times change and continuity in japanese corporate governance key: cord- - bn wgd authors: sun, y.; sun, j. title: the effect of non-pharmaceutical interventions (npis) on the spread of covid- pandemic in japan: a modeling study date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: bn wgd non-pharmaceutical interventions (npis) are founded to be effective to delay epidemic spread and to reduce the number of patients. moderate npis took in japan seemed to have reduced the covid- patients and to lower death rates, thus, effects of those npis are worthy of investigation. we used open source data and divided the data into three periods: jan to feb (period i), feb to apr (period ii), and apr to may (period iii). we developed the sird model and applied the monte carlo simulation to estimate a combination of optimal results, including the peak of infected cases, the peak date, and r . for period i, the estimated peak infected cases were smaller than the observed ones, the peak date was earlier than the observed one, and the r was about . . for the other two periods, the estimated cases were more, and the peak dates were earlier than the observed ones. the r was . in period ii, and . in period iii. npis took in japan might have reduced more than % of the daily contacts per people compared to that before covid- . owing to the effects of npis, the japanese society had avoided collapse of medical service. nevertheless, the capacity of daily rt-pcr may have restricted the reported confirmed cases. ܴ . for period i, the estimated peak infected cases were smaller than the observed ones, the peak date was earlier than the observed one, and the ܴ was about . . for the other two periods, the estimated cases were more, and the peak dates were earlier than the observed ones. the (period iii). the japanese government gradually enforced more rigorous countermeasure for covid- containment through these three periods. for instance, on feb , large-scale sports and entertainment events were canceled throughout japan, and organizers of small-scale events were advised to voluntarily cancel events (prime minister of japan and his cabinet, b). on apr , emergency declaration was first declared to seven prefectures by prime minister of japan, and were gradually declared to other prefectures ( figure ) (prime minister of japan and his cabinet, b). to illustrate the effects of npis on the dynamics of the epidemic, we developed a simple susceptible-infectious-recovered-deceased (sird) model (osemwinyen & diakhaby, ) of covid- transmission. this model assumes that the total population of reference is constant. the population is divided into several different compartments (i.e., susceptible, infective, recovered, deceased) and specify how agents move across the separate compartments over time. the model assumes that susceptible individuals can become infectious after contact with an infectious individual, and recovered individuals cannot be re-infected. the sird model uses the following system of differential equations: . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . are the rates of infection, recovery, and mortality, respectively. in addition, a key parameter regulating the dynamics of an epidemic is the basic reproduction number (ܴ ), which correspondes to the average number of secondary cases generated by an index case in a fully susceptible population (zhang et al., ) . using the sird model, we calculated the peak number of existing infected cases and the peak date. we compared the differences between estimated results and official reports, in order to estimate the effects of npis. according to the basis of plausible ranges from the recent reports of covid- data (bhatraju et al., ; geleris et al., ; guan et al., ; li et al., ; mehra, desai, kuy, henry, & patel, ; , we consider that the average incubation period ranges from to days (ߚ = / , / ] ), the recovery period ranges from to days (ߛ = [ / - / ] ), and the deceased period ranges from to days (ߤ = [ / - / ]). for the n, which is the total population of the people who may be exposed to the virus, was assumed to range between a baseline value and a maximum value. the baseline value equals to the number of people who received the reverse transcription polymerase chain reaction (rt-pcr) test in japan. the maximum value equals to the product of "the baseline value * the average daily number of contacts per japanese people". for period i, no rigorous measures were taken to prevent covid- . most of the laboratory-confirmed cases of covid- were reported in large cities such as tokyo, yokohama, and kyoto (japan ministry of health labour and welfare, ). thus, we assume that the daily contacts per people in japan is . , which is the same as that reported in shanghai (zhang et al., ) . the total number of people who received rt-pcr was , until feb (japan ministry of health labour and welfare, ). hence, for period ii, voluntary event cancellation, school and workplace closures were carried out throughout japan. the covid- epidemic has already gradually spread to small cities and towns. thus, we assume that the daily contacts per people in japan is . * . * . . the coefficient of " . " means that the japanese daily contacts are about % of that in shanghai, and " . " means that npis have reduced % of daily contacts. the total number of people who received rt-pcr was , until apr (japan ministry of health labour and welfare, ). hence, for period iii, emergency declaration was declared, and the japanese government aimed to reduce the daily contacts per people to % compared to that before covid- (prime minister of japan and his cabinet, a cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . learning to produce a bevy of results of , and n. we extracted the optimal combination of , and n from groups of simulations. for each simulation, we produced , cases of the sird model. we wrote the code package and run the simulation by python . . based on the official reports (jhu csse, ), the distribution of total confirmed cases and the existing infected cases were shown in figure . the optimal results of is . , is . , is . , and n is , . the is . . based on these optimal results, we produced the estimated number of existing infected cases, shown in figure . our simulation results displayed that the estimated peak number of existing infected cases would be , on may . however, the official reports showed that the peak number of existing infected cases was , on april . these results indicated that ) npis took in japan might have reduced more than % of the average daily number of contacts per people compared to that before covid- ; and ) owing . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint to the effects of npis, the japanese society had avoided collapse of medical service. nevertheless, ) the capacity of daily rt-pcr may have restricted the reported confirmed cases; and ) the official reports may have provided the underestimated figure of covid- . the optimal results of is . , is . , is . , and n is , . the is . . based on these optimal results, we produced the estimated number of existing infected cases, shown in figure . our simulation results displayed that the estimated peak number of existing infected cases would be , on apr . compared to the official reports, which showed that the peak number of existing infected cases was , on april , these results indicated that ) the capacity of daily rt-pcr obviously restricted the reported confirmed cases; ) the of period i was higher than that reported in wuhan prior to the lockdown on jan (tian et al., ) ; and ) rigorous countermeasures were obviously required to contain covid- . the optimal results of is . , is . , is . , and n is , . the is . . based on these optimal results, we produced the estimated number of existing infected cases, shown in figure . our simulation results displayed that the estimated peak number of existing infected cases would be , on may . compared to the official reports, which showed that the peak number of existing infected cases was , on april , these results indicated that ) npis took in period ii might reduce more than % of the daily contacts per people compared to that before covid- ; ) owing to the effects of npis, the japanese society had avoided collapse of medical service, which had been seen in italy during the same period (remuzzi & remuzzi, ) ; ) the of period ii was close to that reported in china before march (chinazzi et al., ) . nevertheless, . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . / . . . doi: medrxiv preprint ) the capacity of daily rt-pcr may have restricted the reported confirmed cases. figure estimated and observed number of existing infected cases from feb to apr . the optimal results of is . , is . , is . , and n is , . the is . . based on these optimal results, we produced the estimated number of existing infected cases, shown in figure . our simulation results displayed that the estimated peak number of existing infected cases would be , on may . compared to the official reports, which showed that the peak number of existing infected cases was , on april , these results indicated that ) npis took in period ii might have reduced about % of the daily contacts per people compared to that before covid- ; ) owing to the effects of npis, the epidemic transmission may mainly have occurred through family contacts . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . https://doi.org/ . other than social contacts such as workplace (zhang et al., ) ; and ) the ܴ of period iii was close to that reported throughout the entire period, which means that, the capacity of daily rt-pcr during period iii may have satisfied the required numbers. we developed an sird model to estimate the peak number of existing infected covid- cases, the peak date, and the ܴ in different periods. for an aging society like japan, the most important prediction is the maximum number of patients who will require medical service. this prediction is of crucial importance to design for policies of covid- containment in japanese hospitals and to calculate the time period in which they need to be available. meanwhile, as it is the first time to declare large-scale emergency declarations of infectious disease in japan, the prediction is of fundamental significance to plan for social countermeasures such as npis. for the monte carlo simulation, instead of using the total population of japan as the n population (sugishita, kurita, sugawara, & ohkusa, ) , we used the number of people who received rt-pcr to estimate the developing trends of epidemic transmission. this method can generate more reliable predictions of the number of existing infected cases than using other methods. reasons are that, first, we consider that the total confirmed cases are less challengeable for medical service than the existing infected cases. studies have reported that, about % of covid- patients did not need medical treatment, and thus do not exert any burdens on medical service (prime minister of japan and his cabinet, a). second, using the number of rt-pcr receivers to predict the peak incident can display the effectiveness of countermeasure policies. for instance, in period ii, the estimated peak incident is much more serious than the observed fact, meaning that the capacity of daily rt-pcr in japan cannot satisfy the required numbers. third, the prediction of peak incident in each period is crucial for the preparation of the minimum number of hospital beds. for instance, based on estimations of the peak incident by japanese experts, the total hospital beds for covid- patients have increased from about , in early february to , in may (japan ministry of health labour and welfare, ). our simulation results showed that despite period i, the estimated peak date could appear to days later than the observed date. the biggest reason might be because of the capability of daily rt-pcr, which directly defines the observed confirmed cases. another reason might be the high level of japanese medical service. on jan , the japanese government announced that the covid- epidemic has been defined as "a designated notifiable disease" according to the law of infectious disease and patient treatment, and medical treatment expenses of covid- patients will be covered by the national budget (prime minister of japan and his cabinet, b). owing to stable and high-qualified medical service, the japanese society seems to successfully have discharged covid- patients and have avoided collapse of hospital service. according to our simulation results, ܴ in period ii is about . , which is similar to that reported by previous studies in china and italy (chinazzi et al., ; remuzzi & remuzzi, ; zhang et al., ) . nevertheless, ܴ throughout the entire period was a smaller number as of . . when considering that we used the number of covid- in critically ill patients in the seattle region -case series faith and global health practice in ebola and hiv emergencies estimating the impact of school closure on influenza transmission from sentinel data report of unexplained pneumonia by wuhan cdc the effect of travel restrictions on the spread of the novel coronavirus (covid- ) outbreak impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand observational study of hydroxychloroquine in hospitalized patients with covid- targeted social distancing design for pandemic influenza clinical characteristics of coronavirus disease in china hospital beds for infectious disease situation report population census fifty years of influenza a(h n ) following the pandemic of novel coronavirus (covid- ) cases effect of non-pharmaceutical interventions for containing the covid- outbreak in china. medrxiv early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia cardiovascular disease, drug therapy, and mortality in covid- non-pharmaceutical interventions during an outbreak of pandemic influenza a (h n ) virus infection at a large public university mathematical modelling of the transmission dynamics of ebola virus basic countermeasure policies for covid- containment prime minister of japan and his cabinet. ( b). meeting report of the headquarters of covid- containment covid- and italy: what next? the lancet effect of voluntary event cancellation and school closure as countermeasures against covid- outbreak in japan. medrxiv an investigation of transmission control measures during the first days of the covid- epidemic in china isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus ( -ncov) outbreak changes in contact patterns shape the dynamics of the covid- outbreak in china evolving epidemiology and transmission dynamics of coronavirus disease outside hubei province, china: a descriptive and modelling study. the lancet infectious diseases clinical characteristics of covid- -infected cancer patients: a retrospective case study in three hospitals within wuhan key: cord- -q xh xts authors: kano, shigeyuki; kimura, mikio title: trends in malaria cases in japan date: - - journal: acta trop doi: . /j.actatropica. . . sha: doc_id: cord_uid: q xh xts just after world war ii, more than , malaria cases per year were reported in japan, including indigenous, imported and induced malaria. malaria has been successfully eradicated since in japan and now only imported malaria cases are encountered. however, as the number of japanese people who are going abroad and also the number of foreigners who are visiting japan increases (about and millions, respectively, in ), so does the chance for japanese doctors to see imported malaria or transfusion-transmitted malaria cases. in fact, the total number of the patients with acute malaria in japan has been around – annually for the last years. of those, about % are japanese and % are foreigners, and about % are male and % are female. the peak age is in the s. recently, about % of patients are plasmodium falciparum and another % plasmodium vivax infections. the former species is likely to be seen in travelers coming back from african countries and the latter is mainly from asian countries. the important issue is that patients in japan have not been diagnosed promptly nor treated properly because doctors in japan are no longer familiar with tropical medicine. therefore, some patients are dying from severe malaria as a consequence. as it is, most of the effective medicines for drug-resistant malaria or severe malaria have not been registered in japan. there is now a need for medical practitioners to focus on travel medicine in japan. in just after world war ii, about , malaria cases including indigenous, imported or transfused malaria were reported according to the surveillance and statistics of the ministry of health and welfare, currently the ministry of health, labour and welfare (mhlw) of japan. owing to the dedicated effort made through the community-based malaria control programs in local settings in japan, indigenous malaria has been eradicated since (ishigaki city office, ) . in time, the japanese economy grew and the density of the responsible mosquitoes for the transmission such as anopheles sinensis and anopheles minimus decreased with the promotion of hygiene. however, the malaria situation in endemic areas, particularly in developing countries in the tropics, has not improved dramatically; in fact, it is reported to be worse partly because of the emergence of drug-resistant malaria and insecticide resistant mosquitoes. deterioration of the current malaria situation has also been caused by global environmental changes and population movements. many people now are traveling in malarious areas, visiting friends and relatives, and coming (back) to regions or countries where there is no malaria. consequently, the spread of malaria is now a very big burden not only for people in malarious areas but also for those in non-malarious areas (schlagenhauf, ) . japan is not an exception to this problem, because the number of international travelers from/to japan is steadily increasing ( , , japanese people traveled abroad and , , foreign citizens came to japan in ). as a result, no fewer than imported malaria cases have been reported annually since (ohtomo and takeuchi, ) . a higher incidence of fatalities than that reported in other developed countries has also been seen in japan, because prompt and proper diagnosis and treatment are not readily instituted by physicians. in this article, we review current trends of imported malaria cases in japan. because of the side effects of chloroquine such as retinopathy among chronic users with renal diseases, administration of the drug has been prohibited for malaria patients as well. since then, oral quinine had been the only licensed drug for malaria for decades. in order to conquer the situation of the shortage of the available drugs in japan for the treatment of malaria and other tropical diseases, a research group on the chemotherapy of tropical diseases has been established since supported by a grant from the mhlw. at last in , "fansidar ® (pyrimethamine/ sulfadoxine)" was registered in japan, and quite recently in october , "mephaquin ® ss tablets (mefloquine)" was allowed to be marketed in japan as a result of the continuing effort of the research group. the research group is importing other nonregistered antimalarials after obtaining permission for their use as orphan drugs from the mhlw and checking their quality at the national institute of health sciences to clear the japanese good manufacturing practice standards. the drugs are then distributed to research group member hospitals or universities throughout japan for the malaria patients have access ready to the drugs. those antimalarials are chloroquine, primaquine, injectable quinine, artesunate (i.v.) (oral/rectal/intramusclar/intravenous), atovaquone/proguanil, and artemether/lumefantrine. the web page of the research group is now a useful one (http://www.ims.u-tokyo.ac.jp/didai/orphan/, at present in japanese only). the research group analyzes demographic data together with questionnaire-based studies on imported malaria cases. purpose of the travels, destination and period of travels, prophylactic antimalarials used, initial diagnosis made, chief complaint, present and past histories, species of the parasites infected, and laboratory data during hospitalization, have been asked and compiled. the research group is also collecting data on the efficacy or adverse effects of the drugs when they are used. table shows the number of malaria cases through - . during those years, - cases were identified annually and, of them, - % were foreign visitors. people coming from india have been the largest in number followed by those from the philippines. the highest number of fatalities was observed in when the number of deaths was four (data not shown). because of the increasing number of japanese travelers to african countries nowadays, the proportion of falciparum malaria cases seems to be gradually increasing. this trend is illustrated in fig. . from , the proportion of falciparum malaria patients became comparable to that of vivax patients, and the available data on japanese patients show that nearly three-fourth of them were suffering from falciparum malaria (data not shown). trends of malaria treatment have been analyzed and precisely reported by one of the present authors (kimura et al., ) . briefly, mefloquine was used for treatment only in . % of cases in , whereas it has now become more widely used at % in . to our surprise, artesunate was administered initially for % of the total number of patients in . on and after april , a new law which is called "the law concerning the prevention of infectious diseases and medical care for patients of infectious diseases" was effective. the law before the revision was enacted in and was quite outdated after years of application. in the new law, infectious diseases are categorized in five groups according to the severity and infectivity: "group " consists of seven diseases namely ebola hemorrhagic fever, lassa fever, crimean-congo hemorrhagic fever, marburg disease, pest (plague, severe acute respiratory syndrome (sars) and smallpox); "group " has six diseases namely typhoid fever, paratyphoid fever, diphtheria, cholera, bacillary dysentery (shigellosis), acute poliomyelitis (polio); "group " has only escherichia coli diarrhea such as o ; "group " is currently made up of diseases including such vector-borne diseases as malaria, yellow fever, and west nile fever; and "group " is of other diseases i.e. amoebiasis, syphilis, and influenza. there is another category of "new infectious diseases" which will include emerging infectious diseases human beings have never before contracted. under the new law, malaria is now one of the diseases to be reported to the governor by the physician who diagnoses a patient within days. the reporting system has been simplified after the revision of the law by submitting a questionnaire in which the following questions are asked: ( ) sex, ( ) age, ( ) how the diagnosis was made (microscopic, serologic, symptomatic, or by other instruments), ( ) description of the chief complaints, ( ) date of onset, ( ) date of admission to hospital, ( ) date of definitive diagnosis, ( ) suspected date of contraction, ( ) date of death (if the patient is already dead when diagnosed), ( ) places of residence for the last few years, ( ) suspected place of where malaria was acquired, ( ) possibility of being bitten by the mosquitoes, ( ) suspected route of infection, and ( ) if there are friends or relatives around who are complaining of similar symptoms at the same time. through this questionnaire, we cannot identify the patient or even know his/her citizenship. doctors are not asked to submit slide smears of the patient to be re-checked by experts, therefore we suspect there might be some misdiagnosis in deciding the species. in fact, there were some symptomatically diagnosed malaria cases reported without our knowing whether the patients were showing parasitemia. severity of the patient is not fully described, drugs used for the treatment are not reported, and, as it is, the outcome is not known at all. however, the number of cases reported has been increasing because, under the new law, doctors not reporting a case will be punished with a fine of no more than , japanese yen (around ). the mhlw of japan announced the annual number of malaria cases reported to the ministry (fig. ) . it seems that the reporting system had not been working effectively under the previous law before the revision in , because the number of the patients is about a half of that reported by the research group each year (table ). in the national epidemiological surveillance of infectious diseases (nesid) under the new law conducted by the national institute of infectious diseases (niid), the number of malaria cases have been officially reported (table ) (niid and idcd, ; niid, ) . the largest number of malaria patients ever reported in may be a reflection of the new regulations on reporting system. however in , as the annual number of japanese people who fig. . the highest incidence is observed in the late s, and this trend is observed every year. young patients whose ages are in the s are likely to be infected during their summer vacation through august to september (data not shown) (niid, ) . the first report of transfusion-transmitted malaria in japan was described by sakai ( ) including two vivax malaria cases; one was a -year and -month-old boy who received ml of whole blood from his father for the auxiliary treatment of bacillary dysentery, and the other was a -year and -month-old boy who received totally ml of whole blood from his father for the auxiliary treatment of pertussis. totally cases of transfused malaria were reported before world war ii, in which most of the blood donors were patients' parents but paid blood donors were included (takada and nakayama, ) . during wartime, those paid blood donors were likely to be veterans coming back from tropical pacific areas where malaria was very endemic. of course, the possibility of transfusion-transmitted malaria was well known to medical technicians in those days, but there was no systematic way to identify the infectious blood. after the war, preserved blood was mainly used for the transfusion, which increased the chance of infection. then, the first case of preserved blood transfusion-transmitted malaria was reported in in a -year-old man receiving totally ml of the blood during lung surgery (takada, ) . twenty-two transfused malaria cases after world war ii were summarized by ito et al. ( ) reporting also that the total number of reported transfusion-transmitted malaria cases in japan was ( plasmodium falciparum case, plasmodium vivax cases, plasmodium ovale case, and cases from unknown species). in , platelet transfusion-transmitted malaria was reported in a -year-old woman patient suffering from thrombocytopenia, who had been unsuccessfully treated as a consequence (kano and suzuki, a) . this case was the last case of transfusion-transmitted malaria case in japan, and is the th reported transfused case so far within the authors' investigation. malaria infection associated with needle injury or usage of contaminated syringes has also been reported in japan since . the first report was of nine cases of malaria among drug users sharing the same syringe (otsuru and kamo, ) . after world war ii, many people including veterans were homeless and jobless, indulging in the intoxication of drugs or narcotics, sometimes selling their own blood as an act of desperation, and consequently spreading malaria parasites. morishita ( ) summarized the syringe-associated malaria cases among these people during - . the first nosocomial malaria case was a -year-old nurse, most probably transmitted by accidental needle injury from a falciparum malaria patient whom she was taking care of at the hospital (amano et al., ) . chloroquine mg, sulformethoxine mg and pyrimethamine mg treatment was administered through a stomach tube after she fell into coma but this could not save her life. another needle-associated falciparum malaria case reported in was a -year-old male who was successfully treated and cured (kimura et al., ) . no iatrogenic malaria was reported in japan transmitted, for example, by non-sterilized surgical operation, through contaminated catheter, or by hypnozoite-infected liver transplantation. so-called laboratory malaria, which may happen among researchers who are cultivating the p. falciparum parasites, has never been reported in japan, either. halofantrine has been reported to be effective since involving more than patients in countries, and particularly for the treatment of multidrug-resistant malaria in the world, because its chemical structure is quite unique and not closely related to any other antimalarials. the first case of malaria successfully treated with halofantrine in japan was reported in , who was a -year-old pakistani male suffering from p. vivax malaria (yodonawa et al., ) . parasite clearance was rapid and fever dropped dramatically. no adverse reaction caused by the drug was reported except slight nausea. following this case, the first author (s.k.) of the present paper and his group reported a total of imported cases who were successfully treated with halofantrine: p.f. and p.v.; all japanese male, p.f. and p.m. mixed; foreign female (masuda et al., ; kano et al., ; obana et al., ; kano and suzuki, b) . it was in when we first experienced recrudescence of falciparum malaria after treatment with halofantrine . in order to meet this resistance, we established in vitro halofantrine susceptibility test (shikado et al., ) . we formed the opinion, as a result of this experience, that halofantrine was very effective, safe and tolerable for the treatment of all species of malaria. however, in , a clinical study involving patients on the thai-myanmar border revealed cardiac effects of halofantrine, including one sudden death after the treatment (nosten et al., ) . then there were some spontaneous reports of serious ventricular dysrythmiasis with prolongation of qt intervals, so we attempted to monitor ecg while we were treating the patients with halofantrine. we instituted the treatment for two japanese imported malaria cases (p.o.: male years old and p.v.: male years old) with informed consent, and monitored the ecgs. consequently, we found that the qt intervals were slightly prolonged with unaffected qrs intervals. no symptoms or signs were observed on their cardiac system other than ecg findings kano, ) . who announced a drug alert for halofantrine in which conditions were described which doctors should abide by (who, ) . further careful studies on individual japanese patients to confirm its efficacy or tolerance are needed before halofantrine can be generally used in japan. artemether is a chemical extract of the herb qinghao (artemisia annua l.) that has been used for about years in chinese traditional medicine for the treatment of fever and malaria. this drug has been reported to be particularly useful for the treatment of severe and complicated falciparum malaria. the first case of severe malaria treated with artemether (i.m.) in japan was a -year-old japanese male who had been traveling in africa (kano et al., ) . on admission, he was already comatose with a parasitemia of %. artemether (i.m.) was administered intramuscularly to the patient with the informed consent of his family. he was successfully cured with no consequent sequelae. in those days, artemisinin derivatives were only distributed by limited authorities in china, from whom the drug had been kindly given to the first author (s.k.). the second case treated with artemether (i.m.) in japan was a much more severe case of a patient with mixed infection with falciparum and vivax malaria, who showed p. falciparum parasitemia at % (hosaka et al., ) . his deep coma improved soon after the administration of the drug. we did not understand why the artemisinin derivatives were so effective, but an experimental study conducted on the morphology of p. falciparum in an owl monkey confirmed the effect of artemether (kawai et al., ) . various regimens of the administration of artemisinin derivatives were introduced in japan using tablet, suppository, intramuscular or intravenous fluid. about that time, we already knew that the recrudescence rate was high when one of the artemisinin derivatives, artesunate, was used as single monotherapy (kondou et al., ) . anyway, artesunate or other artemisinin derivatives have not been officially registered for use in japan, the above-mentioned research group have stocked plasmotrim ® -lactab (artesunate tablet) and plasmotrim ® -rectcaps (artesunate suppository) in case we need to treat severe and complicated malaria in japan. quite recently, several new data have been reported by looareesuwan and his group in mahidol university, thailand, that combination therapy with artesunate (i.v.) followed by mefloquine is quite effective for the treatment of both drug-resistant malaria and severe malaria. this combination has been tried in japan on three patients and the outcomes reported (yasuoka et al., ; itoda et al., , yoshizawa et al., . now, trends in action towards multi-drug-resistant malaria are being developed with new combination therapies that include artemisinin derivatives. artemether-lumefantrine (coartem ® / riamet ® ) will be one of the very promising combinations (ishizaki et al., ) , which will be accepted for general use in japan. although the number of japanese travelers going abroad is increasing, % are visiting europe and north america, % are traveling to asian countries where korea and taiwan are the most popular, and only % are going to african countries. therefore, the chances of contracting malaria will be smaller for japanese travelers than for american or european travelers. moreover, the number of foreign travelers coming to japan is not high (about million) as compared to france (> million) or united kingdom (> million). introduction of the parasites through a migrating population who may be frequently visiting friends and relatives is not a primary concern in japan. nevertheless, we have to be fully prepared for the continuing increase of malaria patients, particularly from areas where drug-resistant malaria is endemic. the total amount of blood donations is around million a year in japan. therefore, if we apply the estimate of incidence of transfusion-transmitted malaria in the united states which is about or in million units (mungai et al., ) , the possibility of parasitic contamination in preserved blood for transfusion in japan may be nil, if we abide by the us guidelines on blood donor selection. in fact, the japanese guidelines are stricter than in the us', so the chance of transfused malaria is very small in japan. a surveillance system of malaria patients in japan has been enforced by the mhlw after the enactment of the revised infectious diseases control law in april . however, the information we get from the survey has to be augmented by that obtained through the activities of the research group. integration of the two independent surveillance systems should be envisaged. another problem to be solved is the drug preservation and distribution system under the research group. the mhlw of japan only allow use of fansidar ® and mephaquin ® officially, but encourages the research group at the same time to be fully prepared with new drug therapy for severe malaria cases. efforts have to be made by the members of the research group, for example, by publishing case reports and guidelines, until mhlw accept the importance of introducing effective new drugs. the significance of chemoprophylaxis has not been well recognized in japan, but after the approval of mefloquine for use by mhlw not only for treatment but also for prophylaxis, physicians are encouraged to prescribe the drug for international travelers. still, not many japanese physicians are confident with tropical or travel medicine, and guidelines on the prevention of malaria using appropriate drugs have to be established as soon as possible. mefloquine resistant malaria has been reported widely in asian malarious areas, and indeed there are already some reports of its resistance in japan. we have to prepare for use of another good chemoprophylactic regimen applying, for instance, atovaquone/proguanil, primaquine, doxycycline or minocycline (lin et al., ) . in japan, there are now some other research groups on travel medicine, on vaccine development, on drug development, and on field survey and control, which are, respectively, supported by grants from the japanese government. integration of the malaria research will yield fruitful results for the control of imported malaria cases in japan. a fatal case of induced cerebral malaria due to plasmodium falciparum a case of severe mixed plasmodium falciparum and p. vivax infection successfully treated with artemether history of ishigaki city (ishigaki-shi shi), special issue on malaria an imported case of falciparum malaria successfully treated with artemether-lumefantrine in japan ovale malaria suspected to be transmitted by blood transfusion severe falciparum malaria with prolonged hemolytic anemia after successful treatment with intravenous artesunate two cases of japanese imported malaria cases who showed transient prolongation of qt intervals on ecg after the treatment with halofantrine prolongation of the qt interval observed in a japanese vivax malaria patient following treatment with halofantrine four imported cases of falciparum malaria successfully treated with halofantrine recrudescence of falciparum malaria following treatment with halofantrine a case of accidental transmission of plasmodium falciparum through platelet transfusion two imported cases of vivax malaria successfully treated with halofantrine a case of severe malaria treated with qinghaosu morphologic effects of artemether on plasmodium falciparum in aotus trivirgatus epidemiological and clinical aspects of malaria in japan recrudescence of falciparum malaria treated with artesunate effect of minocycline against mefloquine-, chloroquine-and pyrimethamine-resistant plasmodium falciparum in vitro a case of mix infection with p.f. and p.m. successfully treated with halofantrine remaining malaria problem in japan transfusion-transmitted malaria in the united states from through cardiac effects of antimalarial treatment with halofantrine ministry of health, labour and welfare of japan ministry of health, labour and welfare a case of plasmodium falciparum malaria successfully treated with halofantrine studies on current trend of imported malaria in japan malaria among drug users precaution for transfusion travelers' malaria: some historic perspectives establishment of an in vitro halofantrine susceptibility test for plasmodium falciparum a case of malaria by the transfusion of preserved blood drug alert: halofantrine-change in recommendations for use a case of falciparum malaria successfully treated with intravenous artesunate an imported case of vivax malaria treated with halofantrine a case of falciparum malaria successfully treated with intravenous artesunate we are grateful to all the members of the research group on chemotherapy of tropical diseases, and also to the following financial support: an international health cooperation research ( c- ) and a grant for research on emerging and re-emerging infectious diseases (h -shinkou- ) from the ministry of health, labour and welfare of japan. key: cord- -hk c authors: imanishi, tamami title: an ethnic model of japanese overseas tourism companies date: - - journal: annals of tourism research doi: . /j.annals. . . sha: doc_id: cord_uid: hk c abstract this study aims to analyze the administrative behavior of japanese travel companies overseas. through case studies and questionnaire surveys, the characteristics of japanese travel multinationals are compared to those of manufacturing companies. an “ethnic model” is developed which explains the unique behavior of these companies. it concludes that they are ethnically characterized in target markets, management styles in foreign subsidiaries, emphases on service, and competitors. though this study focuses on the japanese tourism industry, the result is potentially generalizable and applicable to other countries and other non-manufacturing industries. one of the remarkable social phenomena of the th century in japan was outbound tourism. the number of japanese going overseas expanded rapidly. in , when overseas tourism became liberalized, , went abroad, and in , the annual number had reached . million. while ups and downs followed due to wars, terrorism, and epidemics such as sars (severe acute respiratory syndrome), according to the latest statistics available, the number of overseas tourists was . million in (japan national tourist organization : ) . surprisingly, the outbound market grew approximately times in years from its beginnings. corresponding to this tamami imanishi is associate professor of international business at the university of marketing and distribution sciences (faculty of service industries, - gakuen-nishimachi, nishi-ku, kobe - japan. email <tamami_imanishi@red.umds.ac.jp>). her research areas are international business and multinational strategy, with interests in the internationalization of tourism sectors, and the administrative behavior of multinational companies, especially their global operation in inbound and outbound tourism. expansion, japanese companies began to set up foreign offices and started their overseas businesses. when going abroad, most japanese people use japanese companies and these set their major services for homeland people. at first sight, the firms seem to be internationalized, in view of their overseas transaction and worldwide networks. but observed carefully, they appear to be focusing on their own nation and operating domestically. why are they conducting international business in such a way? it has been noted that nationality does affect tourist behavior (pizam and sussmann ) . race also relates to tourism choice (philipp ) . the contents of package tours differ with countries. various factors, such as the geopolitical situation and social structure of the originating country, perceived and social distance to the destination, experience of traveling abroad, language barrier, and so on, affect the composition of tours (cohen and cooper ; enoch ) . how does the industry respond to these various aspects of tourists' behavior? the purpose of multinationalization has been found to be defending and acquiring markets (vernon ; yoshino ) . when a company tries to keep its acquired overseas market, it may start further overseas expansion, and this movement induces competitors' follow-the-leader action. as a result, many multinational companies today seek opportunities to advance into new fields in their overseas strategy. for the maintenance of strategic control and influence on subsidiaries, headquarters build close relationships with foreign subsidiaries (doz and prahalad , ) . in order to strengthen these relationships, the management styles of multinational corporations become ethnocentric (perlmutter ) . such administrative behavior is observed in both manufacturing and non-manufacturing industries. however, in non-manufacturing industries, the following method of advancing their clientele's overseas expansion is also accepted. they try to provide services worldwide to their customers' companies which have business transactions in their home countries. acquisition of local market share is not necessarily the main purpose for service companies to become multinational. due to the relation with customers, the features of a service industry's international management may differ from those of manufacturing. what is the actual condition? if there is a difference, what is the reason for its formation? even in overseas business, japanese tourism companies take it for granted that japanese people, people of their own descent, and japanese companies compose one unit. this situation puts emphasis on shared traditional culture such as language, customs, habits, and fundamental ways of thinking. people who have this historical and cultural similarity are considered as a unified whole. the range of this unit, however, is not exactly in agreement with race or nationality. since tourism has a close relation to one's lifestyle, companies consider this framework in order to provide a sort of service which tourists can feel comfortable with even if they are traveling abroad and are in a different cultural space. what kind of influence will such customer correspondence have on the international management of the tourism industry? different from existing multinational theories basically developed through research on manufacturing industries, another type of international administrative behavior emphasizes company-customer relationships in the multinationalization of tourism operations. however, the administrative behavior of multinational enterprises has been analyzed mainly from the viewpoint of headquarter-foreign subsidiary relations. in the supply of services there is a need for customer participation and this is achieved through interaction with tourists as consumers of actual services. therefore, one should not disregard the company-customer relationship. this paper will analyze tourism companies' international administrative behavior, bearing the existence of the customers in mind. as japanese firms place emphasis on the company-customer relationships of their own nation-japanese nationals, people, descendants, and companies-their administrative behavior can be analyzed as-''ethnic'' or ''ethnocentric''-the central issue of this paper. the purpose of this study is to examine the international administrative behavior of japanese tourism companies. first, through case studies and questionnaires their characteristics overseas are compared to those of manufacturing companies. second, an ''ethnic model'' which shows the unique type of international business behavior that has developed with japanese multinationals will be constructed. there is a strong incentive for the local suppliers and the destination countries to know about the administrative behavior of the companies which handle their important overseas source markets, and to build continuous business relationships. the conclusion will be helpful for the suppliers to understand and to get on well with the tourism multinationals. though focusing on the japanese industry, the results are considered to have generality and will be applicable to other non-manufacturing industries which have a strong connection between headquarters and foreign subsidiaries like banking and trading; or to other countries where people have difficulty in learning foreign languages and the tourism industry is highly structured and generally dominated by large companies (for example, in korea and taiwan). both qualitative and quantitative analyses were used for this investigation. for the former, three case studies were conducted using onsite interviews with tourism employees. a total of employees were interviewed from to . eleven persons were located at headquarters in tokyo and at local subsidiaries. among the , eight were directors and managers with experience as expatriates in their careers, and three were manager-level employees engaged in corporate strategy with no expatriate experience. among the at the local level, two were expatriate employees (one an executive and one a general manager), and were local employment personnel including general managers, branch managers, section managers, and section chief level members. interview scripts were prepared for headquarter members, expatriates, and general and branch managers. the average length of each interview was two hours and a half for the manager level employees and half an hour for the others. all of the interviews were voice-recorded with the permission of the interviewees and transcribed. this qualitative data which reveals the employees' real words and frank views on their work was used as the material and information to complete the case studies. all three companies, kinki nippon tourist company (knt), tokyu tourist corporation (ttc), and japan travel bureau corporation (jtb) are among the largest tourism companies in japan. so far as japanese tourism is concerned, five major companies take more than half of the market share and this tendency to concentration is getting stronger. most of those leading the industry are general companies. they deal with both domestic and overseas tourism, and function as both retailer and wholesaler. the three companies studied here have these characteristics. major companies also deal with other businesses such as finance, goods sales, publications, tour conductors' employment agency, and so on. their business range is much broader than that of mere travel agencies and they would be better called ''tourism companies.'' moreover, these three companies are multinationals. by setting up local subsidiaries, foreign branches, and liaison offices, they have built worldwide networks. in this research, four local subsidiaries will be considered. they are located in the united states (one of the subsidiaries of jtb), australia (of knt), canada (of ttc), and the netherlands (of knt). field surveys were carried out in the united states in december and in australia in november . observations and interviews were made in the field surveys. corporate culture, atmosphere of the office, and employees' commitment to work were observed. each section's job functions were explained by the section managers. since japanese multinationals consider the bases in europe, north america, and oceania to be the three important supports of their overseas business strategy, it is appropriate to study these local subsidiaries as well. for the quantitative analysis, two types of survey, one for headquarters and the other for foreign subsidiaries, were conducted in january-february, . these consisted of six common themes: office details and the situation of overseas expansion, the target markets, the styles of the local management, the service, the competitors, and the results of overseas performance and future developments. the questionnaire for headquarters, consisting of questions, aimed to grasp their view of overseas expansion and the actual condition of the operation. answer forms included multiple choice questions (single or multiple answers possible) and item ranking. the instrument for foreign subsidiaries consisted of questions, the research purpose being to grasp the actual situation at overseas offices. the answer forms were the same as those for the headquarters. in addition, respondents were asked to write freely their opinions about overseas expansion of a company. telephone interviews were conducted with each company which had a license to handle outbound tourism: companies were found to have entered overseas markets. interestingly, telephone interviews of the top companies starting from the highest ranking by sales revealed a size boundary to multinationalization. most of the top companies were multinationals, while none under the th were. questionnaires were sent to all these multinationals and their local subsidiaries. of sent to company headquarters by mail, were completed and usable, representing a response rate of . %. they were completed by executives and managers of outbound tourism, overseas businesses, or general affairs. similarly, of sent to foreign subsidiaries ( countries), ( countries) were usable, representing a response rate of . % (imanishi ) . most responses were completed by presidents and managers. in this paper the quantitative data complement the qualitative research. knt was established in as a firm owned by an electric railroad company called kintetsu corporation (kinki nippon tourist company ) . while only a few companies are listed on the stock market, knt was the first one to be listed in the first section of the tokyo stock market. it has domestic offices and has set up foreign branches in cities, extending its global network to europe, north america, southeast asia, and oceania. the number of employees is , (corporate profile as of march ). in , expatriates and local staff were working in the foreign subsidiaries (company data as of october ) . among all the local subsidiaries, the three most significant strategic positions are the bases located in north america, europe, and oceania (personal interview with the director of knt in ). the first advance into europe started with the establishment of the amsterdam liaison office in . the objective was to provide services for general tourists such as holidaymakers. local arrangements and operations were entrusted to local tour operators. from doing business in this way, serious problems arose. complaints and grumbles from the customers increased. the tourists were dissatisfied with the service offered by the local operators and wished to maintain their habits and customs during their trips. usually sleeping in their own style bedding called futon, and even when traveling foreign countries with wife or husband, they had a strong preference for twin rooms with bathtubs. they preferred the plain taste and sometimes required chopsticks when eating. they asked for the same service as supplied in their home country. but it was hard for the local tour operators to understand japanese tourists' behavior and ways of thinking. local operators could not explain and manage affairs well. so the headquarters had to manage and administer the local tour operator from the home country. however, reflecting the means of communication in those days, they could not but depend on telephone, letter, and telex, so real time interchange was difficult. from this experience, knt decided to set up local offices of their own and station expatriates to take care of their customers directly. rapid economic growth also caused the overseas market to expand. due to the increasing number of tourists, foreign subsidiaries began to carry out local operations by themselves. collection and accumulation of information and experience on overseas business allowed them to do ''own operation.'' there are three main reasons for tourism companies to start own operation. the first is for customer management. in such firms, a salesperson would use door-to-door approaches to sell and get orders. when a contract was written, the customer became his client and the salesperson was involved in the whole trip, from making the itinerary to conducting the tour. it was natural for them to take care of their customers by themselves. this traditional role and way of thinking has deeply penetrated the business and made it easy for them to perform own operation. the second reason is the prevention of information leakage. by accumulating and analyzing the customer information, knt is able to create original plans and services which will be the company's competitive advantage in the near future. the third reason is cost reduction. by excluding local operators from the distribution system, they no longer have to pay commission. to fulfill the requirements for getting a business license in the overseas market, the form of the office had to change from a liaison operation to a local subsidiary. gaining this status, credit, and bargaining power, knt tried to run their business as a local company in the host society, with equal relationships with area enterprises and suppliers. as a result, in kintetsu international express (europe), besloten vennootschap for example, the major business is inbound tourism dealing with japanese tourists. the principle of management is a mixture of the japanese system and the local style. the latter is used in business practices such as wage scale and documentation. homeland methods are practiced in creating the organizational climate or corporate culture such as time strictness. in amsterdam, % of the employees used to be japanese and % not. however, because of the large volume of japanese tourists, homeland employees are considered to be more appropriate and their proportion is increasing. owing to the small scale of the japanese labor market in europe, it is hard to employ workers from there, so the assignment of human resources is determined by their talent and positioning. for example, for frontline staff-those who meet the customers, such as guides, conductors, and counter staff-japanese and japanese-ancestry staff or at least those who can speak the language are used. for top management who shoulder the decisionmaking, expatriates are used. for back-office staff-those who do not meet the customers but support the service delivery system to make it efficient and effective, such as in accounting and administration-locals are employed (interview with the general manager of international affairs in and the director in ). in the case of kintetsu international express (oceania) proprietary limited that supervises both offices in australia and new zealand, due to a large japanese labor market in the area, . % of the employees are japanese, of which . % are expatriates (field research in ). looking into the case of knt, the following features can be characterized from the overseas development of a company which intends to deal with general tourists. the purpose is to enhance services to tourists. it tries to cope with the problem that they have strong preference for similar standards as in their home country, and are often not satisfied by what local operators provide. to solve this problem, overseas offices are usually established in major destinations. the management style, though containing some local flavors, in principle follows japanese style. this corporation was founded in as the tourism firm owned by an electric railroad company called tokyu corporation. it has domestic and foreign offices in cities mainly located in the pacific rim and europe. while half of the foreign branches are liaisons, two local subsidiaries established in the united states and the united kingdom take the form of affiliated companies. the former has five branch offices (the foreign subsidiary established in guam also took the same form but was closed in ) . ttc changed its name in to toptour corporation, after their main package brand. the number of employees in ttc is , (corporate profile as of april ). since it undertook a large volume of business intended for corporations in the home country, the main purpose of its multinationalization was to handle tourism services generated by japanese corporations' expansion in their overseas business (personal interview with the managers of international business relations in and ). ttc's advancement to canada started in with a liaison office in vancouver. there were two reasons for this. the major one was the influence of the tokyu group: their development plan (the pacific rim plan) was urged by the whole group, and all its companies had to join it. when pan pacific hotel vancouver, owned by the group's hotel company, was established, ttc also opened its office in the same location. being one of the five investment companies, it tried to support the hotel by sending tourists to it. equally, ttc was supported by the hotel. usually a liaison office is managed by at least two employees, one expatriate and one secretary, but the vancouver office could be run by only one expatriate. the hotel secretary held the additional post of secretary of ttc. in light of the overseas advancement in the tokyu group, there obviously existed a strong expectation of synergy. the second reason was the increase in the number of tourists. due to the strength of the yen after the plaza accord-an agreement signed in at the plaza hotel in new york by the then g nations (france, west germany, japan, the united states, and the united kingdom) to devalue the us dollar by intervening in currency markets-many japanese manufacturing companies changed their international strategies from exportation to local production, and set up foreign subsidiaries (yoshihara ) . not only international business trips but also international corporate incentive tours increased in headquarters and their foreign subsidiaries. further, sightseeing tourists increased. the strong yen made japanese overseas tourism more affordable than ever before. it was expected that canada as a country with magnificent natural attractions would become one of the major destinations and that the market would increase continuously. hoping to succeed in capturing a larger number of tourists and serving them, ttc determined to start their business in vancouver. the role of the liaison office was to control and supervise local tour operators. since canadian law prohibited the liaison office from business activities, all local business was entrusted to local operators. it was necessary for ttc to make sure that they were providing services as instructed. however, as with knt, to expand their business and deal with overseas tourism and start wholesale business, after running for seven years as a liaison office, it changed its form to a local corporation in . two expatriates were sent from japan and two local staff were employed. one of these was of japanese descent, proficient in the language, and was assigned to be the operations manager. the other, a non-japanese with legal expertise, took charge of administration. though having obtained a business license and permission to deal in outbound tourism, the local subsidiary's major affairs still remained in the area of inbound tourism. it was responsible for reservation of hotels and buses, but control of guides was assigned to local operators. despite getting the right to initiate outbound business, this subsidiary was too busy with inbound business to tap the local market. though it was at the frontier of a new growth area, it could not bring the opportunity into play. in , ttc closed its subsidiary and withdrew from canada. the major reason was the inefficiency of the operation. the actual number of tourists was less than expected. at present, having no offices in canada, ttc is sending tourists through local operators with an office in japan. through this investigation of the case of ttc, the following features can be remarked in the overseas development of a company which intends to deal with corporate tourists. its purpose was to take charge of business tourism generated as japanese companies expanded overseas. ttc started its overseas business to exercise the advantage in corporate business possessed in its home country. it followed the movement of japanese companies in order to provide them with tourism services. for this reason, ttc has set up offices in areas where many foreign subsidiaries of japanese companies are located. both the purpose and the areas correspond to the multinationalization of companies. this corporation has domestic offices and has set up foreign ones in cities in countries, extending its worldwide network to europe, north america, asia, oceania, and micronesia. the number of employees is , (corporate profile as of march ) . when it started its overseas business, the purpose was to attract foreign tourists to japan. this original intent was different from other companies. however, as the outbound market expanded in the homeland, it over-took the inbound (personal interview with the manager of international travel in ). the first overseas advancement of jtb began in . its predecessor, the japan tourist bureau, set up an information desk at the japan society in new york. its own first foreign unit was established in as the new york branch office. during world war ii international tourism was interrupted, but it revived after the war. in , the new york branch reopened with the function of public relations, followed the next year by the los angeles office. with the establishment of the japan national tourist organization as a foundation, jtb delegated its tourism public relations agencies in and opened new tourism offices in new york and los angeles in . the main business was sending tourists to their home country (japan travel bureau foundation ) . however, to reinforce overseas business, it became necessary to change the formation from an office to a locally incorporated body. business was limited to non-profit activities. in , the homeland parent company had changed to a profit-making enterprise, so the subsidiary was to be treated the same way. the local tax authorities did not accept the overseas business of jtb as non-profit. since most of the japanese tourism companies in the united states had incorporated locally, jtb followed. in , all its us offices were reorganized as japan travel bureau international incorporate (jtbi) so that it could expand its branches and its business field. adapting to the tourism expansion in its homeland, jtbi began actively to welcome tourists and started its own operation. nowadays jtbi is divided into three organizations: jtbi, jtb usa, and jtb travel network. each is individually incorporated with a different business orientation. jtbi handles tourists from the home country, jtb usa treats tourism from the united states, and jtb travel network designs tours which depart from us cities. though each organization differs in its business field, the customers and target markets are nearly the same. they focus on japanese and those of japanese ancestry (field research in ) . the mix of human resources also differs between jtbi and jtb usa. the proportion of homeland/japanese-ancestry personnel to non-japanese in jtbi is seven to three, while in jtb usa the proportion is the reverse. the details of each organization are as follows. in jtbi which deals with tourism from japan to the united states, many japanese are assigned to the frontline. even if not japanese, staff that have japanesespeaking ability will be employed. in the back office, employment depends on job specifications, not ethnic origin. for example, functions which require local legal knowledge, like finance and accounting, have a local employee assigned. a buyer who needs to negotiate with nearby suppliers is also a local person. however, since most of the customers are japanese, japanese employees choose and create the tour for these tourists. japanese staff select services which will compose the trip, and the local staff negotiate and buy them. after purchase, japanese staff form them into a package tour. it is considered that they know japanese tourists' needs and demands and have good ability in arranging all the services. in the organization dealing with tourism into the united states, where the number of japanese/ancestry clients is large, homeland management style appears strongly. in the organization dealing with tourism out of the united states, two aspects can be observed. where the target clientele is japanese/ ancestry, the same features appear as in dealing with tourism from japan. however, where the target clientele is non-japanese, there is no need for the frontline staff to be so too. as before, local people work for finance and accounting, but they can also play an active part in frontline activities, and in choosing and creating a tour. as the number of non-japanese staff is relatively large, local management style will be practiced instead of a japanese one. yet the japanese/ancestry market has a greater weight than others. it can be said that the characteristics of an organization will vary with the affairs it conducts. especially in the frontline, employees are matched to customers. if the clientele is mainly homelanders, japanese staff will be assigned. on the contrary, if the clientele is mainly from elsewhere, non-japanese staff will be assigned. in the back office, locals who possess specific knowledge are assigned. the type of human resource influences the organization's management style, whether to adopt local practices or those of its mother country. by comparing the administrative behavior of overseas advancement between japanese tourism companies and manufacturing multinationals, four differences can be pointed out: target markets, management style, products (goods/services), and competitors. japanese manufacturing enterprises started their overseas business by exportation. in the early stage of international transactions, the form of indirect exportation via trading companies was adopted mainly by textile companies and the like. by the middle of the s, the form shifted to direct exportation, involving companies setting up foreign subsidiaries and building their own off-shore sales network. this was mainly adopted by automobile companies and electrical machinery companies. however, the importing countries began to put japanese companies under pressure. customs duties and quantity limits were applied to exports, for example. for fear of losing their existing markets, manufacturing companies had to change their overseas strategies from exportation to local production in order to defend and maintain them (yoshino ) . in local production, multinational companies manufactured products for the local markets, and nowadays they are also producing for their homeland and for the worldwide markets (yoshihara ) . the products are adapted and developed to meet demand in the international markets. they are sometimes oriented locally and sometimes standardized worldwide. though it is not enough, localization of managerial resources in foreign subsidiaries is proceeding; for example, many natives are employed in them (yoshihara ) . the competitors for the manufacturing multinationals are the companies who target the same markets they do. this would include not only japanese multinationals, but also local companies and the industries of other countries. most of the manufacturing firms are in global competition. by contrast, while manufacturing multinationals intend to develop new global markets, tourism companies start overseas businesses in their target markets. their development can be divided into two types, one for general tourism and the other for business tourism. the former set up foreign offices to improve services for their customers. japanese overseas tourists used to complain about the service provided by local operators, who had trouble in comprehending their needs and complaints. japanese companies had to go overseas to solve these problems themselves. the latter, which rely on corporate travel, start their overseas business with the aim of getting clients from expanding japanese companies. they are often asked to come overseas by japanese multinationals and their expatriates that have business relations in the home country. sometimes there is a request from a group company. this type of company tries to make use of its advantage in corporate sales to obtain clients through business trips, corporate incentive tours, and expatriates' family journeys. however, no matter how the companies differ in their business market, basically both types set their focus on japanese and people of japanese ancestry, and homeland corporation tourism business. according to the questionnaire surveys, for companies which send or welcome tourists from the home country, these amounted to . % in the parent company, and . % in overseas offices. decisionmaking about the location of an office is performed on the basis of the number of their people visiting the spot. hence, foreign offices are located in major destinations and major multinationals' established regions, specifically in europe, north america, oceania, and southeast asia. all these locations are places where large numbers of japanese visit and many people of japanese ancestry live. second, concerning the local management style, . % of foreign offices surveyed were run using a mixed style, with . % based on the local management style and . % on the homeland one. in terms of the employment in foreign offices, the percentage of japanese and ancestry employees is relatively high. according to the quantitative data, expatriates count for . %, japanese and ancestry local staff for . %, and local staff for . % of total employment (table ) . japanese and ancestry people represent more than half of the employees. looking into employment further, the characteristics of each region, based on the classification defined by the world tourism organization, are as follows. in the americas region . % of the employees are japanese or of their ancestry, locally employed. this ratio is relatively higher than in other areas, because hawaii is included in these totals, an area where many japanese and people of their ancestry reside. since the labor market is abundant there, japanese and people of that descent are available for employment by companies in this area. in the east asia and pacific region, however, . % of the employees are non-japanese local staff, except in australasia which is also included in this region, where . % of the employees are composed of japanese and their ancestry local staff and only . % of the employees are expatriates. on the other hand, in southeast asia which is also included in this region, the employment of non-japanese local staff is as high as . % and there are as few as . % japanese and their ancestry local staff. in europe, although . % of the employees are non-japanese, . % are japanese and of their ancestry locally employed. as a result, it can be said that companies tend to employ people of their own nation in any area where they can utilize their people and their ancestry as their laborforce. on the other hand, even with a resident population of japanese/ancestry, non-japanese employment tends to increase where firms are unable to utilize ethnic employees as the laborforce, such as where the japanese residents abroad are expatriates, students, and the like. examining more closely the type of employees, these can be characterized according to the level of responsibility and likelihood of contact with their own people (table ). some . % of the offices appointed expatriates for top management (executives) to communicate and build strong ties with their headquarters, . % assigned local japanese and their ancestry staff, and only . % adopted local non-japanese for executives. progress in localization of top management could not be observed. for frontline staff, . % of the offices employed local japanese and people of their ancestry, . % placed local non-japanese, and . % assigned expatriates. as to back-office staff, . % of the offices assigned local non-japanese because of the need for local knowledge. of the others, . % employed japanese and their ancestry staff, and . % employed expatriates. although there is regional variation reflecting the condition of the local labor market, japanese tourism companies tend to adopt local japanese/ancestry for frontline staff. ability in the use of their mother tongue has become one of the important criteria for establishment of the service. in contrast, within manufacturing companies there is little demand for japanese workers and hence localization is more important in order to reduce costs and to ease local nationalism. third, even in foreign countries, the offering of service is considered significant. ''japanese service'' is of a kind to help homeland tourists keep their habitual ways even when overseas. they make a point of reserving hotel rooms with bathtubs and japanese-speaking guides. when overseas, they usually feel homesick for their own food and begin to feel distaste for local dishes. so in package tours, a japanese meal is often built into the last half of the journey. this device is to reflect their clients' demands. of course, local services which tourists may find exotic are offered, but the companies have a strong focus on a japanese standard of service and they concentrate on supplying it. this is the opposite of manufacturing multinationals which attempt to standardize their goods globally. fourth, while manufacturing multinationals struggle over worldwide competition, tourism companies compete only with japanese enterprises. according to the questionnaire surveys, only . % of the headquarters and . % of the foreign offices answered that they have no competition with others. though there are many non-japanese companies in each location, . % of the headquarters and . % of the foreign offices take it for granted that their competitors are local japanese companies. they perceive their competitors to be the same homeland enterprises that focus on the japanese and their ancestry market. in this situation japanese companies will generally not try to enter and compete in the international market, so they may be said to be acting in niche segmentation. in brief, japanese tourism multinationals operate with a focus on their own nationals who share language, traditions, cultures, and ways of thinking. it is true that whether they belong to manufacturing or non-manufacturing industry, multinational companies operate with the management style of their home country. expatriates are delegated to the offshore sites to keep close relationships between the headquarters and the foreign subsidiaries. however, tourism multinationals differ from manufacturing companies in that their operation is conducted by their own people to achieve adequate correspondence with the needs of their customers. as a result, the international business behavior of japanese tourism companies can be expressed with two dimensions: ''attitudes as fundamental'' and ''markets.'' the first dimension, the attitudes as fundamental, represents the firm's view on global management. this means how executives think about doing business around the world. this concept was first pointed out in the epg profile (perlmutter ) and was revised to the eprg profile (heenan and perlmutter ) . the model indicates the directions of multinationalism by defining four types of headquarters orientation toward subsidiaries in a multinational enterprise. these types are ethnocentric (e), polycentric (p), regiocentric (r), and geocentric (g). while there are several routes to multinationalism (for example, companies highly motivated to multinationalize may proceed directly from ethnocentric to geocentric), most multinational corporations are considered to evolve from ethnocentrism to polycentrism, (and later, in some geographical areas, to regiocentrism) and finally to geocentrism (heenan and perlmutter : - ) . ethnocentric is a preference for putting home country people in key positions everywhere in the world and rewarding them more handsomely for work, along with a tendency to feel that this group is more intelligent, more capable, or more reliable. on the other hand, geocentric is an attitude of attempting to integrate diverse regions through a global systems approach to decisionmaking, and considering headquarters and foreign subsidiaries as parts of an organic worldwide entity (heenan and perlmutter : - ) . ethnocentric and geocentric, the extremes of the attitudes as fundamental, are positioned in the first dimension. the second dimension, the market, represents the firm's target market or handling of it, whether it focuses on the market's ethnic characteristics or not. a posture which tries to confine the market to its own nation is an ethnic approach. on the other hand, the attitude not to confine it and to handle the international market is non-ethnic. by combining these two dimensions, the construct of figure is advanced. the model as constructed classifies the approach of tourism multinationals into four cells. the ethnic model which is the subject of this paper is shown in cell one. it expresses the characteristics of the companies which stand in this position in both the attitudes as fundamental and markets dimensions. there exists a strong relation of ethnocentric-ethnic between the two dimensions. thus, this ethnic model is defined as the international business behavior of a firm whose fundamental attitude is ethnocentric and whose target market is a particular ethnic group. this model explains that by focusing on the firm's own ethnic market, its organization, service, and so on would become ethnocentric. in the case of a japanese enterprise, by bringing its ethnic characteristics into play, its international business behavior will be focused on the japanese and their ancestry markets. describing the other cells, cell two represents the situation with a geocentric attitude on management but focusing on the ethnic market. although it narrows the main target to its own ethnic market like the case in cell one, it differs on the point that it is managed not only by its own nation but also by others. relationships of geocentric-ethnic will be formed. a corporate organization composed of many nations will take all ethnic characteristics and needs into consideration for its one market, but will be able to reduce cost by exploiting local managerial resources, and will transfer and supply superior managerial resources from overseas to cover the resources which do not appear to exist in the local market. since the service will involve deep consideration of the characteristics of that market, personnel education to improve understanding of the ethnic characteristic of that target market would be important to the company operating in this way. though the target market is limited in cell two, the managerial attitude takes other nations into account, so it is separated from cell one and labeled ''semi-ethnic.'' insofar as its operation in japan is concerned, this cell may be applicable to the travel division of american express, because it employs many local people but it mainly deals with non-japanese companies. cell three is the case of a company with an ethnic attitude in management but which does not focus on a single ethnic market. although it performs business activities using its own people, this situation differs from that in cell one in that the company broadens the main target to other people and tries to develop new markets. between these two dimensions, an ethnocentric-non-ethnic relation materializes. since the market is unlimited, a company belonging to cell three is willing to provide services oriented to each ethnic trait of its customers. however, in reality it is not easy for a company with a single nationality to provide multi-ethnic services. in order to provide such service, it is necessary both to master the language and customs of each market and to understand historically formed backgrounds and biases. therefore, when it comes to dealing with multi-ethnic markets, rather than considering the characteristic of each nation, greater weight is placed on the universal or standardized services that make all the people satisfied. that is, although the company of this type tries to deal with global market for the purpose of handling various ethnic ones, still some ethnic elements remain because of its ethnically based management. it has not yet arrived at the point at which it can completely be called a global company. hence, cell three can be named ''semi-global.'' the leisure company club med seems to be of this form. they offer their unique vacation packages worldwide by adapting marketing methods to local practices and utilizing multinational staff at their vacation villages, but usually assign european people to the top management level (hart, arczynski and maher ; imanishi ) . finally, in cell four is the case of the geocentric-multi-ethnic approach in which a company shows a geocentric attitude and does not adhere to a specific ethnic market. in contrast to cell one, a company belonging to the fourth does not stick to its ethnicity on either dimension. further, unlike those in cell two, it functions globally, where there are various ethnic markets. moreover, it differs from cell three and is not ethnic in its administrative behavior. therefore, cell four may be described as ''global.'' in this area, while the company is able to deal with more customers through targeting a wider range of markets, the tourist also has a wide choice over companies and can enjoy the best service he or she can find. to get to this situation a company may incur immense expense. therefore, in a company belonging to this cell, cost reduction will be practiced by setting a wide-ranged market approach and at the same time attaining standardization of service. in this case, the service levels pursued will be irrespective of the ethnic dimension and standardized service takes the lead. with all the four cells now discussed, it is possible to define the ethnic model. the international administrative behavior of japanese tourism companies, the subject of this paper, is characterized by business behavior based on japanese/ancestry people. since they require to be served as they are in their home country even when overseas, dealing with them requires companies to put special emphasis on japanese service and operate by making the most of the noted laborforce. one of the reasons for such operations is that in the background there lurks an ethnocentrism which considers identity of ethnicity of agent and client to result in the best understanding and fulfilling of people's needs. therefore, cell one in figure most correctly describes japanese companies' international administrative behavior. to define cell one further, the following are important. the administrative behavior of the companies is deeply related to the target market and the attitude of the firm. so attempts can be made to describe this behavior from two dimensions, the attitude as fundamental and the markets. cell one is the case where ethnicity is thought to be important in both dimensions. therefore, the ethnic model can be defined as a model of an international administrative behavior within which a company adopts an ethnocentric attitude and puts the main target on its own people. since it focuses on the market of its own ethnicity, this model explains the situation of japanese tourism multinationals' business organization, service, and so forth, becoming circumscribed by national origin. thus, ethnicity is an important concept which determines the international administrative behavior of toursim multinationals. this paper explored the administrative behavior of japanese tourism companies overseas. compared to manufacturing companies, they differed especially on four points: target markets, management style, products, and competitors. the behavior of the tourism group which is deeply involved with their customers' needs is shown to be explained by an ethnic model. the international administrative behavior of such tourism multinationals demonstrates some particular side effects. for example, it strengthens relationships between headquarters and foreign subsidiaries and improves service and efficiency in managerial resources. furthermore, by doing business in this form of niche segmentation, the order of their business will be maintained and international competition will be inhibited. the corporation can reduce uncertainty and reassure its customers. the rapid expansion of the overseas tourism market let companies remain in specified markets. it cannot be denied that these companies were able to get enough profit from their single market, and thus did not try to enter into the international market, which would have introduced cutthroat global competition. however, any tourism service is deeply involved with its own culture and mind. it becomes necessary for the company to satisfy its customers with a little something such as consideration or kindness toward them. while overseas tourists require some exposure to the exotic, they also ask for daily service appropriate to their needs and experience. especially in physiological terms, they demand ethnocentric service. it is natural for a person to decide on the basis of his self-reference criterion, the unconscious reference to one's own cultural values (lee : ) , and people habitually define, select, value, and make references based on their own culture unconsciously. in this context, japanese companies built a system which enabled them to supply their own service worldwide to their customers. at least frontline service had to be similar to the home country. in this sense, ethnic administrative behavior can be said to be suitable for coping with their tourists. however while the model discussed in this paper shows why ethnicbased behavior worked effectively in the developing stage, the overseas tourism market is changing into the next stage of maturity, where the situation for companies may well be different. three important issues can be pointed out. first, the ethnic model does not allow making use of their advantage as a multinational corporation. essentially multinational corporations can think globally, can utilize various resources in other countries (vernon : ) , and are blessed with the opportunity to be exposed to the environmental stimulation of several countries (bartlett and goshal : ) . ethnically-based administrative behavior will not let the company enjoy these advantages. assigning expatriates to top management all over the world would lead to high-cost management and would reduce the motivation of local staff to make real efforts. limiting both management and target market to japanese and those of japanese ancestry will make a company lose the opportunity to be stimulated by other people. further, they are wasting the chance to capture share in the local market. second, ethnocentricity invites a lack of international competitiveness. global homogenization is expanding in the world market (levitt ) . world customers have come to prefer goods and services with low prices, high quality, and good reliability. global companies are trying to achieve competitiveness through discovering the needs and demands all markets have in common. foreign affiliated companies are entering into japanese tourism markets, and are going to induce international competition by trying to attract people with their worldwide standardized service. the growth of the internet makes the transnational approach of companies and customers easy. as overseas tourism experiences increase, people are getting used to foreign service and are not sticking to the companies of their own country anymore. some even have no hesitation about using foreign companies. thus international competition is starting to occur. by persisting with ethnocentric marketing, japanese companies might lose market share. third, they cannot develop new markets by making use of two-waytourism. they are inflexible in responding to movements in the global market. economic and exchange-rate fluctuations frequently lead to offsetting movements in domestic and overseas tourism. it is certainly true that there are many statistics and reports about international market. however, japanese companies are most interested in their own people's tourism trend, so they may not comprehend and forecast tourism worldwide. supported by the same culture, sense of value, and ways of thinking, japanese companies are successful in satisfying japanese tourists. they can relieve their customers from the stress of different cultures. however, the fundamental backgrounds being different, it is difficult for them to understand what non-japanese prefer in tourism services. yet it should be emphasized that while overseas tourism has become generalized and the number of international tourists has increased considerably, japanese companies are still doing business in an ethnocentric way. similar situations may be found in tourism industries of other countries, for example in korea (imanishi ) . in addition, similar conditions may be observed in other industries, like banking, trading, and marketing. hence, the ethnic model can be recognized as a common form of the service industry which is characterized by a close relationship between the headquarters and foreign subsidiaries and by difficulty in standardizing the service to a wider client base in their developing stages. the international administrative behavior of a multinational company follows not only the headquarters-foreign subsidiaries relations but also the company-customer relationships. tap your subsidiaries for global reach language and tourism patterns of strategic control within multinational corporations contents of tour packages: a cross-cultural comparison harvard business school case - - harvard business school case - - international business of travel companies) chichûkai kurabu no genchi keiei (local management of club méditerranée). institute of marketing and distribution sciences monograph series korean travel company and overseas travel to japan: the case of hanatour) japan national tourist organization kinki nippon tourist company kinki nippon tourist nenshi (the history of kinki nippon tourist years). tokyo: kinki nippon tourist company cultural analysis in overseas operations the globalization of markets the tortuous evolution of the multinational corporation a legacy of discrimination? does nationality affect tourist behavior? sovereignty at bay: the multinational spread of u.s. enterprises kokusai keiei, (shimpan) (international business japan's multinational enterprises acknowledgements-the author thanks all the interviewees and cooperators for this research, and wishes to give special gratitude to hideki yoshihara, hideo ishida, junichi azuma, stephen davies, and muriel larvaron. key: cord- -d mpjkxh authors: kayama, misa; yamakawa, naomi title: acculturation and a sense of belonging of children in u.s. schools and communities: the case of japanese families date: - - journal: child youth serv rev doi: . /j.childyouth. . sha: doc_id: cord_uid: d mpjkxh the population of immigrants in the u.s. continues to grow, with more than one million immigrants arriving every year. this study examines the acculturation of new immigrant and temporary resident children and their parents from japan, as they navigate two cultures and seek a sense of belonging. acculturation to a new culture poses various psychosocial challenges, including a loss of a sense of belonging, which can result in social isolation and withdrawal. examination of the experiences of families from japan, where group belonging is highly valued, can illuminate the role of belonging to acculturation. we examined individual interviews with japanese parents in u.s. southern cities. during the interviews, they described the experiences of a total of children from preschool to th grade. parents indicated an absence of a place for children where they feel a sense of belonging (i.e., ibasho) in their local schools, which resulted in social isolation, psychological exhaustion, and a reluctance to seek support. children, however, found their ibasho at a japanese supplementary school, where they attended weekly to receive academic instruction in japanese, enjoyed playing with japanese friends, and gained energy to navigate challenges at their local schools. the supplementary school also served as parents’ ibasho where they exchanged information to navigate cross-cultural experiences. this study has implications for how we can better support acculturation of new immigrant and temporary resident families including those from other cultural groups. the u.s. is a country of immigrants, with more than one million immigrants entering the country each year (pew research center, ) . relocation to a new culture poses various psychosocial challenges to children and their families. new immigrants and temporary residents often experience disruption in their sense of belonging as they acculturate to their host cultures. acculturation is a process in which individuals participate in, and adjust to, another culture while maintaining their culture of origin (e.g., berry, berry, , . differences in cultural practices, values, and behavior patterns, however, can cause psychological distress (i.e., acculturative stress; e.g., berry, ) , which may lead to a loss of a sense of belonging (kwon, ) and social withdrawal (e.g., seto & woodford, ) . how individuals experience acculturation and navigate two cultures likely varies across immigrant groups reflecting their cultural beliefs and practices of origin (tian et al., ) , and social attitudes toward them in their host culture, such as discrimination (e.g., kiang et al., ; kanno, ) . the outbreak of covid- , for instance, has exacerbated discrimination against asian people, which can disrupt their adjustment to the u.s. children, who have not fully developed their cultural identity, are particularly vulnerable and may experience a sense of not belonging to any culture (e.g., fry, ) . yet relatively little research on the cultural and developmental shaping of acculturation has focused on children's sense of belonging, and how parents support their children in finding a place where they belong. this qualitative study examines japanese parents' narratives on their, and their children's sense of belonging as they acculturate to the u.s. the experiences of families from japan, where individuals' sense of belonging is understood as enhancing their psychological well-being (see bamba & haight, ) , can illuminate how children's and parents' sense of belonging affects their acculturation to the u.s., and how parents support their children. their narratives provide insights into how we can better support new immigrant and temporary resident families, including those from other cultural groups. cultural identity refers to individuals' understandings of self that allow them to gain social acceptance and a sense of belonging to a particular culture (schwartz, montgomery, & briones, ) . identities, such as a feeling of being unwelcome and a lack of belonging. confusion in cultural identity in childhood and adolescence may lead to psychological distress such as depressive symptoms (schwartz et al., ; wei et al., ) and engagement in substance use (grigsby et al., ) . in contrast, possessing a stable cultural identity is associated with high self-esteem and life satisfaction (usborne & taylor, ) . thus, examining the experiences of children can broaden our understanding of the developmental and cultural shaping of acculturation, especially with respect to belonging and cultural identity. children acquire shared understandings of self that are necessary to develop cultural identity and function appropriately within a particular cultural group, that is, a cultural self (see author et al., ; shweder et al., ) , through their everyday social interactions with others, including adults' socialization practices at home and school, and in the community (e.g., azuma, ; clark, ) . acculturation, which requires a balanced engagement with both the host and home cultures (berry, (berry, , , fundamentally changes the way children are socialized. as a result, they may face challenges in navigating two cultural selves. children, for instance, may develop a new cultural-self through their interaction with peers, educators, and other adults in the host culture (akiyama, ; berry et al., ; choi et al., ; rhee et al., ) . a newly acquired cultural self in the host culture helps children navigate and adjust to the new culture, and gain acceptance within their local communities and schools. yet the new cultural self poses challenges in adjusting back to their culture of origin, especially for children from temporary resident families, or "third culture" children (fry, ) , who are expected to return to their home countries. many children, indeed, experience a feeling of being "lost" and perceive that they do not belong to any culture after they return to their home countries (e.g., fry, , kanno, kwon, ) . thus, maintenance of a cultural-self of origin is important for temporary resident children. children's school adjustment in their host cultures is also influenced by their association with the culture of origin, as well as their sense of belonging to local schools (e.g., choi et al., ; endo, ; paik et al., ; shwartz et al., ; zhang et al., ) , including friendships with children from the same culture (e.g., kanno, ) . a strong orientation to their culture of origin, for instance, can help them handle stigmatization and social exclusion at local schools (e.g., dimitrova et al, ) , reduce psychological distress (akiyama, ; rogers-sirin & gupta, ; schwartz et al., ) , and motivate them to adjust to their new culture, including acquisition of language. for example, the involvement of some asian youth in their cultural community of origin is associated with higher english proficiency (zhang et al., ) . thus, the existing literature suggests that balanced cultural identities, or a sense of belonging to both cultures, promote healthy integration of immigrant and temporary resident children into their new culture (e.g., burgos et al., ; koga, ; schwartz et al., ) . in other words, both acquisition of skills necessary for participation in activities in the host culture, such as language competency, and adherence to practices of their culture of origin can enhance children's psychological and social adjustment in local peer groups (berry et al., ; koga, ) . in contrast, a failure to develop integrated cultural identities can lead to low self-esteem and withdrawal from social interactions (schwartz et al., ) . japanese immigrant and temporary resident families are important cases to examine the cultural shaping of acculturation. the number of immigrants and temporary residents from asian countries, including japan, has increased substantially in the u.s. (pew research center, ; u.s. department of homeland security, ) . currently, the asian population is the second largest immigrant group ( % of the u.s. population), and expected to become the largest group by (pew research center, ) . further, as of , % of temporary residents and their families who have entered the u.s., for instance, for work and study, were from asian countries (u.s. department of homeland security, ). likewise, the number of japanese immigrants and temporary residents in the u.s. has increased about . times over the past two decades (ministry of foreign affairs, ). the japanese population, including immigrants, comprises . % of the asian population in the u.s. (u.s. census bureau, ) . japanese people also comprise . % of the asian temporary residents in . among them, % were temporary workers and their families (u.s. department of homeland security, ). thus, supporting the acculturation of asian immigrants and temporary residents, particularly school-aged children, is essential, as they grow to become contributing members of the increasingly globalized society. acculturation of new japanese immigrants and temporary residents has received increasing attention as an exemplary case, although current studies of east asian immigrants in the u.s. primarily focus on those from china and south korea (see endo, ) . the available empirical literature, which also includes a number of dissertation studies that are not peer-reviewed, suggests that cultural support networks for new japanese immigrant and temporary resident families are critical to their adjustment to local schools and communities (e.g., akiyama, ; harkins, ; kanno, ; nozaki, ; sakamoto, ; seto & woodford, ; toyokawa, ; yeh et al., ) . yet the japanese population in the u.s. may face greater challenges in locating and accessing cultural support relative to other east asian immigrants due, in part, to its smaller size and dispersion (see akiyama, ; endo, ; paik et al., ) . the japanese population, including japanese americans and immigrants, is spread across the u.s., mostly in major cities in california, hawaii, and metropolitan areas such as seattle, new york, washington dc, and chicago (ministry of foreign affairs, ). this dispersion limits new immigrants' and temporary residents' access to japanese communities and resources, especially those living in small cities (e.g., akiyama, ) . for example, many japanese temporary workers and their families are sent from japanese companies to work in their u.s. branches (shima, ; takimoto, ) , many of which are located in midwestern and some southern states that do not have welldeveloped japanese communities (see akiyama, ) . children and spouses of temporary residents who do not have adequate english proficiency are particularly vulnerable (akiyama, ; izumi & gullón-rivera, ; takimoto, ) . the limited access to cultural resources by japanese families makes them an exemplary case to examine their sense of belonging and acculturation. the japanese case is particularly important to understanding how a sense of belonging and the access to cultural resources affect the acculturation of new immigrants and temporary residents, because japanese culture generally places a high value on group belonging (markus & kitayama, ) . disconnection from japanese cultural groups, as well as challenges in adjustment to the new culture, can make japanese immigrant and temporary resident families vulnerable to acculturative stress (e.g., akiyama, ; kanno, ; seto & woodford, ) . in order to interpret japanese people's sense of belonging and its impact on acculturation, some understandings of japanese concepts, such as ibasho and self, are necessary. ibasho [i(ru): be present; basho: place)] is a physical and psychological "place" where individuals feel acceptance, belonging, comfort, security, and freedom (bamba & haight, , see also author et al., , . according to japanese folk psychology, ibasho can enhance individuals' psychological well-being (see bamba & haight, ) , and provide relief and aid in recovery from stress (author et al., ) . ibasho also provides protection from anticipated challenges, and serves as a source of motivation to overcome them (author et al., ; bamba & haight, ) . in contrast, an absence of ibasho negatively affects individuals' functioning. people may feel emotionally stressed, engage in problematic social interactions (bamba & haight, ) , and isolate themselves from others (author et al., ) . thus, we expect that the presence or absence of ibasho may also affect japanese immigrants' and temporary residents' experiences of acculturation (see akiyama, ; authors, ) . further, some understandings of a japanese cultural self allow us to understand children's psychosocial functioning within and outside of their ibasho. japan can be characterized, broadly, as a group-oriented society (markus & kitayama, ) . thus, a japanese cultural self, in general, emphasizes the self as a social agent that is interdependent and flexibly responsive to the social context, that is, social experience of self, jibun [one's share of something beyond oneself] (lebra, ) . beginning in early childhood, learning beliefs and behaviors that support the maintenance of social relationships is considered as a primary socialization goal at home and school (azuma, ). yet japanese people also retain a strong sense of "self", that is, the individual experience of self, kokoro [heart and mind] (lebra, ) . kokoro reflects the inner world, such as desires, interests, and feelings (okamoto, ) . within their ibasho, individuals may express their kokoro freely, for instance, toward those with whom they have mutually accepting relationships, such as family members and friends (bamba & haight, ) . japanese children are socialized to balance these two selves, the kokoro and jibun (e.g., hosaka, ; lebra, ) . challenges arise when the individual (kokoro), and social (jibun) experiences of the self are in conflict (shimizu, ) . for example, explicitly disclosing their kokoro outside of their ibasho can disrupt individuals' relationships with others (jibun). yet, a total involvement in groups can result in a loss of the individual experience of self (kokoro; doi, doi, / lebra, ) . to balance these two selves, japanese people prefer to express their thoughts and desires implicitly, including through facial expression and tone of voice. they, in turn, are required to be sensitive to, and understand, others' indirect expressions, and accommodate them without disrupting their relationships (azuma, ; lebra, ) . in contrast, a mainstream u.s. cultural self generally emphasizes the self as an individual with stable traits (rogoff, ; shweder et al., ) . in u.s. socialization practices at home and school, for instance, individual children's strengths, rights, and independence generally are emphasized relatively more than in japan, including through adults' encouragement to express their thoughts and feelings explicitly (e.g., shweder et al., ) . in such a context, children and their families from japan, who are not accustomed to explicit self-expression outside of their ibasho, and who are sensitive to others' responses to their struggles, may become reluctant to express their needs of support and isolate themselves to protect their "self." these cultural differences can delay their acculturation (seto & woodford, ; harkins, ). although few empirical studies examine a sense of belonging with respect to acculturation (but see kwon, ) , the literature on cultural identity of immigrants, including those from asian countries, provides some insights into japanese immigrants' and temporary residents' sense of belonging (e.g., akiyama, ; fry, ; koga, ; takimoto, ). how exposure to a new culture and separation from individuals' culture of origin affect immigrants' and temporary residents' perceived sense of belonging and acculturation may vary depending on whether or not their relocation is voluntary (e.g., garcía coll & marks, ; schwartz et al., ) , the extent to which they maintain social relationships in their country of origin, and the availability of, and access to cultural resources and communities within their host culture (garcía coll & marks, ). for recent japanese immigrants and temporary residents, who typically maintain relationships with relatives and friends in japan, physical distance and a lack of japanese cultural resources can exacerbate psychosocial distress, and delay their acculturation to the host culture (seto & woodford, ; see also schwartz et al., schwartz et al., , . children's perceived feeling of belonging also affects their acculturation to local schools, motivation to engage with school activities, and their relationships with peers and educators (ham et al., ) . school-aged immigrant children from various cultural groups reported lower levels of acceptance at school than the general population, in part, because of school policies and environments that are not inclusive of students from other countries (ham et al., ) . immigrant and temporary resident children, including those from japan, also reported discrimination and social isolation (akiyama, ; kanno, ; kiang et al., ; see also tobin et al., ) , and language barriers (nozaki, ; seto & woodford, ; yashima & tanaka; yeh et al., ) . some japanese children's sense of not belonging also led to social isolation and withdrawal (seto & woodford, ; see also ham et al., ) . for children from japan, particularly, relationships with teachers and classmates at local schools affect their school functioning (miyamoto & kuhlman, ) . as children gain language competency, acquire interpersonal skills expected in the host culture, develop friendships with local children, and adjust to their local schools (akiyama, ; takimoto, ) , they report reduced levels of psychological distress (rogers-sirin & gupta, ). differences in cultural values and practices between individuals' home and host cultures, in general, are considered barriers to acculturation (e.g., schwartz et al., ; tian et al., ) . consistent with confucianism and buddhist beliefs, asian people, generally, are sensitive to others' responses to their actions, including any individual differences (e.g., author et al., ) . they may become reluctant to seek formal support that could ease acculturative stress, which increases their vulnerability to acculturation (harkins, ; yen et al, ) . further, in asian cultures, children's selfexpression of their feelings and preferences, especially towards adults, is considered disrespectful (arora & algios, ; rhee et al., ) . asian, including japanese, immigrant and temporary resident youth, for instance, expressed difficulties in disclosing their school struggles to their parents and educators (harkins, ; rhee et al., ; yen et al, ) , and isolated themselves from peers in times of stress (e.g., seto & woodford, ) . asian children who have family support, in contrast, tend to function better at their local schools in the u.s. (rhee et al., ; zhang et al., ) . parents' adjustment to a new culture also affects children's acculturation. japanese temporary resident parents, for example, reported that stress they have experienced due to relocation to the u.s. has led to their children's increased behavior problems and adjustment challenges in the u.s. (izumi & gullón-rivera, ) . japanese parents also reported a perceived feeling of social isolation, which left their children struggling alone at their local schools (seto & woodford, ) . they, however, discussed that their engagement with a group of japanese parents within their local communities provided mutual social and emotional support, necessary for their adjustment to the u.s. (e.g., sakamoto, ; toyokawa, ) . further, stereotypical labels of the asian population as a "model minority" (e.g., high achievers, studious, respectful, quiet, etc.; kiang et al., ; nozaki, ) also interfere with children's acculturation. such labels may motivate children to conceal their struggles to fulfill expectations of the host culture, and prevent them from receiving necessary support (kiang et al., ) . educators also may misunderstand their school struggles. some elementary school teachers, for instance, considered japanese temporary resident children's behaviors that did not fit the stereotype of model minority, such as inattention, as problematic and referred them to specialized services. children, however, reported that their challenges were due to language barriers (nozaki, ) . school-aged japanese, and other asian, children also reported racial discrimination, including unfair treatment and social exclusion at school (kanno, ; kiang et al., ) , which can lead to low self-esteem and depressive symptoms (kiang et al., ). japanese supplementary schools provide an important context to examine children's and their parents' sense of belonging within the u.s. similar to many language and cultural heritage schools across the u.s., including for children from asian (e.g., china, taiwan, and south korea), and western (e.g., france, and germany) countries (e.g., lee & shin, ; liao, technology, ) . the day at school is structured, as much as possible, in accordance with a typical school day in japan, such as academic instruction, lunchtime, and cleaning their classrooms . children are also exposed to japanese culture through their participation in seasonal events typically implemented in japanese public schools, such as a sports festival. through participation in these academic and non-academic activities, children are socialized to learn japanese culture (e.g., endo, ) , including a value of group belonging. overall, the empirical literature on japanese immigrant and temporary resident families suggests that japanese communities serve as important resources that help them overcome acculturation challenges, but with relatively less attention to how their sense of belonging to japanese communities helps them navitage two cultures. this study, thus, aims at examining japanese families' experiences of acculturation, and how their ibasho, where they feel a sense of belonging, in local and japanese communities, including japanese supplementary schools, supports their acculturation. their narratives can provide insights into our understanding of the roles of cultural resources and belonging in supporting acculturation of children and their families. this study is part of a larger study examining acculturation of japanese immigrant and temporary resident families in the u.s., particularly on their cultural identity and self (authors, ), and how parents support children at home. this report focuses on parents' narratives on their, and their children's sense of belonging, or ibasho, in japanese and local communities. through qualitative approaches, we consider: ) what are japanese parents' observations of their children's acculturation to local schools and their sense of belonging? ) how do parents understand their children's ibasho in their local schools and japanese supplementary school as they acculturate to the u.s.? ) what are japanese parents' perceptions of their own ibasho? as detailed in (authors, ), our research site is a relatively small-sized japanese supplementary school in a southern city, serving children from st through th grades. these children attend local schools and receive instruction in english during the weekdays. the state in which our research site is located is among those that have the lowest percentage of foreign-born population (u.s. census , and english language learners in public schools (pew research center, ) . we expect that the relative lack of japanese cultural resources allows parents to elaborate challenges they and their children have experienced in local schools and communities, and their experiences at the supplementary school as their ibasho. most of the families commute to the supplementary school from cities approximately - hour's drive away on saturdays. despite the long distance, this supplementary school serves as one of a few community resources that allow children and their parents to access japanese language and culture. typically, parents take their children to the supplementary school by am in the morning, and spend a day, until pm, at the school or in the community while their children receive instruction. every week, about parents and their younger children stay in a waiting room in the school. the waiting room serves as a place for parents to exchange information with other parents and share their experiences, and for younger children to learn japanese with other children and adults outside of their families. table ). nine families moved to the u.s. one to three and half years prior to the interview due to the fathers' work obligations. they were sent from japanese companies to work in their u.s. branches, and expected to return to japan in a few years. parents from two other families have been in the u.s. for more than years, and do not have a plan to return to japan. they came to the u.s. as students, and obtained permanent resident status after their graduation. their children were born and raised in the u.s. parents described the experiences of a total of children from preschool to th grade. parents participated in semi-structured, audio-recorded individual interviews, which lasted approximately - minutes. all interviews were conducted in japanese by [author ] in a private, empty classroom. in two families, both parents participated in interviews together. two other parents from the same family participated in the interviews individually. during the interveiws, parents were prompted to discuss their, and their children's experiences at local schools, communities, and the japanese supplementary school; and their sense of belonging, including where they and their children found their ibasho in the u.s. all interviews were transcribed verbatim, and analyzed in japanese by the authors whose native language is japanese. through repeated readings of transcribed interviews, [author ] induced emic codes capturing participants' experiences, particularly of ibasho, using analytic induction techniques (lincoln & guba, ; schwandt, ) . the initial codes were critiqued by [author ], and the coding system was revised. we, then, independently coded all interviews. any disagreements between coders were resolved through discussion. we continued to revise our coding system so that our definition of each code was consistent with participants' descriptions, including through negative case analysis (lincoln & guba, ) . perceptions of [author ] were used to reflect on her day-to-day experiences with participant parents and their children for the purpose of triangulation. both her insider perspectives enhanced the credibility of our analyses. yet her administrative role at our research site may affect parents' voluntary participation and narratives. her primary roles, thus, focused on analyzing data and writing. parents described the absence and presence of ibasho, where they and their children felt a sense of belonging, and how their ibasho affected their experience of acculturation. parents, generally, described the absence of ibasho for their children at school, in both the u.s. and japan, where they were treated as "visitors". most children considered the japanese supplementary school as ibasho. yet some children who had local friends also considered their local schools as ibasho. parents described their ibasho primarily at the supplementary school. the majority of parents from temporary resident families described the absence of ibasho in local communities, and noted that their children were not fully involved in activities at local schools. parents discussed that how others treat their children at local schools, as well as their limited english proficiency , have affected their children's ibasho and acculturation to their local schools. some parents, for instance, perceived that their children were treated as visitors, and that educators had lower expectations for their children because of their status as "foreigners". emi's ( nd grade) mother, living in the u.s. for two and half years, described: my child is talkative at home, but she doesn't talk to others outside much. [her teacher] says, "she is always smiling. she is doing fine, although she doesn't speak much." i thought teachers didn't take [my child's challenges in communicating in english seriously]. how they treat her looks to me like, "she has done good enough as a foreigner, but not [as good as other children]." the teacher's comment on emi's school functioning may reflect a common practice in the u.s., at home and school that focuses on children's strengths (miller & cho, ; shweder et al., ) , which the majority of participant parents report is not typical in japan. emi's mother, rather, interpreted the teacher's comment as reflecting her lower expectation for emi. several other parents also expressed anxiety regarding their children's school functioning in response to their teachers' positive comments. all parents, regardless of their plans to return, or not to return to japan prioritize their children's english proficiency. english proficiency gained in the u.s. can become their children's strength after they return to japan. nozomi's ( th grade) and yuuki's ( rd grade) mother elaborated her anxiety: parents' anxiety also may reflect japanese socialization practices at home and school that consider challenges children face as opportunities for them to learn from solving problems (author et al., ; lewis, ) . from the japanese perspective, u.s. educators' responses may be interpreted as a sign of lost educational opportunities for their children. nozomi's and yuuki's mother also described her children's strategies to "fit" in the classroom by remaining quiet, even if they do not understand: [many japanese] children can't say easily to teachers that they don't understand, unless teachers ask them, "do you understand this? did you get it?" they may just let it go. my children can't speak to [teachers] in english well. thus, they try to be quiet, so they don't have to [bother their teachers]. i tell them "ask your teachers," but they say, "i'm good." this strategy, however, may lead to educators' confusion, over whether their reluctance is due to language barriers or a lack of understanding. their hesitation to speak up can also be interpreted through the lens of stereotypical labels of asians as a model minority, such as smart, quiet, and studious (kiang et al., ; nozaki, ) . educators' confusion and misunderstandings can delay the identification of children's struggles and further isolate the children at school. parents expressed concern that their children would also be treated as "outsiders" in japan, and lose their ibasho. after their relocation to the u.s., for instance, their children have already learned some behavioral patterns expected in the u.s., even if they may still struggle at their local schools. their children's behaviors, thus, may be considered "different" in japan. one father, for instance, described his third grade son, ryota, after years in the u.s.: he's never been to schools in japan, and started building personal relationships with others in the u.s., at school. so, how he expresses himself is almost like american. his self-expression has become very strong. other parents also described their children as losing japanese communication and interpersonal skills, such as sensitivity and thoughtfulness, that are important to maintain a japanese cultural self. as ryota's father described, ironically, skills children have acquired to fit in at u.s. local schools can stand out in japan. japan is a relatively homogenous country, and the concept of diversity is still new. any differences from others can lead to teasing, bullying, and social exclusion (sueda & wiseman, ; see also fry, ) . in order for their children to maintain japanese language and interpersonal skills, some parents sent them to schools in japan during the summer . the experiences at japanese schools, however, reinforced their "visitor" status. hiroto's ( th grade) and kenji's ( nd grade) parents, who planned to permanently return to japan in a year, temporarily sent them to the school hiroto attended as a st grader. their mother expressed concern about hiroto's parents reported that educators in japan also treated their children as visitors, knowing that they would leave in a few weeks. taichi's ( rd grade) and emi's ( nd grade) mother described their experiences at a while children's perception of being treated as visitors may limit their full participation at school, parents discussed that their visitor status, in both the u.s. and japan, can lessen the pressure on children. some parents even made use of this status to support their children. rina's ( th grade) and runa's ( nd grade) mother, after living in the u.s. for years, described: in the u.s., we feel "easy", at times, because we can be "foreigners. hazuki's ( th grade) and aki's ( th grade) mother, after year in the u.s., described: my younger child still is in middle school. similarly, some parents reported that their children enjoyed their visitor status in japan. according to parents, michi ( nd grade) and sachi (kindergarten), who were born in the u.s., enjoyed visiting japan because they were treated as visitors. although this family does not plan to move back to japan, they consider that maintaining their relationships with relatives in japan is important, and occasionally visit them. parents from temporary resident families also described similar challenges. takahiro's father observed his son at a japanese elementary school, "going to school and having to finish homework everyday, he seemed to find that it was hard, [not just fun]." the majority of parents from temporary resident families reported the japanese supplementary school as their children's ibasho, a place where they feel comfortable, safe, and accepted. until children acquire english fluency and begin to actively participate in activities at local schools, the supplementary school is the only place, outside of their homes, they can freely express their thoughts and feelings. parents discussed that talking and playing with other japanese children who share similar experiences help them recover from psychological exhaustion and relieve stress they have accumulated in their local schools. nozomi's and yuuki's mother described: the presence of, and their time in ibasho appear to encourage, and motivate children to exert efforts in navigating challenges (see author et al., ) , including at their local schools where they do not consider themselves as full members (see also authors, ). even after their children have become fluent in english and adjusted to their local schools, typically in several years, most of the parents observed that the supplementary school remains as their children's ibasho. they feel comfortable and accepted when they are with japanese, rather than local, children due, in part, to cultural differences in what children enjoy talking about, and playing, with their peers. hiroto's ( th grade) mother described his failure to understand jokes at his local school, which made him feel that he was an outsider: for him, having japanese friends means a lot. in contrast, at the japanese supplementary school, parents observed that their children enjoy talking about japanese cartoons and tv characters familiar to japanese children. parents also discussed that ibasho at the japanese supplementary school provides a safe place where children do not have to be conscious about adjusting to u.s. expectations. children, especially those who were upper elementary school-age or older when they came to the u.s., have already acquired a japanese cultural-self, such as being attentive to what other people may say (jibun) and carefully expressing their thoughts (kokoro; see lebra, ) . these children appear to be pressured in u.s. local schools, where explicit self-expression is expected (see markus & kitayama, ; shweder et al., ) . haruka's ( th grade) father, for example, described, "if they communicate like when they were in short, children's shared interests in play and communication styles, including jokes, made them feel comfortable (see kwon, ) , and helped them find their ibasho at the japanese supplementary school. haruka's ( th grade) and takahiro's ( rd grade) mother noted, "the same value they are sharing is making it easier to understand each other." despite japanese people's preference of implicit expression of self (e.g., lebra, ) , parents' narratives illuminate that similar to children in yet parents discussed that some children, especially those who came to the u.s. before the school age may find their ibasho at their local schools, as well as at the supplementary school. these children are not exposed to japanese culture, including toys and tv characters as much as older children. younger children also have relatively more common experiences with their local classmates than older children, and tend to have friends at both their local schools and the supplementary school. a mother described her son, takahiro, who came to the u.s. when he was in kindergarten, "he's completely [adjusted to] his local school. it doesn't matter for him if he plays with his local or japanese friends. he's enjoying playing with any friends." these parents' narratives suggest that the presence of friends with whom they can play and enjoy together regardless of their languages is also important for their ibasho in local communities and schools. as akiyama ( ) described, friendship, particularly, with local children can facilitate their acculturation to u.s. local schools. likewise, parents reported that children who were born in the u.s., relative to those from japan, recognized their local schools as their ibasho. they were exposed to the english environment and u.s. culture earlier than children from temporary resident families. although their parents spoke to them in japanese at home, once they started preschool, children quickly learned english, and adjusted to u.s. culture through their interactions with preschool teachers and peers. parents of these children reported that local schools are where their children fully enjoy playing with other children and freely express their thoughts. for them, studying in japanese at the supplementary school has become a burden. sara's ( st grade) mother described: when i asked her which school she likes, she says, her local school. at the supplementary school, she has friends, but for her, it's more like a place to study, which has become stressful for her… she speaks english [better than japanese]. yet sachi's and michi's parents noted that the presence of japanese friends motivated their children who were born in the u.s. to go to, and find their ibasho at the supplementary school. similar to children, many parents perceived themselves as "visitors" in both the u.s. and japan, and described the absence of ibasho. in the u.s., primarily because of the language barriers, they are reluctant to participate in community activities. further, a few parents considered social isolation in their local communities as an opportunity to excuse themselves from responsibilities and obligations within the communities. one mother described: in the u.s., we are foreigners and not involved in any local communities. i feel free. in the absence of ibasho (see bamba & haight, ) , parents, as well as children, may become less motivated to fulfill their social responsibilities and participate in activities as full members. the use of their visitor, or "nonmember", status as an excuse, however, further excludes them from local communities and schools, particularly, in the u.s. where they are expected to express their interests and needs more explicitly (see shweder et al., ) than in japan. many parents also described an absence of ibasho in japan. some parents noted that they were no longer full members of the community to which they belonged in japan. they are reluctant to share their experiences in the u.s. with their friends in japan, who may not fully understand them. some parents also were concerned that telling them about the u.s., even challenges, may be considered "showing off." in japan, relocation to western countries is considered a privilege (see fry, ; koga, ). any sign of differences, such as their children's westernized attitudes and english fluency, may cause others' rejection and envious attitudes toward them (fry, ; kanno, ; sueda & wiseman, ) . rather, participant parents feel closer to parents they see at the japanese supplementary school who have similar experiences and can understand each other. parents described that weekly meetings with other japanese parents not only allow them to relieve stress and enjoy talking freely in japanese, but also help them adjust to u.s. systems and culture. they exchanged information about their children's local schools; and provided, and asked for, suggestions to solve any problems. just like children, parents have to manage challenges in adjusting to the u.s. (izumi & gullón-rivera, ; seto & woodford, ) . participant parents observed that their common experiences of navigating challenges in the u.s. have strengthened their relationships (see sakamoto, ; toyokawa, ) , and created ibasho for them. one mother, for instance, described, "every week, as soon as we get to the thus, the supplementary school is an important place, their ibasho, where they feel comfort and acceptance, free from critique regarding their differences in the local communities. this report is one of the first empirical studies examining a sense of belonging among japanese immigrant and temporary resident families (but see akiyama, ; koga, ; takimoto, ) , in particular, their ibasho (see authors, ). japanese parents, who value group belonging, articulated how their children's japanese supplementary school has become their, and their children's ibasho, a place that provides them with a sense of belonging and eases acculturative stress. parents, however, indicated an absence of ibasho at local schools and communities, in both the u.s. and japan, where they perceive that they are treated as "visitors", which is devastating to their psychosocial well-being. in the absence of ibasho, japanese families not only continue to struggle, but also may withdraw from fulfilling responsibility as "full members", which also is a loss of opportunity for local schools and communities to benefit from their participation. japanese parents' narratives draw our attention to belonging and ibasho as a source of support for acculturation of new immigrant and temporary resident families, possible relevance to those from other cultural groups. although there is no english term that refers to ibasho, japanese parents' narratives on ibasho and belonging have some theory, policy, and practice implications. the existing literature on immigrant and temporary resident families from various cultural groups suggests the importance of access to cultural resources of origin in their adjustment to the host culture (e.g., dimitrova et al., ; kanno, ; koga, ; zhang et al., ) , including at supplementary schools (see endo, ), but has disregarded how a sense of belonging affects their social and psychological functioning, and cultural adjustment. the absence of ibasho at local schools increases vulnerability of children, particularly those from temporary resident families. the presence of ibasho at the supplementary school, in contrast, can strengthen children's sense of belonging, and help them gain energy to overcome challenges. according to japanese folk theories, ibasho cannot be created by others (bamba & haight, what is missing in japanese parents' narratives is collaboration between cultural resources, including the supplementary school, and local schools that also may become ibasho for their children. indeed, the literature on korean culture schools indicates a lack of collaboration with local public schools (lee & shin, ultimately, the presence of ibasho, either within cultural or local communities, supports children's acculturation, and facilitates their development into adults who can contribute to the increasingly diverse society. existing research on children from various cultures suggests that the experiences of overcoming challenges arising from cross-cultural adjustment have some positive impacts on children's development (haneda & monobe, ; kwon, ; see also fry, ) , such as, acquisition of global perspectives, including languages, cultural knowledge, and skills to adjust to new cultures. these perspectives and skills have become even more important in this rapidly globalized society, and can expand children's future career path (kwon, ) . limitations of this study include our focus on parents' perceptions to examine their children's experiences. in order to further discuss children's sense of belonging and ibasho, it is necessary to examine children's voices. further, we relied on parents' narratives to understand their children's experiences at their local schools and the supplementary school. observations at school, and perceptions of educators can deepen our understanding of children's sense of belonging at school. the second limitation arose from the relatively small size of the japanese population, particularly immigrant families, in our research site. due to the small size, immigrant parents' perceptions do not represent japanese immigrant families in general. rather, we used their perceptions to illustrate the continuum of experiences, from families who recently relocated to the u.s. to those who have already acculturated. finally, we purposively selected this supplementary school located in a community that has limited cultural resources for japanese families. the experiences of families who participated in this study may be considerably different from those living in metropolitan cities that have a larger japanese population. language, culture, and identity negotiation: perspectives of adolescent japanese sojourner students in the midwest, usa (publication no. proquest dissertations and theses global school-based mental health for asian american immigrant youth: perceptions and recommendations nihon jin no shitsuke to kyo-iku: hattatsu no nichi bei hikaku ni motozuite child welfare and development: a japanese case study a psychology of immigration conceptual approaches to acculturation immigrant youth: acculturation, identity, and adaptation protective factors of family life for immigrant youth advancing understanding of acculturation for adolescents of asian immigrants: person-oriented analysis of acculturation strategy among korean american youth all together now: american holiday symbolism among children and adults the anatomy of dependence heritage identity and maintenance enhance well-being of turkish-bulgarian and turkish-german adolescents realities, rewards, and risks of heritage-language education: perspectives from japanese immigrant parents in a midwestern community the educational aspirations and expectations of japanese immigrant mothers: narratives of raising bicultural nikkei children in the post- diaspora. diaspora, indigenous, and minority education perspective shifts and a theoretical model relating to kaigaishijo and kikokushijo, or third culture kids in a japanese context immigrant stories: ethnicity and academics in middle childhood cultural stressors, identity development, and substance use attitudes among hispanic immigrant adolescents immigrant integration policy for future generations? a crossnational multilevel analysis of immigrant-background adolescents' sense of belonging at school bilingual and biliteracy practices: japanese adolescents living in the united states understanding the acculturation process for kaigaishijo who are our heritage language learners? identity and biliteracy in heritage language education in the united states school absenteeism, bullying, and loss of peer relationships in japanese children family resilience among sojourning japanese mothers: links to marital satisfaction and children's behavioral adjustment bilingualism and identity: the stories of japanese returnees model minority stereotyping, perceived discrimination, and adjustment among adolescents from asian american backgrounds growing student identities and school competences in sojourning: japanese children's lived experiences across japan and the u.s. (dissertation no. ) [doctoral dissertation proquest dissertations and theses global third culture kids: growing up with mobility and cross-cultural transitions. diaspora, indigenous, and minority education japanese patterns of behavior korean heritage language education in the united states: the current state, opportunities, and possibilities educating hearts and minds: reflections on japanese preschool and elementary education bridging two worlds: experiences of chinese and taiwanese americans attending chinese heritage schools in houston naturalistic inquiry culture and the self: implications for cognition, emotion, and motivation self-esteem in time and place: how american families imagine, enact, and personalize a cultural ideal ethnographic methods: application from developmental cultural psychology ministry of education kaigai shijyo kyouiku no gaiyou guro-baru jinzai ikusei ni kansuru kaigai-shijyo, kikoku-shijyo tou kyouiku ni kansuru jittai chousa [research on the current status of education for children living abroad and those who have returned from abroad, concerning the global human resource development sho-gaikoku no kaigai-shijyo, kikoku-shijyo ni kansuru chousa kenkyu [research on education for children living abroad and those who have returned from abroad in other countries -united states ameliorating culture shock in japanese expatriate children in the u essentializing dilemma and multiculturalist pedagogy: an ethnographic study of japanese children in a u education of the rising sun : an introduction to education in japan ( nd ed.). national federation of social education ethnic afterschool programs and language schools in diverse asian american communities: varying resources, opportunities, and educational experiences (part : how they differ) modern immigration wave brings million to u.s. driving population growth and change through : views of immigration's impact on u.s. society mixed facts about english language learners in u.s. public schools key findings about u.s. immigrants acculturation, communication patterns, and self-esteem among asian and caucasian american adolescents the cultural nature of human development cultural identity and mental health: differing trajectories among asian and latino youth when family enters the picture: the model of cultural negotiation and gendered experiences of japanese academic sojourners in the united states the sage dictionary of qualitative inquiry the role of identity in acculturation among immigrant people: theoretical propositions, empirical questions, and applied recommendations developmental trajectories of acculturation: links with family functioning and mental health in recent-immigrant hispanic adolescents rethinking the concept of acculturation: implications for theory and research helping a japanese immigrant family cope with acculturation issues: a case study japanese sojourners learning english: language ideologies and identity among middle school students (publication no. aai ) [doctoral dissertation proquest dissertations and theses global japanese cultural psychology and empathic understanding: implications for academic and cultural psychology the cultural psychology of development: one mind, many mentalities sheltered ethnic identity: the effects of education on japanese adolescent sojourners in the united states (dissertation no. ) [doctoral dissertation proquest dissertations and theses global the function of the social network formed by japanese sojourners' wives in the united states an examination of ethnic identity, self compassion, and acculturative stress in asian international students children crossing borders: immigrant parent and teacher perspectives on preschool the role of cultural identity clarity for self-concept clarity, selfesteem, and subjective well-being the asian population america's foreign burn in the last years nonimmigrants residing in the united states: fiscal year taiwanese immigrant mothers' childcare preferences: socialization for bicultural competency minority stress, perceived bicultural competence, and depressive symptoms among ethnic minority college students roles of social support and social skills in the international adjustment of japanese adolescent sojourners in the usa the cultural adjustment and mental health of japanese immigrant youth the role of family and community bicultural socialization in the bilingual proficiency of immigrant young adults acculturation poses various psychosocial challenges to children and their parents. . the presence of ibasho supports acculturation of japanese children and parents japanese children found ibasho in their relationships with friends from japan. . the absence of ibasho increases vulnerability of immigrant children author statement misa kayama: conceptualization, methodology, validation, formal analysis, investigation, writing -original draft preparation, writing -reviewing and editing naomi yamakawa: validation, formal analysis: writing -reviewing and editing title of the manuscript: acculturation and a sense of belonging of children in u.s. schools and communities: the japanese case conflicts of interest: none key: cord- - qyee authors: mase, m.; tsukamoto, k.; imai, k.; yamaguchi, s. title: phylogenetic analysis of avian infectious bronchitis virus strains isolated in japan date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: qyee to define the origin and evolution of recent avian infectious bronchitis virus (ibv) in japan, a genetic analysis was performed. by phylogenetic analysis based on the s gene including the sequence of the hypervariable regions, ibv isolates in japan were classified into five genetic groups, which included two already-known groups (mass and gray). among them, three major genetic groups were associated with the recent outbreaks of ib in japan. one group is indigenous to japan and could not be placed within the known existing groups in other countries. the remaining two groups, which have emerged recently, are related to isolates in china and taiwan. m. mase et al. post-translational cleavage of two separate polypeptide components, designated s and s [ ] . of these, the s glycoprotein is associated with virus attachment and is a major target of the neutralizing antibodies in chickens, so serotypic evolution in ibv is associated primarily with the sequences of the s glycoprotein [ , ] . hence, the molecular characterization of ibv is based mainly on analysis of the s gene [ , ] . classically, ibv strains have been placed into serologically related groups on the basis of virus neutralization tests (vnt). however, vnts are very laborious and time-consuming. in addition, there is considerable evidence that serologic relationships among ibv isolates, determined by in vitro vnts, have not been reflected by the results of in vivo cross-immunity studies [ ] . moreover, the antigenic variants emerging from wild-type or vaccine viruses by point mutation or rna recombination cannot be detected by vnts [ ] . recently, genetic grouping of ibv on the basis of nucleotide sequences have been applied for virus classification [ , ] . since anyone can apply the genetic information of viruses deposited into the genbank, genetic comparison with other virus strains for epidemiological analysis can be performed quickly. since the first isolation of ibv was recorded in japan in [ ] , the outbreaks have been ongoing. however, the epidemiological analysis of ibv isolates in japan has not been thorough except for a few strains [ , ] . the relationships between japanese ibv isolates and foreign ibv isolates have also remained unknown. we were particularly interested to know whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, to define the origin and evolution of recent ibv in japan, we determined the nucleotide sequences of ibv isolated in japan using the reverse transcriptase-polymerase chain reaction (rt-pcr) method coupled with direct sequencing, and analyzed the sequences phylogenetically with viruses isolated in other countries. this information is important for determining strategies to control ib. the ibv isolates employed in this study are listed in table . most of the ibv isolates were obtained from regional laboratories in japan. most of the specimens of ibv were isolated by two or three passages using embryonated specific pathogen-free (spf) eggs. the materials were submitted to our laboratory, and were propagated once in spf eggs. the presence of ibv in the inoculated embryos was initially determined by immunofluorescence assay of allantoic cells using anti-ibv chicken serum and observation of characteristic embryo changes such as dwarfing, stunting or curling, according to the procedures of a previous report [ ] . the following commercial attenuated live vaccine strains in japan were also used in this study: c- , miyazaki, on/ , kita- , ku and tm . they were derived from field cases of ib in japan. viral rna was extracted from infected allantoic fluids using a kit (isogen-ls, nippon gene, tokyo, japan). a reverse transcriptase (rt) reaction was carried out with superscript ii (life technologies, gaithersburg, md, u.s.a.) using random mers. the n-terminus of the s glycoprotein, which includes [ ] : country/region of origin/year of isolation except jp/kh/ strain. jp, japan important structures such as hypervariable regions (hvr) associated with antigenic properties [ , , ] , was selected for this analysis. the following primers were used: ibv-s (forward), agg-aat-ggt-aag-ttr-ctr-gtw-aga-g , and ibv-s (reverse), gcg-cag-tac-crt-tra-yaa-aat-aag-c . (the expected product was about base pairs). these were designed based on a comparison and alignment of the genbank sequences of several known ibv strains. pcr products were purified using the qiaquick pcr purification kit (qiagen, hilden, germany) according to the manufacturer's instructions. purified pcr products were used as a template for sequencing on an applied biosystems s automated dna sequencer using dye terminator cycle sequencing chemistry (perkin-elmer/applied biosystems, foster city, ca, u.s.a.). purified pcr products were sequenced from both directions. the derived nucleotide sequences were analyzed using genetyx-mac ver. . (software development corp., tokyo, japan), and through genbank searches. the phylogenetic analysis with available sequences from genbank was conducted using the clustal [ ] , and the tree was constructed by the neighbor-joining (nj) method [ ] . the expected sizes of dna fragments were successfully amplified by rt-pcr from all the employed ibv samples with our designed primers. the determination of nucleotide sequences of obtained pcr products revealed the diversity of those lengths ( - bp) ( table ) . by phylogenetic analysis, the ibv isolates in japan used in this study were divided into five genetic groups (fig. ). in particular, recent isolates in japan were assigned to three major genetic groups. among them, one group (jp-i) is indigenous to japan and is not present in other countries. the two other groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. some isolates that were mainly isolated in the s, such as jp/ishida/ or jp/nerima/ , and two local attenuated vaccine strains, kita- and ku, were classified into the mass group. another local attenuated vaccine strain, c- , was classified into the jp-i group, and miyazaki and tm strains were classified into the jp-ii group. only one strain (on/ ), which was also a local attenuated vaccine strain, was classified into the gray group. the deduced amino acids from obtained nucleotide sequences of pcr products were aligned with various ibv strains isolated in different geographic regions. the results are shown in fig. . many insertions or deletions of amino acids among ibv strains including the japanese isolates were found in the amplified region. the generated pcr products were employed in restriction endonuclease analysis for the development of a simple and rapid classification of genetic groups. after comparing the obtained sequences in this study, we selected two endonucleases, hae ii and ecor i (takara, tokyo, japan), for this analysis. the restriction profiles fig. . dendrogram of the s glycoprotein of infectious bronchitis viruses. amino acids - (in genbank accession number p ) of the beaudette strain s glycoprotein were used for phylogenetic analysis at the amino acid level. the dendrogram is rooted to murine hepatitis virus ml- strain. horizontal distances are proportional to the minimum number of amino acid differences required to join nodes and sequences of the pcr products of the five genetic groups in this study, as revealed by two restriction endonucleases are shown in table and fig. . each japanese genetic group has a specific profile pattern. the other genetic groups represented, such as connecticut, d , uk/ / , and ark , do not have sites for these two enzymes in their reported sequences. hence, the three major genetic groups in japan were easily differentiated from other genetic groups. recently, novel ibv strains have been proliferating abroad, and genetic analysis of these strains have been mainly based on the s gene [ ] . ibv strains in japan have been considered variants different from foreign ibvs such as massachusetts or connecticut types by vnt or pcr-restriction enzyme fragment length polymorphism based on the s gene [ , , ] . still, it remains unknown whether the current ibv isolates in japan were newly introduced from other countries or whether they arose by mutations of circulating japanese ibv strains. in this study, it was revealed that five genetic groups, which could be differentiated by simple restriction endonuclease analysis, were present in japan. although sequencing is recommended to obtain precise genetic information, restriction endonuclease analysis is simple, easy and convenient for primary characterization in routine diagnosis. among these genetic groups, three major genetic groups were associated with recent outbreaks of ib in japan. a previously published study divided ibv strains into several distinct genetic groupings by analyzing the s gene [ ] . however, most ibv isolates in japan formed genetic groups distinct from these. among three major genetic groups of ibv in japan, one group (jp-i) has not been found in other countries; this group may be indigenous and has been prevalent in japan for a long time, from at least the s. the other two groups that have emerged recently (jp-ii and jp-iii) are related to isolates in china and taiwan. recent ibv isolates in china and taiwan also form distinct genetic groups [ , ] . it is unknown whether these genetic groups originated in japan or in these neighboring countries. interestingly, recently isolated newcastle disease viruses in japan were also closely related, genetically, to those isolated in taiwan and china [ ] . furthermore, it was recently revealed that the coronavirus isolated from pheasants was genetically closely to ibv [ ] . one possibility for the migration of ibv strains into this region is dissemination by some kind of wild birds close to chickens, such as pheasants. the virus neutralization antibodies that form the basis for comparison of ibv isolates are induced largely by the n-terminus in the s glycoprotein [ , ] . in this study, prevalent ibv genetic groups in japan were shown to be completely distinct from other known serotypes from europe and north america by comparing amino acid sequences of the n-terminus in the s glycoprotein. on the basis of the relationships between genotypes and serotypes of ibv [ ] , it was suggested that the serotypes of recent ibv in japan are novel. further analysis would be required to examine the antigenic property of isolates for establishment of a control strategy for ib outbreaks in japan. on the other hand, the variation of ibv may also be attributed to recombination following co-infection of a few distinct strains [ , ] . previously, the kb strain isolated in japan [ ] was revealed to be genetic recombinant [ ] . in japan, in addition to major massachusetts (h strain) and connecticut (l strain) type live vaccines, local attenuated vaccines isolated from outbreaks of ib in japan have been developed and used. they proved to derive from the genetic groups, mass, gray, jp-i or jp-ii, although the genetic backgrounds of these attenuated vaccines have not been clarified. so far, it has been suggested that the recombination events occur between vaccine strains and field strains [ ] . it is unknown whether or not vaccine strains in japan are associated with the emergence of recombinant viruses. to detect recombinant viruses, it would be required to analyze two or more different genetic regions of isolates. further analysis of viruses would clarify whether recombinant variants have become prevalent in japan. all sequences used in this study were sent to ddbj, and their accession numbers areab to ab . completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus ibv: structural characterization of the spike protein amino acids within hypervariable region of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes infectious bronchitis a nomenclature for avian coronavirus isolates and the question of species status coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys use of allantoic cells for the detection of avian infectious bronchitis virus humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus serotypes of avian infectious bronchitis virus isolates from field cases in japan cross-immunity in chickens using seven isolates of avian infectious bronchitis virus a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains location of antigenic sites defined by neutralizing monoclonal antibodies on the s avian infectious bronchitis virus glycopolypeptide phylogeny of antigenic variants of avian coronavirus ibv sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus differentiation of infectious bronchitis virus serotypes using polymerase chain reaction and restriction fragment length polymorphism analysis typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the s gene a new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism typing of recent infectious bronchitis virus isolates causing nephritis in chicken phylogenetic analysis of newcastle disease virus genotypes isolated in japan a virus isolated from infectious bronchitis-like diseases of chickens the neighbor-joining method: a new method for reconstructing phylogenetic trees ibv genotypes in japan cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools evidence of natural recombination within the s gene of infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus relationship between serotypes and genotypes based on the hypervariable region of the s gene of infectious bronchitis virus molecular epidemiology of infectious bronchitis virus isolates from china and southeast asia author's address: dr. masaji mase, department of infectious diseases, national institute of animal health, - - kannondai redaktion: sachsenplatz - , wien, austria. -satz und umbruch we thank drs. t. imada and n. yuasa, national institute of animal health, japan, for valuable discussions and suggestions. we would like to thank the veterinary officials of miyazaki, akita, mie,yamanashi, nagano, shizuoka, shimane, chiba, toyama, aichi, fukui, okayama, kanagawa, osaka, and ibaraki prefectures for their cooperation in the collection of the viral samples. key: cord- -nkr x authors: yokomichi, hiroshi; tanaka-taya, keiko; koshida, rie; nakano, takashi; yasui, yoshinori; mori, masaaki; ando, yuka; morino, saeko; okuno, hideo; satoh, hiroshi; arai, satoru; mochizuki, mie; yamagata, zentaro title: immune thrombocytopenic purpura risk by live, inactivated and simultaneous vaccinations among japanese adults, children and infants: a matched case–control study date: - - journal: int j hematol doi: . /s - - - sha: doc_id: cord_uid: nkr x this case–control study investigated immune thrombocytopenic purpura (itp) risk following live, inactivated, and simultaneous vaccination, with a focus on infants aged < years. we matched case patients with itp to one or two control patients with other diseases by institution, hospital visit timing, sex, and age. we calculated mcnemar’s pairwise odds ratios (ors [ % confidence interval]) with case–control pairs. the case group had ( %) males and ( %) infants, and the control group included ( %) males and ( %) infants. for all age groups, the mcnemar’s or for itp occurrence was . ( . – . , p = . ) for all vaccines. among infants, these were . ( . – . , p = . ) for all vaccines, . ( . – . , p = . ) for live vaccines, and . ( . – . , p = . ) for inactivated vaccines. sex-adjusted common ors for simultaneous vaccination were . ( . – . , p = . ) for all vaccines, . ( . – . , p = . ) for inactivated vaccines only, and . ( . – . , p = . ) for mixed live and inactivated vaccines. in infants, these were . ( . – . , p = . ), . ( . – . , p = . ) and . ( . – . , p = . ), respectively. these limited data suggest no significant itp risk following vaccinations or simultaneous vaccination in any age group, including infants. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. immune thrombocytopenic purpura (itp) is a haemorrhagic disorder characterised by thrombocytopenia, a purpuric rash, normal bone marrow and the absence of signs of other identifiable causes of thrombocytopenia [ ] [ ] [ ] [ ] [ ] . previous studies have shown that itp risk increases after measles, rubella, chickenpox and influenza infection [ , [ ] [ ] [ ] . recently, live measles-mumps-rubella (mmr) [ ] [ ] [ ] [ ] and varicella [ ] vaccines have been reported to potentially increase the risk for itp. studies have also suggested that inactivated hepatitis b [ ] and diphtheria-tetanus-acellular pertussis (dtap) [ ] vaccines may increase itp risk. evidence suggests that itp risk after vaccination increases through the same mechanism as that by which microbial infections induce antiplatelet autoantibodies [ ] . because vaccines are designed to induce protective immunity by mimicking infections in the human body, both live and inactivated vaccinations can theoretically trigger the development of itp [ ] . researchers have investigated associations between multiple vaccinations and itp risk, but findings regarding inactivated vaccinations were inconsistent [ ] [ ] [ ] [ ] [ ] . however, because many vaccines are administered to children in infancy, an epidemiological study for vaccine safety conducted some time after a single vaccine dose is difficult to design. therefore, more studies are needed to clarify the itp risk associated with live and inactivated vaccines. a combined vaccine is defined as a single product in which equivalent component vaccines are administered as a single vaccine to prevent more than one disease or to protect against multiple strains of infectious microbes [ ] . for example, mmr, diphtheria-pertussis-tetanus and multivalent pneumococcal conjugate vaccines have been licensed for large-scale supply. the nature of combined vaccines means it is difficult to separately measure the adverse reaction rate of each component. simultaneous vaccination, which is commonly conducted for children in europe [ ] , north america [ , ] and asia [ ] , is defined as administering more than one vaccine at different anatomic sites during the same clinic visit, without combining these vaccines in the same syringe [ ] . early research reported that parents were concerned about the efficacy of simultaneous immunisation [ ] . however, experimental and clinical epidemiological studies provided a scientific basis for the efficacy of this practice [ ] . recently, the focus of interest in simultaneous vaccination has moved from questions of efficacy to questions of safety [ ] [ ] [ ] . if a vaccination has adverse reaction risks, these risks may accumulate in simultaneous vaccination [ ] . however, no research has compared the risks when administering vaccines simultaneously versus separately in the same population. studies conducted in the s indicated rates of adverse reactions were similar between simultaneous and separate vaccine administration [ ] [ ] [ ] . correspondingly, the centers for disease control and prevention issued a guideline recommending that parents use simultaneous vaccination for children younger than years to avoid missing the appropriate vaccination timing [ ] . however, some studies suggested that rates of several adverse reactions may increase with simultaneous vaccination [ , ] . therefore, the risk associated with simultaneous vaccine administration needs to be investigated more comprehensively. in this case-control study, we aimed to determine the itp risk after live, inactivated and simultaneous vaccination in japan. we also examined the risk associated with simultaneous vaccination among subjects aged < years, referred to in this study as infants [ ] , because this age group are frequently immunised using this method. we requested physicians from paediatrics and internal medicine departments at seven university hospitals and regional centre hospitals in japan to recruit inpatients and outpatients for this study. recruitment occurred from october to march . during this period, participating physicians enrolled in all new cases with itp that attended their hospital and met the inclusion criteria for this study. these physicians filled in the study questionnaire with the requested data for the case and control patients. we defined exposure as vaccination within days before the onset of itp. to measure this exposure, participating physicians who treated case (itp) and control (other diseases) patients completed questionnaires covering retrospective information on vaccination history and other characteristics. in japan, vaccination history for infants and preschool children is recorded in the maternal and child health handbook, which is provided by the municipality. vaccination history for other children and adults was captured in a medical interview conducted by physicians or obtained from medical records. additional information was obtained from hospital medical records. in japan, the immunization law defines itp as an adverse event following immunisation that occurs within days after vaccine administration [ ] . based on this law, we considered ≤ days after administration as the duration in which vaccinated people could potentially develop itp as a result of vaccination. the live vaccines investigated were the bacillus calmette-guérin (bcg), rotavirus, varicella, mumps and measles-rubella (mr) vaccines (in japan, mr and mumps vaccines are administered separately). inactivated vaccines were the influenza, diphtheria-pertussis-tetanus-polio (dpt-ipv), hepatitis b, following japanese and american guidelines [ ] [ ] [ ] [ ] , cases with itp were identified when patients met all of the following conditions. ( ) peripheral blood platelet count ≤ , /µl [ ] [ ] [ ] . ( ) without anaemia unless the patient was bleeding as a result of itp. ( ) without deformation of red or white blood cells. ( ) without aplastic anaemia, myelodysplastic syndrome, leukaemia, malignant lymphoma, paroxysmal nocturnal haemoglobinuria, systemic lupus erythematosus, disseminated intravascular coagulation, bone marrow metastasis, myelofibrosis, thrombotic thrombocytopenic purpura, haemolytic uraemic syndrome, liver cirrhosis, hypersplenism and megaloblastic anaemia. ( ) without bernard-soulier, wiskott-aldrich, may-hegglin and kasabach-merrit syndromes. ( ) condition was not pseudo-thrombocytopenia due to ethylenediaminetetraacetic acid. ( ) condition was not thrombocytopenia caused by pharmacological agents, radiation or measles. because this study included patients with new onset itp, all itp cases were considered to be as acute [ ] . each patient with itp (case) was matched with one or two control patients [ , ] . participating physicians matched controls with case patients by the institution, timing of hospital visit (within a -month difference), sex and age. in the matched study design, we permitted overlapping use of case patient datum to be paired with two different control patient data; within an institution, one control datum was paired with two different case patient data. we requested that participating physicians reduced the difference in timing of hospital visit between case and control patients to within weeks where possible. age was matched as age in months for case patients aged under year, and within years for case patients aged - years. for case patients aged ≥ years, age was matched within years where possible, with a maximum difference of years. all participating patients were of asian ethnicity. sex, age at hospital visit and consulted department were measured; height and body weight were not measured. to exploit the matched study design, we calculated mcnemar's odds ratios (ors) and their % confidence intervals (cis) [ ] for the primary outcome. mcnemar's or is suitable for matched case-control design. briefly, in this calculation method, vaccination histories for case-control pairs were grouped into four profiles: ( ) vaccination ( +) for a patient with immune thrombocytopenic purpura (case) and vaccination ( +) for a patient with other diseases (control); ( ) vaccination ( +) for case and vaccination ( −) for control; ( ) vaccination ( −) for case and vaccination ( +) for control; and ( ) vaccination ( −) for case and vaccination ( −) for control. in calculating mcnemar's ors, only two of these profiles ( and ) were used based on an assumption of the binary distribution. for example, if the number of the case-control vaccination profile equalled that of profile , mcnemar's or = (null hypothesis) [ ] . if the former number of profile doubled the latter number of profile , mcnemar's or = . this calculation method originated when mcnemar's or was developed to evaluate the results of a matched case-control study. subsequently, conditional logistic regression was developed to further adjust for covariates. we also calculated mcnemar's ors stratified by age group. because vaccination for measles, rubella and varicella are known risk factors for itp onset [ ] [ ] [ ] [ ] [ ] , we calculated those ors as the reference values. tests for p values were based on a binary distribution with the null hypothesis being the same pair of numbers between the two vaccination history profiles, which is usually used in calculating ncnemar's or [ ] . we also calculated common ors to exploit all available data for unmatched case and control patients. data for those patients whose paired partner was excluded from the matched data analysis were included in the unmatched analysis. common ors were the secondary outcome. we aimed to measure confounders of history of infection with helicobacter pylori [ , ] , other viruses and bacteria, but only obtained these data for a subset of patients. therefore, in estimating common ors, we only adjusted for sex [ , , ] , and calculated ors stratified by age group. the age groups were < , - , - and ≥ years, based on the vaccination schedule for children, adults and older adults recommended by the japan pediatric society and the infectious disease surveillance center (english versions) [ , ] . because the risk associated with simultaneous vaccination could not be assessed in the matched analysis because of the small sample size, this risk was only assessed in the unmatched analysis. we calculated sex-adjusted common ors for itp occurrence in the simultaneous administration of two or more vaccines. we performed all statistical analyses using sas version . (sas institute, cary, nc, usa). all reported p values were two-sided, and p < . was considered significant. in total, pairs were included in the matched analyses, and patients in the unmatched analyses. most patients with itp were enrolled from paediatrics departments. in the itp case and control groups, varied infectious diseases were reported month before the hospital visit. there were no underlying diseases involving an immunocompromised status, but there was an epilepsy case in the itp case group and an asthma case in the control group. table shows the characteristics of the matched case and control patients. in total, participants ( . %) were under years of age, ( . %) were aged - years and ( . %) were male. for the cases on whom data was provided by their physicians, the mean (standard deviation) of duration from vaccination to itp onset was . ( . ) days. among case patients, there were cases of purpuras, three of bleeding in the oral cavity and difficulty stopping bleeding, three of hypermenorrhoea and one of haematochezia. diagnoses recorded for hospital visits among control patients included respiratory tract infections, nine digestive tract infections, three urinary tract infections, nine allergic diseases and four malignant neoplasms. there were positive results for any helicobacter pylori test in the case group, but no positive results in the control group. figure presents a histogram of the ages of case patients. most case patients with itp were aged < - years. for simultaneous vaccination, we investigated children aged months to years: six children had two vaccines, four had three vaccines, two had four vaccines and one had five vaccines. table shows the sex-adjusted common ors for itp occurrence in the simultaneous administration of two or more vaccines. the mcnemar's and common ors for itp occurrence for all, live and inactivated vaccines ranged from almost null to more than among people of all ages, children aged < years and children aged < years, but these results were not statistically significant. the common ors for simultaneous administration of two or more vaccines were greater than for all, live and inactivated vaccines; these results were not statistically significant. previous research suggests that people may develop itp as an immune reaction to vaccination in combination with a genetic predisposition [ , [ ] [ ] [ ] [ ] [ ] . because vaccines are designed to mimic real infections and trigger immune reactions, clinicians have been concerned that vaccinationparticularly the administration of live vaccines-may cause autoimmune disease [ ] . mmr-associated acute itp has been frequently investigated in studies on this topic [ , [ ] [ ] [ ] . in , the united states institute of medicine acknowledged that evidence had established a causal relationship between mmr vaccination and thrombocytopenia [ ] . in our study, the common or for mr vaccination was . , although this result was not statistically significant (supplementary table ). in summary, the present study did not find many significant effects, but the mcnemar's and common ors estimated for live vaccination ranged from null to high across age groups. itp risk associated with inactivated vaccination has not been sufficiently investigated in previous studies. a postlicensure retrospective study in the united states did not find any itp cases among -month-old infants vaccinated with the dtap-ipv/hib or other dtap-containing vaccine from october to july [ ] . a canadian surveillance study ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) identified eight itp cases after dtp or dtap ± ipv ± hib vaccinations, three after hepatitis b vaccination and two after influenza vaccination in paediatric hospitals [ ] . an italian post-licensure prospective study involving a large number of older adults conducted from to reported cases of itp after seasonal influenza vaccinations [ ] . a study using a taiwanese national database found an incidence rate ratio for itp of . ( % ci . - . ) following pandemic influenza (h n ) monovalent vaccination without adjuvant versus non-vaccination among people aged - years, and of . ( % ci . - . ) among children [ ] . the united states vaccine adverse event reporting system database reported that from to , influenza vaccination had a proportional reporting ratio (an index of identifying unexpectedly frequent reports) for thrombocytopenia occurrence of . ( cases, lower bound of % ci of the empirical bayesian geometric mean < . ) [ ] . however, there is a lack of consistent evidence about the itp risk associated with inactivated vaccines. in our study, the estimated mcnemar's ors for inactivated vaccines varied from under to over , and were not statistically significant in the examined age groups. calculation of mcnemar's ors used discordant case-control pairs in terms of vaccination history; therefore, calculating mcnemar's ors required a much larger sample size than would have been required to calculate common ors [ ]. because of insufficient power, we were unable to clarify whether itp risk accompanied inactivated vaccination. care should be taken in the interpretation of the ors reported for inactivated vaccines in this study. in the context of japan, many inactivated vaccinations are scheduled for children aged < year [ ] , meaning that infants of this age have a high chance of being immunised with inactivated vaccines. previous research suggests that among japanese children, the peak age of itp onset is < years [ ] . the ors for inactivated vaccination could, therefore, be overestimated in this study because most data used for their estimation included data for infants aged < year, who frequently receive inactivated vaccines and have a relatively high incidence of itp. therefore, we interpreted the mcnemar's or of . (table ) and common or of . (table ) among children aged < years as indicating that this study did not detect itp risk in inactivated vaccination. vaccination is frequently criticised in the media [ , ] . however, vaccination has three types of benefits: direct effects (immunity of the vaccinated person), indirect effects (herd protection of non-vaccinated people) and reduction in the risk for developing itp caused by infection with viruses or bacteria. the third type of benefit could be rephrased as the observation that the incidence of itp following vaccination is lower than the incidence of itp after infection with wild viruses [ ] . the incidence of itp development in children immunised with mmr is reportedly around one in , , whereas it is approximately one in following natural rubella infection and approximately one in following measles infection. another report suggested that the incidence of itp caused by mmr vaccination is - times lower than the incidence of itp following natural infection [ ] . as is inherent with a single outcome (in this case, the development of itp), the present findings of ors greater than do not consider the third type of vaccination benefit. in other words, it is difficult to investigate the long-term effect of vaccination on preventing itp development from the perspective of the third type of benefit-if it exists-in cohort or case-control studies. the present study is notable for investigating itp risk following simultaneous vaccination. although we did not find statistically significant results, the sex-adjusted ors were relatively high (or . among children aged < years and . among children aged < years; table ). the point estimate of the or was larger than that of single vaccination ( . among children aged < years and . among children aged < years; table ) in children of each age group. few studies have investigated the risks associated with simultaneous vaccination. a randomised controlled trial involving people receiving simultaneous influenza vaccination reported increased haemagglutinin inhibition titre, seroconversion and seropositivity rates, suggesting that the immune response was boosted [ ] . because vaccine efficacy and immune-related adverse reactions have the same origin [ ] , the rate of itp development may increase depending on the administration method. table suggests that there is potential for a relatively higher risk for itp with simultaneous vaccination compared with separate vaccination. however, further analyses using large databases are needed to confirm this finding. there are several limitations inherent to this study. first, the sample size meant the study was underpowered because we adopted a matched design to adjust for several confounders between cases and controls. this sample size also made it difficult to estimate ors separately by age group. to compensate for this limitation and present additional results, we calculated common ors using all available data, although adjustment for confounders might have been insufficient for this analysis. second, we could not adjust for infection history as a confounder. although we aimed to measure infection history in the questionnaire, we did not obtain sufficient data. third, because the investigation depended on the voluntary replies of physicians by post, reporting bias might have occurred. physicians who were interested in potential adverse events might have been more likely to participate in this study, which could have led to an overestimation of the ors. a national surveillance system of adverse events following vaccinations in japan is needed in an era when people are concerned about drug-induced diseases. fourth, each case was matched to one or two controls. this method might have resulted in an imbalance in the weight of samples for the primary outcome. because several matching factors may restrict selecting potential control patients, we chose a design to counter this limitation. in terms of study strengths, the detection of itp cases by physicians was a main advantage of the present study. the use of physician-registered cases enhanced the internal validity of this investigation. this validity would not be possible in a large database study. second, the data were gathered from hospitals across japan. the reported itp risks, therefore, reflect a broad area, suggesting relatively high external validity. third, the analyses included detailed results stratified by age group and type of vaccine. this information will be informative for researchers and clinicians seeking to assess the potential risks for adverse events for their patients. based on the limited data available in this study, high itp risk was not found following inactivated or simultaneous vaccination. future investigations of itp risk should include analyses using large databases. idiopathic thrombocytopenic purpura recurrent immune thrombocytopenia after influenza vaccination: a case report prediction of response to first-line therapy with itp 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japan: revision thromboembolism in patients with immune thrombocytopenia (itp): a meta-analysis of observational studies : the analysis of case-control studies the mcnemar test for binary matched-pairs data: mid-p and asymptotic are better than exact conditional high-dose dexamethasone therapy as the initial treatment for idiopathic thrombocytopenic purpura risk of immune thrombocytopenic purpura after measles-mumps-rubella immunization in children. child care health dev a nationwide survey of newly diagnosed childhood idiopathic thrombocytopenic purpura in japan routine/voluntary immunization schedule in japan vaccination and autoimmune disease: what is the evidence? idiopathic thrombocytopenic purpura and mmr vaccine recurrent thrombocytopenic purpura after repeated measles-mumps-rubella vaccination effect of live measles vaccine on the platelet count persistent changes in circulating white blood cell populations after splenectomy adverse events associated with childhood vaccines other than pertussis and rubella: summary of a report from the institute of medicine on the sample size for studies based upon mcnemar's test safety and immunogenicity of a pandemic influenza a h n vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the - influenza season: a multicentre, randomised controlled trial publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to express our deep gratitude to the participating patients. for their cooperation as physicians in this study, we would also like to thank dr. katsuko maeda, yamagata city hospital saiseikan; dr. yuya sato, dokkyo medical university hospital; dr. noritaka furuya, saitama citizens medical centre; dr. yoshinori kobayashi, national hospital organization yokohama medical centre; conflict of interest tn received honoraria from daiichi sankyo co., sanofi k.k. and mitsubishi tanabe pharma corporation. mmochizuki received honoraria from pfizer inc. mmori's department received unrestricted research grants from abbvie gk; ayumi pharmaceutical corporation; chugai pharmaceutical co., ltd.; csl behring k.k.; japan blood products organization; nippon kayaku co., ltd.; ucb japan co., ltd.; and asahikasei pharmaceutical corporation. the other authors have no conflicts of interest to declare. the study protocol was reviewed and approved by the ethics committee of the national institute of infectious diseases (the principal institution of this project) in accordance with the ethical guidelines and regulations of the declaration of helsinki (approval number: h - ).informed consent physicians in hospitals explained the study to their patients and obtained written informed consent from the parent(s) or legal guardian(s) of each child and/or assent by the child (when applicable) before initiating medical interview and record review. hiroshi yokomichi · keiko tanaka-taya · rie koshida · takashi nakano · yoshinori yasui · masaaki mori · yuka ando · saeko morino · hideo okuno · hiroshi satoh · satoru arai · mie mochizuki key: cord- -fd qmr authors: slepushkin, vladimir a.; staber, patrick d.; wang, guoshun; mccray, paul b.; davidson, beverly l. title: infection of human airway epithelia with h n , h n , and h n influenza a virus strains date: - - journal: mol ther doi: . /mthe. . sha: doc_id: cord_uid: fd qmr three subtypes of influenza a virus cause human disease: h n , h n , and h n . although all result in respiratory illness, little is known about how these subtypes infect differentiated airway epithelia. therefore, we assayed a/pr/ / (h n ), a/japan/ / (h n ), and x (h n ) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air–liquid interface. in this model system, viral infectivity was highest when virus was applied to the apical versus the basolateral surface; japan was most infectious, followed by pr . the x strain showed very low levels of infectivity. confocal microscopy and fluorescence-resonance energy transfer studies indicated that japan virus could enter and fuse with cellular membranes, while infection with x virions was greatly inhibited. japan virus could also productively infect human trachea explant tissues. these data show that influenza viruses with saα , gal binding specificity, like japan, productively infect differentiated human airway epithelia from the apical surface. these data are important to consider in the development of pseudotyped recombinant viral vectors for gene transfer to human airway epithelia for gene therapy. influenza viruses comprise some of the most common pathogens of the human upper and lower respiratory tract ( ) . the virus lipid envelope surrounds a segmented rna genome, with the envelope protein, hemagglutinin (ha), responsible for binding and fusion to host cells ( ) . another viral surface glycoprotein, neuraminidase, is important in both budding and entry ( ) . humans are infected by influenza a virus subtypes with the classification of h n , h n , and h n . the ha proteins bind to sialic acid-bearing receptors on host cells. specificity for sialic acid residues terminating in ␣ , -galactose ␤ , -n-acetyl glucosamine (sa␣ , gal) or sa␣ , gal is determined by ha protein sequence ( ) ( ) ( ) ( ) ( ) . earlier studies showed that many human isolates preferentially bind and agglutinate red blood cells bearing oligosaccharides terminating in sa␣ , gal while the avian strains often exhibit specificity for erythrocytes bearing sa␣ , gal receptors ( , , ) . in subtypes cultured in the laboratory, the host cell used to culture the virus can direct ha-coding sequence alteration ( , - ) and alter sialic acid-binding specificity. cells in the human trachea express both types of oligosaccharides ( ) . using specific lectins it was shown that ciliated cells express sa␣ , gal moieties on their surface, while mucus-secreting goblet cells stain positive for sa␣ , gal. the ability of influenza to infect differentiated airway cells expressing one, both, or neither oligosaccharide is not currently known. however, an earlier report showed that an h human virus strain with sa␣ , gal specificity could bind to formaldehyde-fixed human respiratory epithelium ( ) . experiments that examined influenza virus-host cell interactions, using dog renal epithelia ( ) ( ) ( ) , showed that virus binding and budding occur preferentially at the apical surface of polarized cells. binding to cultured airway cells or tissues has also been examined. prior reports demonstrated that an h n strain could bind and propagate in dissociated airway epithelia cultured from fetal lung tissue explants ( ) . the strain pr was also shown to productively infect airway epithelial cells grown as dissociated cells on plastic dishes ( ) . thus these experiments together with earlier studies ( ) do not directly address the polarity of infection or budding of influenza strains on differentiated respiratory epithelia. a culture system which closely resembles the airway epithelium both morphologically and physiologically has been described ( - ) . in this model, airway epithelia cells are isolated from human trachea or bronchus and the cells dissociated. the cells are grown on filter inserts and cultured at the air-liquid interface. in approximately weeks, the culture is multilayered and polarized, with basal cells, intermediate cells, and columnar cells present. the most apical layer contains both ciliated and nonciliated cells. this model has been used to demonstrate the polarity of infection of recombinant retroviruses ( ) , recombinant adenoviruses ( , ) , and recombinant adeno-associated viruses ( ) . we examined the polarity of influenza virus binding, fusion, and budding in human airway epithelia cultures. the h strain, x , with sa␣ , gal specificity; a/japan/ / , an h n influenza strain with sa␣ , gal specificity; and a strain with mixed specificity (a/pr , h n ) were tested. our results demonstrate that influenza a strains bind to the apical surface of the epithelia. for h n and h n this results in a productive infection. we also demonstrate entry and fusion of the h n virus with cellular membranes and show that x virions can bind but do not productively infect differentiated airway epithelia. primary culture of human airway epithelia. primary cultures of human airway epithelia were prepared from bronchi by enzymatic dispersion according to established methods ( , ) . briefly, epithelial cells were disassociated and seeded onto collagen-coated, semipermeable membranes with a . -m pore size (millicell-ha; surface area, . cm ; millipore corp., bedford, ma). millicell inserts were placed into -well plastic cell culture cluster (costar, cambridge, ma). twenty-four hours after seeding, the mucosal medium was removed and the cells were allowed to grow at the air-liquid interface as reported previously ( ) . the culture medium, which consisted of a : mixture of dulbecco's modified eagle medium and ham's f- with % ultroser g (sepracor, inc., marlborough, ma), u of penicillin/ml, and g of streptomycin/ml, was added to the wells to feed cells from the basolateral surface. only well-differentiated cultures Ͼ weeks old were used in these studies, with approximately ϫ cells/millicell. the presence of tight junctions was confirmed by transepithelial resistance measurements (resistance Ͼ ⍀ ⅐ cm ). influenza viruses. influenza virus preparations were purchased from spafas laboratory (storrs, ct). preparations were provided as concentrated and partially sucrose-gradient-purified preparations. three strains of influenza a virus were used in this study: a/pr/ / (h n ) (pr ), a/japan/ / (h n ) (japan), and x (h n ). the latter strain is a reassortment with surface glycoprotein genes from a/aichi/ / (h n ) and internal genes from a/pr/ / ( ) . the total protein concentration of each virus preparation was mg/ml. viruses were titrated by serial dilutions on mdck cells in the presence of g/ml tpck-treated trypsin (sigma, st. louis, mo) ( ) . infected wells were determined by the presence of hemagglutination activity in the culture medium. the tissue culture infectious dose % (tcid ) was calculated using previously described methods ( ) . for measurement of the kinetics of influenza virus production from infected airway cells in one infectious cycle, l of the virus preparation was diluted in dulbecco's pbs (life technologies, inc., grand island, ny) and applied to the apical surface of the epithelia (m.o.i. of . ). after incubation for min at °c the cells were washed three times with pbs and incubation at °c was continued for various time periods with l of pbs left on the surface of epithelia. release of the progeny virus into pbs or basolateral cell culture medium was assayed by hemagglutination with human donor erythrocytes ( ) . there was no hemagglutination activity in the third pbs wash. to infect airway epithelia with different influenza virus strains from the basolateral side, the millicell culture insert was turned over and virus preparations diluted in pbs were applied in a -l volume to the bottom of the membrane (m.o.i. ϭ . ). after incubation for min at °c, the suspension was removed and the membranes were washed three times with pbs. the millicell insert was returned to its regular upright position with l of pbs added to the apical side. after incubation for h at °c, virus hemagglutination titer was assayed in both the apical pbs and the basolateral cell culture medium. for testing viral infectivity during a multiple cycle infection, l of the virus preparations was added to the apical side of the airway epithelia at an m.o.i. of . and incubated for min at °c. the viral suspension was then removed and the cells were washed three times with pbs. epithelia were incubated at °c for h to allow for initial virus production. to assay for virus release, l of pbs was added to the mucosal surface and cells were incubated at °c for h. to test the effects of protease on influenza virus ha, tpck-treated trypsin was added to the pbs (final concentration of g/ml) in some experiments. after incubation, surface liquid was collected and assayed by hemagglutination for the presence of the virus. the cells were incubated further at °c without medium on the apical side. these steps were repeated every h for days. after h of incubation the cells were lysed in laemmli sample buffer ( ) , and viral proteins were analyzed by immunoblot assay. metabolic labeling of virus proteins. airway epithelia were infected with different influenza virus strains from the apical side, at an m.o.i. of . , for h at °c. after the initial infection epithelia were incubated in methionine-and cysteine-free cell culture medium (life technologies, inc.) for h at °c to accommodate the initial steps of the virus replication cycle. [ s]methionine (nen life science products, boston, ma) was then added to the basolateral culture medium ( ci/ml final) and incubation continued for h at °c. cell-free virus was collected from the apical side and cells were lysed in laemmli sample buffer. analysis of radioactively labeled proteins was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) with subsequent autoradiography. antiviral polyclonal antibodies. polyclonal antibodies against japan and x strains were generated by inoculation of sheep with the corresponding partially purified formalin-inactivated virus by elmira biologicals (iowa city, ia). the antibodies were purified from serum using a combination of ammonium sulfate precipitation and deaecellulose chromatography ( ) . for immunostaining, purified antibodies were labeled with either texas red or fluorescein isothiocyanate (fitc) using fluoreporter protein labeling kits (molecular probes, inc., eugene, or). reactions were performed according to the manufacturer's recommendations. immunoblot assay. following page, proteins were transferred to nitrocellulose membranes (immobilon-nc; millipore corp.) using a semidry transfer cell (trans-blot sd; bio-rad laboratories, hercules, ca), . h at ma. membranes were blocked with % fat-free dry milk, h at room temperature, followed by overnight incubation at °c with anti-japan sheep serum diluted : in . % tween /pbs solution. specific virus proteins were stained with peroxidase-conjugated affinipure donkey antisheep igg (jackson immunoresearch laboratories, inc., west grove, pa), used at : , dilution for h at room temperature. the bands were visualized on x-ray film after development with the ecl kit (amersham pharmacia biotech uk ltd., little chalfont, england). staining of membranes with preimmune serum was negative. influenza virus interactions with airway epithelia. partially purified preparations of influenza virus were labeled with n-(lissamine rhodamine b sulfonyl) diacyl phosphatidylethanolamine (rh-pe) (avanti polar lipids, inc., alabaster, al) by passive insertion into the virus envelope according to earlier described methods ( ) . cellular membranes were labeled with n-octadecyl-nЈ-( -(fluoresceinyl))thiourea (f ; molecular probes) by addition of the dye into the basolateral culture medium at a final concentration of m and incubation for h at °c. f -labeled airway epithelia were infected with rh-pe-labeled influenza virus by adding l of virus suspension to the apical surface (m.o.i. ϭ ). virus was allowed to internalize for min at °c. cells were then washed three times with pbs and fixed with % paraformaldehyde for min at room temperature. cells were then washed two times with pbs and the millicell inserts removed and mounted onto slides for microscopy. cells were scanned using a mrc confocal microscope (bio-rad). images were acquired using two photomultipliers, set for a double-labeling protocol (fluorescein and pe-rhodamine), and analyzed using cut plane program software, allowing for xy and xz axis stacking of sections. for fluorescence-resonance energy transfer (fret) assays cells were scanned using customized settings with the excitation wavelength set for fluorescein only. images were acquired using the same double-labeling protocol as above. at this excitation wavelength, detection of red indicates fret. airway epithelia were cooled on ice and influenza virus preparations diluted -fold in pbs were added to the apical side (m.o.i. of ). the virus was allowed to adsorb for min at °c. virus was then removed, cells were washed three times with cold pbs, and °c-warm fitc-dextran solution ( mw, anionic, lysine fixable; molecular probes) . mg/ml in pbs was added. cells were incubated at °c for min, washed three times with pbs, and fixed with % paraformaldehyde for min at room temperature. cells were counterstained with texas red-labeled lectin from maackia amurensis (sigma), specific for sialic acid. infection of tracheal explants. blocks of human trachea, approximately . cm in size, were placed in airway culture medium in a -well plastic cluster dish. explants were infected with the influenza virus preparations diluted to the same concentration as for evaluation of fluid-phase endocytosis (above). virus was incubated for h at °c. excess virus was then removed by four washings with pbs, with the last wash tested for virus by hemagglutination assay. infection of airway epithelia was allowed to proceed overnight at °c in the airway culture medium, and the release of the progeny virus was assayed by hemagglutination. twenty-four hours after infection explants were washed two times with pbs and fixed with % paraformaldehyde. fixed tissues were cryoprotected and embedded and -m sections obtained for staining. the sections were stained with fitc-or texas red-labeled sheep igg against japan or x strains. photomicrographs were acquired using a leica dm rbe fluorescence microscope equipped with a sony digital camera and captured with adobe photoshop software. we first compared the abilities of x (h n ), japan (h n ), and pr (h n ) strains to infect cultures of primary human airway epithelia. this model culture system, in which human airway epithelia are cultured at the airliquid interface ( , ) , has been invaluable in testing how other viruses can or cannot access intracellular compartments when applied to the mucosal surface ( , , ) . the titers of the strains tested were determined in mdck cells, and sa-gal specificities were confirmed on erythrocytes. titers and sa-gal specificities are listed in table . the viruses were applied to the apical side of the airway epithelia at m.o.i. equal to . . the amount of the virus released to the apical surface fluid was measured at different time points by hemagglutination. the results of a representative experiment are shown in fig. . in all cases, virus production in airway epithelia was highest for the japan strain. the number of virus progeny produced by pr was considerably lower than that of japan, but higher than that of x . for japan, virus release started h after virus inoculation and continued to rise throughout the -h time course. the virus production was somewhat delayed compared to the mdck cell model, in which maximal virus production was reached in approximately h ( ). however, the kinetics of virus production observed in our experiments were very similar to previous reports for human airway cell lines infected with the h n influenza a strain ( ) . there was no drop in transepithelial resistance measurements throughout the study (data not shown), indicating that the epithelia remained intact. our first experiment tested the ability of influenza a viruses to productively infect airway epithelia when applied to the mucosal surface. we next compared the amount of virus produced after basolateral or apical application. the virus released into the basolateral or apical compartments was measured by hemagglutination. apical infection of the airway epithelia produced -to fold more virus than basolateral infections (fig. ) . when japan strain was allowed to infect from the top of the cultures, approximately -fold more virions were generated than when pr was applied and approximately fold more viruses than following x infection. in similar studies, we found no cross-release of the virus (data not shown). cleavage of hemagglutinin is an important, and often a host-limiting, step of influenza virus infection. in most cell culture systems used for replication of influenza virus the addition of exogenous trypsin is required for multiple cell infection. we asked whether a multiple-cycle infection could proceed in airway cells without the addition of proteases. to perform this experiment, airway epithelia were infected with x , pr , or japan strains and then washed with pbs with or without addition of trypsin. virus production was monitored by hemagglutination. protease treatments and assays were repeated on days , , and after infection. it was found that the difference in tcid between japan virus-infected filters treated and untreated with trypsin did not exceed log (fig. a) . infectivity of x strain under either condition was very low (data not shown), precluding our ability to test for cleaved ha. immunoblot assays were done to confirm that japan virus hemagglutinin was cleaved in the absence of exogenously added proteases (fig. b) . japan strain hemagglutinin precursor (ha ) in the absence of trypsin can be seen in fig. b (lane Ϫt) . the addition of trypsin facilitated more complete digestion of the hemagglutinin protein (fig. b, lane ϩt) , but as noted this additional cleavage did not dramatically increase virus production. the x strain is a reassortment virus generated for vaccination purposes ( ) . prior work showed that h n strains similar to x could productively infect nasal polyps grown as organ cultures ( ) . also, other work showed effective binding of x to formaldehyde-fixed tracheal tissues. to determine if the block in x infection was after entry or after binding, both metabolic labeling and binding assays were performed. cultures of airway epithelia were infected with x or japan strains and metabolically labeled with side were analyzed by sds-page. the data in fig. a show accumulation of influenza nucleoprotein (np) in apical washings from japan-infected cells (fig. a , lane js) but not x -infected cells (fig. a, lane xs) . the authenticity of viral np protein was confirmed using an immunoblot with anti-japan antiserum (fig. b) . this result indicates that the hindrance to x infection occurs at early stages of the infectious cycle. japan and x strain infection was monitored by confocal microscopy after application of fluorescently labeled virions to the cultures (m.o.i. ϭ ) to determine if the x strain was capable of entry into human airway epithelia. to distinguish between viruses and cells, the rhodaminelabeled lipid probe rh-pe was introduced into the viral envelope, and cellular membranes were labeled with f (fluorescein lipid probe). thirty minutes after application of virus, cells were washed, fixed, and observed by confocal microscopy. the photomicrographs in fig. show an en face view (figs. a and b) or x-z stacked series (figs. c and d) from the acquired images of influenza virusinfected airway epithelia. japan virions were noted both apically and internally (fig. c ). in contrast, the x strain is localized to the apical surface only (fig. d) . we next tested for evidence of fusion using the fret assay. the assay was designed to test for fret between the fluorescein label in cellular membranes and the rhodamine fluorophore in the viral envelopes. this process can take place only if the distance between the two dyes is in the range of to Å ( ), as can be accomplished during membrane fusion processes. figures e and f are representative of the level of fret occurring in the presence of excitation of fluorescein only. fret, detected as excitation of the ph-re label, was observed throughout the thickness of the culture after application of labeled japan strain (fig. e ). for cultures to which rh-pe-labeled x was applied, fret was rarely detected (fig. f ). fluid-phase endocytosis is minimal in fully differentiated airway epithelia ( ) . to investigate if application of influenza virus would increase endocytosis, fitc-labeled dextran, as a fluid-phase marker, was applied to the cells after their infection with japan or x strains. after incubation with virus for min at °c (m.o.i. ϭ ), cells were washed and intracellular fluorescence was observed using confocal microscopy. the results of this experiment show that endocytosis in the cells infected with japan strain was significantly enhanced, probably as a result of virus uptake (fig. a) . in contrast, fitc-dextran uptake in the cells infected with x was very low (fig. b) and was indistinguishable from that of control, uninfected cells (not shown). to confirm the ability of japan to productively infect human airway cells, experiments were done on freshly explanted human trachea. after overnight infection with japan strain, culture fluid was collected and assayed by hemagglutination for the presence of the virus. the japan strain produced a ha titer of : . in similar studies with pr and x , the titers were : and undetectable, respectively. thus qualitatively, in both the cell culture and the explant models, japan and pr influenza strains were capable of infection, while x was not. the infection of the tracheal epithelium by japan virus was also confirmed by immunostaining (fig. ) . notably, only the apical surface was positive for influenza proteins. the detection of virus proteins was replication-dependent, as staining of control tracheal explants, fixed immediately after adsorption of the viruses, was negative (data not shown). we compared the infectious activity of three influenza a strains in differentiated primary human airway epithelia grown at an air-liquid interface. each of the viruses tested was representative of a different ha subtype. x (h n ) has binding specificity for sa␣ , gal, japan (h n ) binds preferentially to sa␣ , gal, and pr (h n ) is able to utilize both types of oligosaccharides ( ) . we confirmed the sa-gal specificity ( , ) of our viruses in hemagglutination inhibition tests with normal horse serum (table ) . based on studies of virus binding to fixed sections of human trachea ( ) , and human respiratory tissue ( ), we initially hypothesized that application of viruses with sa␣ , gal specificity would be most likely to result in a productive infection in human airway epithelia. however, our data show that influenza strains with preferential binding to sa␣ , gal receptors productively infect human airway epithelia. this was recently confirmed by clinical observations; avian influenza viruses isolated from infected patients in hong kong were found to be sa␣ , gal specific ( ) . prior studies using specific lectin staining showed that tracheal epithelia express sialyloligosaccharides with both sa␣ , gal and sa␣ , gal linkages ( , ) . in earlier work by baum and paulson ( ) , goblet cells and their corresponding mucus droplets stained with lectins specific for sa␣ , gal, while ciliated cells stained predominantly with lectins specific for sa␣ , gal. these data supported studies done by liu ( ) , showing binding of dye-labeled human isolates with sa␣ , gal specificity to ciliated epithelia of the ferret trachea. similar studies, using paraformaldehyde-fixed preparations of human trachea, confirmed binding of viruses with sa␣ , gal specificity to ciliated cells ( ) . interestingly, the authors of this study also noted labeled virus with sa␣ , gal specificity situated over goblet cells, but attributed this binding to tissue preparation artifact. in our experiments binding and infection were tested using viable cells, with clear evidence of sa␣ , gal-specific strain binding and fusion with the apical membranes of polarized airway epithelia. like japan, x could bind to the apical surface of airway epithelia. however, it could not enter the cells; there was minimal evidence of membrane fusion within the cells as was found with the japan strain, and there was little to no virus produced after h of infection. the block in infection was not due to defective virus, since the titer in mdck cells was similar to those of the other viruses tested. also, we did not find an overall block in infection by other h n strains with sa␣ , gal specificity, since other h n human isolates could productively infect human airway epithelia from the apical surface ( ) . one possibility for the inability of the x strain to infect airway epithelia could be mutations within ha that do not alter binding specificity, but do inhibit interaction with a secondary receptor or entry of the virus into cells. sequencing of the ha rna encoding the ha subunit from x and a/aichi/ / virions revealed only a conservative mutation: ile in aichi to val in x (data not shown). however, we cannot discount that mutations in ha or other proteins were responsible for the low infectious activity of x in airway epithelia. a second possibility may relate to the susceptibility of japan or x ha to inactivation ( ) . work by korte and colleagues demonstrated that x ha is more sensitive to inactivation after preincubation in low-ph buffer (ph . - . ) than japan virus. those studies showed that a -min preincubation of x at ph . reduced the fusogenic capacity of the virus by %. japan was more stable under these conditions. when similar experiments were done using pr , the rate and extent of inactivation were greater than for x . however, we noted that pr was more infections than x in airway epithelia, suggesting that inactivation of x ha may not be the sole determinant for its inability to infect airway epithelia in this study. finally, the observed differences between the parent strains (pr and a/aichi) and their reassortant (x ) could be due to altered interactions between surface glycoproteins derived from the h n strain and the membrane proteins (m and m ) originating from h n virus. how the requirements for these interactions would differ between kidney cells and airway epithelia is unclear at this time. infection of epithelial cells by most viruses is polarized in terms of entry and release of virus particles (reviewed in ( ) ). we noted previously that entry of moloney murine retrovirus vectors with amphotropic or xenotropic envelopes into human airway epithelia was restricted to the basolateral surface ( ) . in contrast, influenza virus infection resulted from binding to the apical plasma membrane (( ) and this study), similar to coronavirus ( , ) and measles virus ( ) . researchers investigating viral vectors for application to the treatment of cystic fibrosis have shown that one of the major limitations to infection of airway epithelia from the apical side is receptor inaccessibility ( ) ( ) ( ) ( ) . recently, methods have been developed to transiently disrupt the tight junctions to allow envelope viruses access to all cell layers of the epithelia ( ) . an alternative method to target retroviral vectors to airway epithelia from the mucosal side may be to pseudotype them with envelopes shown to allow for productive infection when applied to the apical surface. our data suggest that japan, but not x , could be a candidate envelope protein for such an approach. for consideration of gene therapy for cf or other airway diseases, it would be advantageous to use an envelope not currently circulating in the human population (like japan). this would preclude problems of existing neutralizing antibodies. such envelopes, coupled with lentivirus vectors, which we previously showed to result in longterm expression ( ) , may be ideal. in conclusion, we confirm the previously reported polarity of influenza virus binding and budding, but using a model of well-differentiated airway epithelia cultured at an air-liquid interface. our data revealed interesting differences between x infectivity in mdck vs airway epithelia and suggest that the block in x infection was due to inhibition of infection after binding to the cell surface. we demonstrate that japan virus can bind and fig. . fluid-phase endocytosis in human airway epithelia infected with japan or x strain of influenza virus. influenza virus preparations were added to airway epithelia in the cold, followed by addition of a °c solution of fitc-dextran to detect fluid-phase endocytosis. after a -min incubation cells were fixed, stained with texas red-labeled lectin to label the apical surface, and observed by confocal microscopy. the photomicrographs are a stacked x-z series. background levels of endocytosis (not shown) in the absence of added virus were uniformly low as previously published ( ) . cryosection of trachea explant infected with japan virus. trachea explants were left uninfected or were infected with the japan influenza strain as described under materials and methods. cryosections ( m) were stained with fitc-labeled antibodies against japan virus. the photomicrograph shown is representative of the level of anti-japan immunoreactivity seen in three independent experiments using blocks of trachea from different donor lungs. sections from control tissues, or mock-infected tissues, were negative (not shown). the arrow points to the apical side of tracheal epithelium. nuclei were detected using dapi staining. fuse with apical cellular membranes, increase fluid-phase endocytosis, and replicate in viable cultures of well-differentiated epithelia. finally, we speculate that the ha from japan, or from similar strains shown to productively infect airway epithelia models, may be useful for targeting other enveloped viruses, such as recombinant lentiviruses, to the apical surface of airway epithelia for gene therapy of lung diseases. orthomyxoviridae and 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airway epithelia from the apical surface entry and release of measles virus are polarized in epithelial cells increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect key: cord- - z sr authors: kuniya, toshikazu title: prediction of the epidemic peak of coronavirus disease in japan, date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: z sr the first case of coronavirus disease (covid- ) in japan was reported on january and the number of reported cases has increased day by day. the purpose of this study is to give a prediction of the epidemic peak for covid- in japan by using the real-time data from january to february . taking into account the uncertainty due to the incomplete identification of infective population, we apply the well-known seir compartmental model for the prediction. by using a least-square-based method with poisson noise, we estimate that the basic reproduction number for the epidemic in japan is [formula: see text] ([formula: see text] ci, [formula: see text] – [formula: see text]) and the epidemic peak could possibly reach the early-middle summer. in addition, we obtain the following epidemiological insights: ( ) the essential epidemic size is less likely to be affected by the rate of identification of the actual infective population; ( ) the intervention has a positive effect on the delay of the epidemic peak; ( ) intervention over a relatively long period is needed to effectively reduce the final epidemic size. in december , the first case of respiratory disease caused by a novel coronavirus was identified in wuhan city, hubei province, china. the outbreak of the disease is ongoing worldwide and the world health organization named it coronavirus disease (covid- ) on february [ ] . in japan, the first case was reported on january and the number of reported laboratory-confirmed covid- cases per week has increased day by day (see table ). as seen in table , the number of newly reported cases per week has increased and a serious outbreak in japan is a realistic outcome. one of the greatest public concerns is whether the epidemic continues until summer so that it affects the summer olympics, which is planned to be held in tokyo. the purpose of this study is to give a prediction of the epidemic peak of covid- in japan, which might help us to act appropriately to reduce the epidemic risk. the epidemic data as shown in table would have mainly twofold uncertainty. the first one is due to the fact that asymptomatic infected people could spread the infection [ ] . the second one is due to the lack of opportunity for the diagnostic test as sufficiently simple diagnostic test kits have not been developed yet and the diagnosis in the early stage in japan was mainly restricted to people who visited wuhan [ ] . in this study, taking into account such uncertainty, we apply a simple and well-known mathematical model for the prediction. more precisely, we assume that only p ( < p ≤ ) fraction of infective individuals can be identified by diagnosis. we apply the following well-known seir compartmental model (see, e.g., [ ] ) for the prediction. where s(t), e(t), i(t) and r(t) denote the susceptible, exposed, infective and removed populations at time t, respectively. β, ε and γ denote the infection rate, the onset rate and the removal rate, respectively. note that /ε and /γ imply the average incubation period and the average infectious period, respectively. let the unit time be day. based on the previous studies [ , ] , we fix /ε = , and thus, ε = . and γ = . , respectively. we fix s + e + i + r to be so that each population implies the proportion to the total population. we assume that one infective person is identified at time t = among total n = . × number of people in japan [ ] . that is, denotes the number of infective individuals who are identified at time t. thus, we obtain i( ) = /(p × . × ). we assume that there is no exposed and removed populations at t = , that is, e( ) = r( ) = , and hence, it was estimated in [ ] that cases were confirmed among the possible infected population in february in hokkaido, japan. based on this report, we assume that p ranges from . to . . the basic reproduction number r , which means the expected value of secondary cases produced by one infective individual [ ] , is calculated as the maximum eigenvalue of the next generation matrix fv − [ ] , where thus, we obtain ( ) it is obvious that the basic reproduction number r is independent from the onset rate ε. the sensitivity of r to other parameters β, γ and p are calculated as follows: where a β , a γ and a p denote the normalized sensitivity indexes with respect to β, γ and p, respectively. we see from equation ( ) that the k time's increase in β (resp. γ) results in the k (resp. k − ) time's increase in r . in particular, we see from the third equation in equation ( ) this implies that the identification rate p in a realistic range almost does not affect the size of r . let y(t), t = , , . . . , be the number of daily reported cases of covid- in japan from january (t = ) to february (t = ) . we perform the following least-square-based procedure with poisson noise to estimate the infection rate β. (p ) fix β > and calculate the numerical value of y(t), t = , , . . . , by using model equation ( ). where (t), t = , , . . . , denote random variables from a normal distribution with mean zero and variance [ ] . note that for the reason stated above, the value of . ≤ p ≤ . does not affect this estimation procedure. by (p )-(p ), we obtain a normal distribution with mean . and standard derivation . . thus, we obtain an estimation of β as . ( %ci, . - . ) (see figure ) . moreover, by equation ( ), we obtain an estimation of r as . ( %ci, . - . ) (see table ). we define the epidemic peak t * by the time such that y attains its maximum in year, that is, y(t * ) = max ≤t≤ y(t). we first set p = . . in this case, we obtain the following figure on the long time behavior of y(t) for β = . , . and . . we see from figure that the estimated epidemic peak is t * = ( %ci, - ). that is, starting from january (t = ), the estimated epidemic peak is august (t = ) and the uncertainty range is from july (t = ) to august (t = ). we next set p = . . in this case, we obtain the following figure. we see from figure that the estimated epidemic peak is t * = ( %ci, . that is, starting from january (t = ), the estimated epidemic peak is july (t = ) and the uncertainty range is from june (t = ) to july (t = ). in contrast to r , the epidemic peak and the (apparent) epidemic size are sensitive to the identification rate p. note that the essential epidemic size, which is characterized by r , is almost the same in both of p = . and p = . . we next discuss the effect of intervention. in japan, school closure has started in almost all prefectures from the beginning of march [ ] and many social events have been cancelled off to reduce the contact risk. however, the exact effect of such social efforts is unclear and might be limited as the proportion of young people to the whole infected people of covid- seems not so high ( % of , reported cases in china [ ] ). in this simulation, we assume that such social efforts successfully reduce the infection rate β = . to % during a period from march (t = ) to a planned day (t = t ≥ ). in what follows, we fix p = . . first, we set t = , that is, the intervention is carried out for month (from march to april). in this case, the epidemic peak t * is delayed from ( july) to ( july). however, the epidemic size is almost the same. on the other hand, if t = , that is, the intervention is carried out for months (from march to september), then the epidemic peak t * is delayed from ( july) to ( september) and the epidemic size is effectively reduced (see figure ). more precisely, we see from figure a that the epidemic peak t * is delayed almost linearly for ≤ t ≤ and fixed to t * = for t ≥ . this implies that the intervention has a positive effect on the delay of the epidemic peak, which would contribute to improve the medical environment utilizing the extra time period. on the other hand, we see from figure b that the number of accumulated cases at t = , which is calculated as pr( ) × . × , is monotonically decreasing and converges to . × as t increases. however, it almost does not change for small t ≤ . this implies that the intervention over a relatively long duration is required to effectively reduce the final epidemic size. in this study, by applying the seir compartmental model to the daily reported cases of covid- in japan from january to february, we have estimated that the basic reproduction number r is . ( %ci, . - . ) and the epidemic peak could possibly reach the early-middle summer. of course, this kind of long range peak prediction would contain the essential uncertainty due to the possibility of some big changes in the social and natural (climate) situations. nevertheless, our result suggests that the epidemic of covid- in japan would not end so quickly. this might be consistent with the who's statement on march that it is a false hope that covid- will disappear in the summer like the flu [ ] . the estimated value of the basic reproduction number r in this study is not so different from early estimations: . ( %ci, . - . ) [ [ ] . in addition, in this study, we have obtained the following epidemiological insights: the essential epidemic size, which is characterized by r , would not be affected by the identification rate p in a realistic parameter range . - . , in particular, p ≥ . × − . the intervention exactly has a positive effect on the delay of the epidemic peak, which would contribute to improve the medical environment utilizing the extra time period. intervention over a relatively long period is needed to effectively reduce the final epidemic size. the first statement implies that underestimation of the actual infective population would not contribute to the reduction of the essential epidemic risk. correct information based on an adequate diagnosis system would be desired for people to act appropriately. available online covid- ) situation reports transmission of -ncov infection from an asymptomatic contact in germany japan sets up emergency measures for coronavirus age-structured populatin dynamics in demography and epidemiology incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data tracking and predicting covid- epidemic in china mainland population estimates monthly report japan's hokkaido may have infected, researcher says on the definition and the computation of the basic reproduction ratio r in models for infectious diseases in heterogeneous populations reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission parameter estimation and uncertainty quantification for an epidemic model the japan times. nearly all prefectures in japan shut schools amid coronavirus outbreak characteristics of and imoprtant lessons from the coronavirus disease (covid- ) outbreak in china it's a 'false hope' coronavirus will disappear in the summer like the flu, who says report : transmissibility of -ncov novel coronavirus -ncov: ealry estimation of epidemiological parameters and epidemic predictions preliminary estimation of the basic reproduction number of novel coronavirus ( -ncov) in china, from to : a data-driven analysis in the early phase of the outbreak the reproductive number of covid- is higher compared to sars coronavirus the author would like to thank the associate editor and the anonymous reviewers for their helpful comments that allowed me to improve the manuscript. the author declares no conflict of interest. key: cord- -fnb yc authors: humphreys, brad; watanabe, nicholas m. title: the history and formation of east asian sports leagues date: - - journal: the sports business in the pacific rim doi: . / - - - - _ sha: doc_id: cord_uid: fnb yc this chapter considers the creation and growth of professional sport leagues throughout east asia. in this, the different leagues and sport are examined, and noted for their hybrid use of both north american and european methods of business and regulation of sport leagues. notably, prominent japanese, chinese, south korean, and taiwanese sport organizations are covered in their emergence as the top sport businesses within the region. the creation of these leagues varies from the old (japanese baseball) to the new (chinese football). as the dynamics of political and economic power has shifted in east asia in the last several decades, so has the popularity and importance of many of the sport leagues in the region. at the same time, as these leagues have grown, many of the top stars have begun to leave for more popular and competitive leagues in north america and europe. this chapter concludes in considering the future potential of sport leagues in asia, and whether the teams and leagues will be able to continue to survive in their current formats. relatively little attention has been paid to the professional east asian sports leagues in the academic literature. in part, this is because professional sports leagues in east asia developed long after similar leagues in north america and europe. humphreys and watanabe ( ) noted that east asian professional sports leagues follow a "hybrid" model of organization where these leagues adopt the rules, regulations, and structure used by prominent north american or european leagues playing the same sport. for example, the nippon professional baseball (npb) league, the top professional baseball league in japan, employs a league structure similar to that used by major league baseball (mlb) in north america. likewise, many of the football (soccer) leagues in east asia adopted promotion and relegation ladders like professional football leagues in europe and south america. although east asian sports leagues emulate north american and european league structures, they exhibit radically different ownership forms, with the predominant ownership form being significant corporate investment or outright ownership of sport franchises by industrial corporations. although some major league baseball teams are owned by large corporations-notably the seattle mariners, purchased by japanese consumer electronics giant nintendo in -some other major north american professional sports leagues, and some european football leagues, explicitly ban corporate ownership of teams. the east asian "hybrid" model of league organization has important implications for league outcomes and team operations. in this chapter we discuss this model, the operation of professional sports leagues in east asia, and also discuss some uniquely east asian professional sports like sumo and attempts by north american and european leagues to gain footholds in the large and growing east asian professional sports market. there are many reasons for the existence of corporate ownership in east asian professional sports leagues, and it is common for this ownership method to extend across different sports, no matter what ownership structure is common in that sport in other parts of the world. one reason for corporate ownership of professional sports teams in japan and south korea is that, prior to the creation of professional leagues, the strongest sport clubs often played in industrial leagues. corporations in japan and south korea formed these industrial leagues in order to help develop and manage players as if they were semi-professional athletes. in this manner, industrial leagues helped develop much of the structure in regards to professional sport franchises, as well as to foster connections to local communities in sports such as baseball, football, and even basketball. china, on the other hand, did not allow private corporations to own and operate sports teams before the s. thus, teams were often part of state backed organizations. beijing guoan, a popular football club in china, were owned by a state owned enterprise, and were often considered to be one of the teams which was to develop talent for the chinese national team. when business regulations were changed in china to allow organizations to operate sport teams as a business, the chinese football leagues moved from clubs of state-owned enterprises to clubs owned and operated by private (industrial) businesses. once private companies were allowed to purchase sport teams, many of the largest corporations in china invested in sport teams as their counterparts had done in japan and south korea. while there have been different paths to corporate ownership of sport teams in east asia, this ownership form is now widely employed throughout the region. in this chapter, we survey the state of play in professional sports leagues in east asia. the chapter focuses on football, baseball, and basketball leagues in japan, korea, china, and taiwan. note that we follow the convention of referring to the sport north americans call "soccer" as "football." while other professional sports leagues exist in east asia, these are the oldest and highest profile leagues in this region. we also discuss some factors common to east asian professional sports leagues, and the attempts by north american leagues to expand their footprint into the region. figure shows total attendance in the most recent season ( or ) for eight professional sports leagues in east asia: nippon professional baseball, j-leagues and (football in japan), the chinese super league (football in china), the chinese basketball league, the k-league (football in korea), the korean baseball championship, and the chinese professional baseball league (baseball in taiwan). professional baseball in japan attracted almost as many fans as the other leagues combined, and professional baseball in korea is the second most popular team sport in the region. nippon professional baseball is the oldest of these leagues (founded in ) . after the npb, the next three largest leagues, in terms of attendance, are the korean baseball championship and the professional football leagues in japan and china. although sumo operated professionally in japan from the mid-seventeenth century, when baseball became a professional sport in japan, initially at the club level in , and then as a full-fledged league in , the organization of nippon professional baseball (npb) did not adopt the structure of sumo, probably because sumo originated in edo period ( - ) japan and had its roots in samurai culture. rather, as had been the practice during the modernization projects in japan during the meiji period , the country turned to industrial experts to learn how to best operate their organizations and business institutions. as mlb was the predominant professional baseball league in the world at that time, it was only natural for the npb to try to emulate the success of mlb in north america. in , the npb began play with teams divided into two different leagues, similar to the american league and national league in mlb. the pacific league of npb followed the structure of the american league employing a designated hitter, and the central league of npb followed the national league by forcing pitchers to bat. since its founding, there have always been teams in the npb, with teams in each league. while the total number of teams has remained constant over time, there has been significant turnover in the composition of teams in the league. in all different teams have played in the npb; teams merged with other teams because of financial difficulties. this happened as recently as when the kintetsu buffaloes were forced to merge with orix bluewave to form the team that is now known as orix buffaloes. naturally, when these two teams merged, a vacancy was created in the league leading to the creation of the expansion tohoku rakuten golden eagles in order to have six teams in each league and maintain a balanced schedule. npb teams currently play a game regular season. salaries in the npb are relatively high. in , the average salary was about $ , (jane et al. ) . top players earn millions of dollars per season. research on asian sports economics has mostly focused on the npb, as researchers are able to draw parallels between the npb and mlb (leeds and sakata ) . prior studies into the npb focused on attendance in the league, and have found that the central league has been more popular with fans (lacroix and kawaura ; yamamura and shin ; leeds and sakata ) . there is some debate as to whether the larger attendance in the central league is predominantly because of the tokyo giants (yamamura and shin ) , or whether the positive boost in attendance provided by giants is only marginal at best (leeds and sakata ) . in this group of research studies, the npb has provided a context through which to examine an asian sport league, however it is worth noting that despite the popularity and long existence of the league, there is still but these few examinations in western research journals. the npb has strived to ensure that the league has stayed balanced and operated in an organized manner. however, while the league tries to operate like mlb, there has been significant criticism of the business practices of japanese baseball franchises. specifically, the public perception is that most of the franchises in japan are not run as independent business entities, but rather as marketing and public relation arms for the corporations which own the teams. this is evident when teams win championships or league titles; these events often coincide with commemorative sales in the stores and/or products produced and sold by the parent corporation. considerable disparity exists in team payroll and broadcast rights fees in the npb. figure shows total team payroll (major and minor leagues) for npb teams in , in billions of yen. the tokyo yomiuri giants spent around . billion ¥ on player salaries, while the next closest team in the central league, the chunichi dragons, spent only . billion on player salaries. the lowest spender in the npb, the hiroshima carp, spent only . billion. many npb clubs receive subsidies from their controlling corporations to fund payroll and operations. the tokyo yomiuri giants are believed receive an annual subsidy of around billion ¥ from the youmiuri group, the giants' owner and japan's largest media conglomerate ( rosner and conroy ) . this makes it difficult for other npb teams to compete with the giants for talent. broadcast rights fees are also unequally distributed in the npb. because the tokyo giants are owned by the yomiuri newspaper and broadcasting group, the team has been able to dictate how much all npb teams receive for each home game broadcast (rosner and conroy ) . the tokyo giants receive around million ¥ for each game broadcast, while their nearest competitors receive only ten percent of that amount for each game. rosner and conroy ( ) reported that the seibu lions only received , ¥ per game, and the chiba lotte marines , ¥ per game in . the tokyo giants received more than times the revenue from media broadcasts of their games than other teams in the league. the npb championship is decided by the japan series, where the top team from the pacific league faces the top team from the central league, much like mlb's world series. while this postseason format was used for decades, eventually the league realized that expanding its playoff structure would provide more excitement, as well as generate additional revenue for clubs and the league. this expansion of the postseason came about because regular season attendance in the league had been on the decline for several years, with the season having especially poor attendance. again, the npb emulated the playoff expansions in mlb, by adding what is called the "climax series" in each league. the climax series first made its appearance in , with the second and third place teams meeting in the first stage of the climax series. the winner of the first stage (best-of-five series) advances to the second stage where they meet the first place team in each respective league. the climax series system is somewhat complicated by the fact that the second series, between the league champion and the winner of the first-stage playoff, is a best of six format, where the team that finished in first place in the league starts with a "free" win and a - advantage. in other words, the first team to get four wins advances to the japan series, with the team that won the regular season title given the advantage of a free win. the npb postseason has been criticized for years, first for not allowing enough teams into the postseason, and second for changing the system in a way that does not give the underdog an equal chance to advance and win the japan series. despite this criticism, the climax series playoff system has been successful to some degree in creating interest around in the league, and allowing more teams to compete for the postseason championship. because of the natural ties to their parent corporations, professional baseball teams in japan are often subsidized heavily in order to make payroll and keep operations running smoothly. it is believed that only a few of the teams in npb actually turn a profit. the tokyo yomiuri giants, often compared to the new york yankees, are the dominant team in japanese baseball in terms of history, tradition, past performance, and star power. because of this, the giants have a large following through the country, and are able to profit from this popularity. in addition to yomiuri, the hanshin tigers are the only other profitable team in the league. in you gotta have wa, whiting ( ) noted that the seibu lions may be the team which are run most like a business. that is, despite the profitability of the tokyo giants and hanshin tigers, it could be inferred that these franchises are able to earn profits because of their popularity, and not because they are operated like a successful professional sports team. in his profile of the seibu lions owner, whiting ( ) notes the owner's disinterest in the sport of baseball, and demand that the seibu lions be run like a business, in the same way that the seibu railway company is operated. curiously, while the lion's owner is against having the lions being fully subsidized by seibu railway company to the same extent as other teams in the npb, the owner has heavily subsidized a professional, industry-sponsored ice hockey league, as he is fan of that sport. the business of professional baseball in japan is dependent on the economic success of the parent companies. in this sense, it is possible that economic downturns and recessions have a very negative effect on japanese professional baseball. like north american leagues, the npb employs a reverse-order draft to allocate top amateur talent to the teams. in this familiar system, the team with the worst record is given the first pick in the amateur entry draft. the idea behind this is that the worst team will naturally select the best available talent and hopefully improve the quality and talent level of the franchise. in japanese baseball, the tokyo giants have long been seen as the dominant team, and some critics claim that the team has too much control over npb rules, regulations, and policy. this claim is supported by recent changes in the npb draft system. the league recently determined that the reverse-order draft system was not fair, and decided to install a new system for allocating incoming talent to teams. under this new system, called "gyakyushimei," or reverse designation, npb teams jointly decide which player is the best amateur player in japan before the draft. this player is then allowed to choose which team he would like to play for. after the top player picks his team, the rest of the teams get to pick from the remaining eligible players in the standard reverse-order format. one problem with this change in draft regulations is that it often allows the best players to go to the championship or high paying teams like the tokyo giants. this system has again changed in recent years, with teams being picked via a lottery system to select players in the draft. for example, in the npb draft, the overall consensus was that the first player to be picked would be high school pitcher yuki matsui. five teams put themselves into a lottery to be able to draft matsui. in the end, the tohoku rakuten golden eagles, who were also competing in the japan series, gained the rights to matsui. under this lottery system, successful teams still able to obtain the rights to the best incoming player in the league, as the golden eagles were able to do in this case (nagatsuka ) . one function of sports leagues is to standardize rules and the condition of play across the league. the npb experienced a controversy in because of the characteristics of the balls used during games, suggesting that the league has problems standardizing playing conditions. was one of the worst years in the npb history in terms of the total number of home runs hit, and the league was concerned that the lack of offensive output might adversely affect attendance and revenues. the lack of offensive production, combined with increasing numbers of the best players moving to north america to play in mlb, led the npb to try to reinvigorate the game. to do this, the league decided to use game balls that were uniform across all clubs in the league; these balls were livelier than ones used in the past. in previous seasons, the npb did not have a standard league-wide sanctioned ball. rather, individual franchises were able to pick and choose which balls they would use for games as long as they met certain standards set by the league. the initial complaints over using different balls first occurred when japan was competing in the world baseball classic (wbc). players returning from the wbc noted that the balls used in the wbc were consistent, and that many of the teams in japan were using baseballs that behaved differently depending where games were played. when the npb decided to adopt a standard game ball, it made this decision without the knowledge of the teams or players, and made no official announcement about this change. in the early part of the season, offensive production increased significantly in the league, with more home runs being hit. when players, coaches, and the media became suspicious about the increase in offense in the npb, the league admitted that they had changed the balls without informing anyone. the public backlash against the league was strong, including calls for the head of the league to resign. commissioner ryozo kato resigned in september, several months after it was determined that the game balls had been surreptitiously changed by the league (calcaterra ). as evidence of the effect that the new "juiced" balls had on offensive production, wladimir balentien set a new npb record for homeruns in a season, hitting out of the park. while some noted that balentien had benefitted from the juiced ball as he had only hit combined homeruns in the previous two seasons, the npb denied that the new balls had any effect on setting a new home run record. another way the npb has copied changes made in mlb, has been to adopt interleague play, or "kouryusen" as these games are called in japan. interleague games began in as a move by the npb to try and combat declining attendance, and recapture the interest of fans, which started to follow star japanese players like ichiro suzuki and hideki matsui who moved from the npb to the usa to play in mlb. the initial interleague games were between pairs of teams in home-and-away three-game series between every team in each league. the npb found that this format created too many interleague games and diminished the traditional rivalries that exist between league members, and reduced all of the series to two games. to further add excitement to the interleague games, the npb decided that, instead of spacing interleague games out through the season, as is done in mlb, all interleague games would be played in a span of about two months. under this system, all teams would play their interleague games simultaneously, and the team with the best record would be awarded the "interleague" title. from through , pacific league teams have won the interleague title every year, indicating that there may be a lack of balance between the leagues. in either case, the npb has become a more dynamic organization within the last decade. where in earlier times the organization had been very resistant to change, it is now the case that the organization has attempted many changes in the structure, scheduling, rules, and play to try and make the game more attractive. in this, the npb has realized that while they still be the most attended sport in asia in regards to total attendance, the support for their organization has been worn down by the popularity of other foreign leagues on satellite television, as well as the growth of domestic soccer in japan in the form of the j-league. the j-league, the top tier of professional football in japan, began play in . the j-league was rushed into existence when japan was competing for the rights to host the fifa world cup, which were awarded in . the j-league (like major league soccer in the usa) was created because fifa said they would only allow countries to host a world cup if they had an established professional football league in operation. the j-league was initially composed of clubs of teams from an industrial league known as the japan football league. the j-league began its inaugural season with ten clubs; most of these clubs were located in the tokyo/kanto region in eastern japan. when the j-league began play, it lacked a lower division because of the push to get the league established for the world cup bid. thus, in the early years, the j-league did not have a promotion and relegation system in place. the first season of the j-league proved to be a great success with an average attendance of almost , fans per game. however, this initial novelty effect dissipated quickly, and by the mid- s the j-league was averaging , fans per game. though much of the structure of the j-league was modeled on european football leagues, the league actually divided the season into two parts, like the apertura and clausura ("opening" and "closing" in spanish) employed throughout professional football leagues in latin america. the champions of the first half of the j-league season would face the champions of the second half of the season to decide the league champion. if the same team won both the first and second half, that the team would be named the league champion with no playoff. the j-league eliminated this system in , adopting a single season format in to better emulate the european league structure. additionally, by moving to this format, j-league teams had schedules that better matched the rest of the professional football leagues in asia. this coincided with the asian football confederation (afc) schedule, and also providing easier scheduling for j-league teams competing in international competitions like the asian champions league. since parent corporations subsidized j-league teams, the league needed a longterm strategy for operation of franchises and the league as a whole to compete with professional baseball. the j-league instituted several measures to help expand the popularity, reach, and sustainability of the league. first, in , the league created a two-tier system with the top league renamed j and the second tier named j . at this time, j expanded to teams, and j had teams. j would eventually expand into its current -team format, with teams in j . additionally, as part of the growth plan for the league created by the japan football association (jfa), the j-league started a plan to establish and expand lower level football leagues to create more players and clubs at the grassroots level. the japanese promotion and relegation system is now setup so that the teams can move from low-level prefectural leagues, up to japanese regional leagues, up to the japan football league, then to j and finally to j . in this way the j-league and jfa created stronger ties and links to regions across japan, and fully adopted a european promotion and relegation ladder. in , it was announced that the j-league would make additional structural changes to the league. in september of , it was decided that starting in , the league would return to a two-part season, to heighten interest. this change comes as many young japanese football players have begun to leave the league for better, higher paying leagues in europe; the j-league is trying to create interest in the game, but not trying to promote and retain domestic talent in the league. the japanese professional sports leagues are clearly some of the more successful leagues in east asia. while they have set the standard for business practices, league organization, and professionalism, the model of corporate subsidization and financing may not be an ideal model for other asian leagues to follow. additionally, as the chinese economy has continued to grow, the corporations owning and sponsoring japanese professional teams have found themselves competing, and losing ground to professional teams in china. once many star players throughout east asia would head to japan to play baseball or football, but now young asian players set their eyes on moving to the usa to play professional baseball, and europe to play professional football. this movement of young talent presents a great challenge for professional sports leagues in japan, korea, and china. while japanese leagues clearly understand that losing young players to us and european leagues is an issue, the response by league administrators to date has not been adequate to stem the outflow of players. one of the biggest problems facing professional sports leagues in asia is to retain domestic talent. the history of professional sport in japan can be traced to the traditional sport of sumo wrestling. in this highly ritualized sporting event, two men compete to push the other out of a ring drawn on the ground, or down into the ground inside the ring. sumo is a professional sport, as the winner of sumo matches win large cash prizes. sumo also employs a complex ranking structure where it is important to have a winning record in order to move up into higher tiers, with stiffer competition and larger prizes. the current format of professional sumo competition dates back to the s, when it was decided that there would be six sumo grand tournaments each year. three of these tournaments are held in the main sumo hall, known as ryogoku, in tokyo; the other three tournaments are held in the cities of osaka, nagoya, and fukuoka. a sumo grand tournament runs for days, with all of the higher-ranking wrestlers scheduled to fight once a day on each day of the tournament, and lower ranking wrestlers fighting about seven bouts during the competition. professional sumo tournaments use a ranking structure, where individuals who win eight or more matches become eligible to move up in rank, while those who lose eight or more matches risk demotion. through this system, wrestlers are able to advance from lower divisions such as the "makushita" (literally, below the curtain), all the way up to the top makuuchi division. in makuuchi, wrestlers who win the matches advance through a series of titles from komusubi to seikwake up to ozeki. above the ozeki title is the rank of yokozuna (grand champion), which is reserved for only an elite few wrestlers who are able to constantly display championship-level sumo wrestling in every grand tournament. after becoming an ozeki, a wrestler must usually win at least two tournaments in a row to become a yokozuna. this feat is made especially difficult because an aspiring yokozuna will face many other ozeki and potentially one or more yokozuna to win a grand tournament. once a wrestler has attained the rank of yokozuna, they are expected to continue to win bouts, and if their performance falters, they are expected to retire from sumo rather than continue wrestling. sumo wrestlers are compensated for their performance in several ways. sumo wrestlers in the makuuchi division are paid a salary of around $ , a month for low-ranked wrestlers to $ , a month for yokozunas. in addition, wrestlers earn income from bonuses provided by corporate match sponsors. notably, sponsors will pay more for matches involving highly ranked and more popular wrestlers; so much of this bonuses money goes to higher-ranking wrestlers. in some cases, a match may have no sponsors; in this case there will be no payout to the winner. finally, sumo wrestlers earn other bonus money based on their annual performance, especially the number of tournaments in which they have winning records. while organizers claim this system encourages high performance, it has also generated incentives to fix matches. in , japanese police seized the cell phones of many coaches, trainers, match organizers, and some wrestlers (hongo ) . the ensuing investigation uncovered evidence of a large match-fixing ring. the japan sumo association previously denied all allegations of match-fixing, including allegations dating back decades. as the investigation continued, rumors that several wrestlers were involved in match-fixing sent the japan sumo association into crisis mode. with the evidence of match-fixing surfacing, closely following a national scandal where sumo wrestlers were found to be betting on baseball games with bookmakers run by the yakuza (japanese organized crime syndicates), sumo's image took a beating in the eyes of fans, sponsors, the government, and media. the march grand sumo tournament was cancelled, only the second tournament was cancelled since the end of world war ii. eventually, a group of wrestlers admitted they had fixed matches, and were forced to retire from the sport. despite this positive development, the japan sumo association continued investigations into match fixing and ultimately concluded that they were possibly more wide spread than previously thought. these scandals have led to a decline in the popularity of sumo. with the rise of other professional sport leagues, sumo has lost much of its attraction, especially among younger japanese who have grown up watching both professional baseball and football. compared to japan, professional baseball in south korea is relatively new. the league, previously the korean professional baseball league, now called the korea baseball championship, was founded in , just year after the founding of the korea baseball organization (kbo). it is a bit confusing in translation but "kbo" is now used as the brand name of the league as well; it now means both the league as well as the governing body. we will use kbo throughout and the correct context as needed. the kbo was initially composed of six teams, and was not able to divide into divisions, like the npb and its model, mlb. like in japan, kbo teams are all owned or affiliated with corporations or industrial groups. in , the average salary for players in kbo was $ , per year. foreign players can be paid up to $ , per year, and rookie players earn about $ , . foreign players and rookies are subject to a salary cap. the kbo employs a playoff system different from that used in mlb or npb. because of the single league structure, the league holds several rounds of playoffs. in the first round, known as the "semi-playoff" the third and fourth place teams play each other in a best-of-five series. the winner of the first series then plays the second place team in the league in another best-of-five series. finally, the winner of the second playoff series faces the first place team in a best-of-seven series, the korean series. the growth of baseball in korea includes growth in the number of teams in the kbo. while many expansion franchises have joined the league, there have also been changes in the sponsor company of many of the existing teams, as well as teams leaving the league. the kbo has grown from the original six teams, to nine teams currently in the league. the league plans to expand to ten teams in . currently, teams in the kbo play regular season games, a number that has been reduced because of expansion. because of its small size, kbo teams play each other many times in the regular season. before the league expanded to nine teams, the league played a game regular season. after expanding, each team played every other team times for a game regular season schedule. if teams played each other times, this would add an extra eight games into the schedule, which would have made the regular season longer than the kbo wanted. founded in , the k-league is the second oldest professional football league in east asia (after one formed in hong kong in the early s). originally this league was known as the korean super league and was composed of five corporate sponsored or owned teams. the k-league maintained this structure until , when the league was renamed the k-league, and expanded from to teams. in , the k-league moved from a single league structure to a two-league format with promotion and relegation. the top division was renamed to the k-league classic, and the second division the k-league challenge. the average salary in the k-league was about $ , in . before , k-league salaries were not public. by adding a promotion and relegation component in , the k-league moved to a more open structure, but has yet to become a fully open league like the j-league or european leagues. currently, teams are promoted from a semi-professional league called the national league to the k-league challenge, and from the k-league challenge to the k-league classic. the concept of promotion and relegation is not entirely new to the league in . in earlier years, the league employed a promotion and relegation system under which teams in the national league could be promoted under certain circumstances. despite this possibility, many eligible teams rejected promotion. like other east asian leagues, the k-league has experienced periodic match fixing scandals. while the k-league was not the first league in east asia to experience match fixing, it was one of the most widely publicized cases of corruption in sport. in the k-league match-fixing case, several players, coaches, and referees were found to be the part of a group that conspired to fix matches, in cooperation with bookmakers and organized crime. fifa ultimately helped with the investigation (duerden ) . multiple matches in both the k-league and k-league cup were fixed in and . the scandal started with the suicide of a player, yoon ki-won, involved in the match-fixing ring (kim ) . at the conclusion of the investigation, former and current k-league players were sentenced to prison and fined, and another individuals were forced to pay fines. duerden ( ) noted that south korea has one licensed sports book, but that there were between and illegal gambling sites in the country at the time of the match-fixing scandal. while the police and league have worked to crackdown on these sites, the reorganization of the k-league could partly be traced to the need to rebrand the league after these match-fixing scandals. the chinese professional baseball league (cpbl) is the top professional baseball league in taiwan. the league was founded in , and currently has four teams. like the npb and kbo, large taiwanese corporations own all teams in the cpbl. the cpbl has undergone contraction over the last years, and had six teams in the s. like mlb teams, taiwan has a minor league system with four teams; each of these teams is owned and operated by a cpbl team as a "farm" team. cpbl teams play a game regular season. in , the average salary for a taiwanese player was $ , per year and the average salary for foreign players, mostly former us major or minor league players, was $ , per year. in the late s, the cpbl experienced a match fixing scandal. three members of the china times eagles were convicted of match fixing, many other players on other teams were banned from the league, and the eagles were disbanded. attendance fell to half its pre-match fixing level for years after this scandal. like many other professional sports teams in east asia, some evidence suggests that cpbl teams operate at a loss and can only survive financially through subsidies provided by their parent corporation. interestingly, the cpbl faced a rival league, the taiwan major league (tml) that formed in with four teams and competed with the cpbl for players. the owner of the media conglomerate that broadcast cpbl games from - formed the tml. the rival league was formed after a contractual disagreement about future cpbl broadcast rights. a total of players under contract to cpbl teams left to play in the tml. all four teams in the tml were owned and operated by the media conglomerate that formerly televised cpbl games. in two of the four tml teams were merged into the cpbl. in , former mlb star manny ramirez signed a contract to play for the eda rhinos of the cpbl. his salary was $ , per month. attendance increased significantly while ramirez played in the cpbl; home attendance at games played by the rhinos increased from about per game to more than per game for the three months that he played in taiwan, and the other three teams saw large increase in attendance at games against the rhinos. however, ramirez returned to the usa after months, and attendance declined more than % league-wide after his departure. mao zedong was reportedly a basketball fan, and his interest in the game may have played a significant role in the development of the sport in china. there exists rare video footage of mao attending a basketball game and appearing to enjoy himself. as basketball was a sport of interest for the founder of the people's republic of china, it was only natural that a governing organization for the sport was created in , shortly after the establishment of the people's republic. this organization, the chinese basketball association, has the same name as the chinese basketball association (cba), a professional basketball league that began play in . for simplicity, this paper will refer to the professional league as the cba, and the overarching organization governing basketball in china as the chinese basketball association. the popularity of basketball in china reportedly comes not only from chairman mao's love of the game, but also from rules governing the congregation of individuals in the country. in order to prevent large groups of people from congregating together, china once had stringent rules about the number of people who could congregate together in a group. under this rule, enough people could congregate to play a game of basketball without running afoul of the law, but a full -a-side football match constituted an illegal gathering. since only ten people are needed to play a game of basketball, and the laws of the country allowed the sport to be played without fear of repercussion, basketball flourished in china. professional and semi-professional basketball leagues in china emerged over the last three decades. in , the cba began play as china's top professional basketball league. as of , the cba has teams divided into two divisions. cba teams play a -game regular season. cba teams have corporate sponsors that appear as a part of the name of the team, but these corporations do not own the teams, and team sponsors change frequently. the cba does not follow the common east asian practice of direct corporate ownership of teams, probably because of the planned nature of the chinese economy. the cba postseason is modeled exactly on the nba. the top eight teams in the regular season advance to the postseason, which consists of two rounds of best-offive series (quarterfinals and semifinals) and a best-of-seven championship series. playoff teams are seeded - and higher seeds play lower seeds. cba player salaries and team revenues and expenses are not public information. however, a player for the shanghai sharks ' " center zhang zhaoxu became a free agent after the season and has reportedly been offered a multi-year deal by the xinjiang flying tigers that would pay him $ . million per season. together with the fact that many foreign players, including former nba stars stephon marbury and gilbert arenas have played full seasons in the cba suggests that average salaries are substantial in the league. other leagues that were not necessarily rival leagues also existed at various points in time. these other leagues include the national basketball league (nbl), which is still in existence and will soon have a two-division structure, as well as the now defunct chinese new basketball alliance (cnba). the nbl has teams that once constituted single tier structure, but recently was reorganized by the chinese basketball association into a semi-professional league with ten teams in the top division, and six teams in the bottom division. like the system in europe, these two tiers use promotion and relegation to move teams between them. although the nbl is sanctioned by the chinese basketball association and follows a european-style promotion and relegation model, the nbl is a minor, semiprofessional league. the cba has adopted a north american closed league model like the national basketball association (nba). this is part of a national plan for developing basketball talent and promoting domestic growth in the sport. the chinese basketball association has developed an organizational model adopted directly from the usa. in the usa, players are developed through a system of high school teams, amateur athletic union (aau) teams, and college teams. in china, the best players had been developed under the direction of the chinese basketball association or by local basketball clubs. by operating in this manner, chinese basketball was following a structure that took parts of the other communist regimes, as well as european club sport. while the cba still develops a lot of players through club youth teams (for example, nba players yi jianlian and yao ming were both developed on cba club rosters), the country has begun to experiment with different methods to develop basketball talent. the nbl operates as a semi-professional league, providing china with a lower level system that can be used to develop young players. at the same time, the chinese university basketball association (cuba) was created to emulate the national collegiate athletic association (ncaa) basketball system in the usa, using universities instead of clubs and the organizations that develop basketball talent. the cuba represents an interesting organizational case study. for most of its existence, cuba was an organization that allowed college students to compete against teams from other schools in basketball, but was not viewed as a mechanism to develop high level basketball talent. cuba observed the success of the ncaa in developing young talent, and in generating large revenue streams, and decided to emulate the ncaa approach. the cuba is an independent organization, and technically is not controlled or related to the chinese basketball association. despite this, the cuba has focused on increasing revenues and developing talent like the ncaa, including holding an annual bracket style knock-out tournament. the last four teams in this tournament play in a nationally televised event called the cuba final four, just like in the ncaa. the cuba hopes to continue to develop basketball talent and promote domestic basketball competition at all levels. cuba's long-term goal appears to be a system exactly like the ncaa-nba relationship in the us. like japanese baseball and football, the cba faces the problem of top chinese players moving overseas to play in the nba and other professional leagues around the world. yao ming started this migration. however, no chinese player has fully reproduced the success yao ming had in the nba. the cba competes directly with the nba for fans watching games on television. although the cba features a high level of competition, it is hard for any league to compete against the star power and excitement of the nba. in order to build popularity, the cba has also begun to import foreign basketball players to raise the level of domestic play. the cba has signed many former nba stars including stephon marbury, gilbert arenas, tracy mcgrady, and steve francis. through these star players, the cba has managed to develop a large following among the basketball fans in china by providing some nba level talent, while still being including top domestic players. although mao zedong was an avid fan of basketball, he passed away two decades before any of the professional chinese leagues came into existence. mao's successor deng xiaoping is reportedly a fan of football. it may be a coincidence, but it is interesting that after the development of basketball in china, the next sport to develop professionally was football. the chinese super league (csl), is the top professional soccer league in china. the league was formed from the remnants of the jia-a, an earlier professional league that was rebranded because of concerns about the business practices and management of clubs and the league as a whole in the early s. jia-a, the original professional football league in china, was formed from club teams that were primarily controlled and owned by government-backed businesses. in this sense, the jia-a was formed as a semi-industrial league, which was backed by the chinese central government. jia-a appeared to be headed towards success until a series of match fixing scandals rocked chinese professional football in the late s. the csl has a structure similar to the other football leagues in asia, following a hybrid model that copies the organization of professional football in europe, while following the east asian practice of corporate ownership of teams. in addition to the csl, professional football in china includes two lower leagues, china league and china league , on the national promotion and relegation ladder. china also holds an open domestic competition, the fa cup. csl teams are either owned by state-owned industrial conglomerates, like beijing guoan which is owned by citic group, a state-owned investment company, or by individual industrial tycoons, as is increasingly common in european football leagues. salaries appear to be high in the csl. like other east asian leagues, player salaries are not public information. but media reports indicate that the average salary of a domestic csl player in - was . million yuan, $ , at yuan to the dollar (compton ) . the average salary of international players in - was . million yuan, or $ , . a number of high-profile international football players have signed contracts to play in the csl for significant sums of money. didier drogba, a former striker for chelsea of the english premier league, reportedly signed a contract with shanghai shenhua paying him $ . million per season for - ; a number of other international players have csl contracts paying more than $ million per year. payrolls have increased significantly in the csl in the last few years. compton ( ) reported that league wide payroll doubled from $ . to $ million dollars from to . predictably, most teams in the league reportedly lose money. guangzhou evergrande, a very successful csl team owned by billionaire real estate tycoon xu jiaying, reportedly spent million yuan on salaries in and reported only million yuan in ticket sales (compton ) . media rights fees in the csl are not known, but the league recently signed an agreement with european sports broadcaster eurosport to televise csl games in europe. the csl has also experienced match-fixing scandals. in the past, csl teams, players, and officials of the chinese football association (cfa) have been convicted of fixing matches in csl league play. the most famous csl match-fixing scandal is known as the "black whistle" scandal, took place in . even in , there are still trials ongoing and punishment being handed out to the teams and individuals across china. the match-fixing scandal reached as far up as the head of the cfa, who was dismissed from his post and subsequently arrested in , when police found evidence of his role in match fixing in the csl. these scandals significantly reduced fan support and interest in the league. to regain the trust of chinese football fans, the central government instituted a series of reforms in the mid- s. the league was renamed the chinese super league in order to break ties with the past corruption in jia-a. since these reforms, the csl has witnessed historic growth in terms of performance of clubs, attendance, and investment into professional football. several large corporations have purchased or sponsored csl teams. the most prominent example is the purchase of a guangzhou-based club by the evergrande real estate group. jiayin xu, a regular on the annual forbes top richest people in the world list, controls the club, renamed to guangzhou evergrande, as well as the real estate corporation that oversees it. with a multi-billionaire's backing, guangzhou evergrande rocketed from the second division to the top of the csl table in a single year. the financial support was so great that the team went the entire season without losing a game. this did not satisfy xu, who fired his coach, brought in even more player talent, and hired former italian world cup winning coach marcello lippi to manage the team. from this point, guangzhou has become one of the top teams in asia, despite being relatively unknown just three years ago. additionally, guangzhou evergrande became the first chinese team to win the afc champions league final in . the influx of money into chinese professional football has been a blessing to the domestic game which has witnessed its largest crowds ever; the csl now has the highest average attendance of any football league in east asia and has surpassed the j-league and k-league in terms of domestic popularity; of course china has a much larger domestic population than either japan or korea. with that said, attending csl matches appears to be more of a middle-class activity in china, in terms of the cost of attending a game and the time it takes to travel and attend a match. while the csl has grown in popularity in recent years, the status of the chinese national football team has gone in the opposite direction. unlike in korea and japan, where growth in domestic leagues was accompanied by increased success by the national teams, in china the national team reached its apex when qualifying for the fifa world cup, and has enjoyed little international success since. the rise of the csl, and the concomitant decline of the chinese national team, raises an interesting question. what factors caused the domestic league to improve yet had no impact on the success of the national team? one possible answer is that china still has not put an effective grassroots youth development system in place that generates homegrown talent. the improved performance of the csl in continental competitions has come alongside increases in the number of foreign players allowed on csl rosters. in this, the drive for success in the csl has potentially come at the cost of developing domestic young talent, as aging international players take roster spots on csl teams that would have gone to young domestic players. as the csl has had more success and invested more money into payrolls, they have brought in famous star players from abroad such as didier drogba. while the drogba deal ended with the player leaving china after only playing a short time, it is one example of how far many csl clubs have gone to attract fans and win league matches. much of the focus in china is placed on domestic professional football, and not on international competition. this emphasis is interesting in light of the importance the chinese central government placed on performing well in international competitions like the olympic games. as revenues in the national football league (nfl) began to grow in the s, the league turned its eyes toward building a global audience for the sport. the nfl realized that expanding beyond the traditional north american audience could translate into increased revenues and enhanced brand values for teams, players, and the league as a whole. the nfl began its international expansion with a series of games called the american bowl, a series of pre-season games begun in in countries outside of the us and canada. the series initially included games in london, but it soon included games in japan. games broadcast during us primetime were held in large venues in japan in the early hours of the morning. this scheduling practice did not attract many japanese fans to the nfl. another problem was that the nfl's attempt to build a global audience was based on playing exhibition games in japan and europe, while other north american leagues like the national hockey league (nhl) were playing meaningful regular season games in these countries. the american bowl ended in . after the cancellation of the american bowl in , the nfl replaced it with the nfl international series in the rest of the world, staging regular season games abroad. but the nfl failed in its only attempt to stage a regular season game in asia, the "china bowl" attempt in beijing. the game was scheduled one year before the start of the beijing summer olympic games to build nfl visibility in china and around the world. interest was high for one of the scheduled competitors, the new england patriots, since they had already established business operations in the country. however, after the game was rescheduled twice, and eventually cancelled in , the patriots and other nfl teams eventually pulled almost all of their operations out of china. the failure to gain footholds in east asian markets by the nfl is not a unique outcome. in a book examining the growth of domestic soccer in china, simons ( ) notes that even large successful european football clubs have been unsuccessful in bringing their sports/leagues to china. however, the nba has adopted a more focused operation in china focusing on building grassroots interest among fans. the joint nba/cba coaching program has trained more than chinese basketball coaches and in an nba run training center, the dongguan training center, opened and has trained more than elite - year old players. in , the asian football confederation (afc) consolidated all three regional competitions and organized them under the single banner of the afc champions league (an earlier attempt under the same name floundered and died in ). modeled after the uefa champions league, this competition had a group stage and teams advancing to knockout rounds after group play, with a home-and-away championship format. the initial launch of the afc champions league appeared to be successful, but the season was canceled because of the sars virus outbreak in east asia, which made travel for the competition impossible. after a year break, the afc champions league returned in with clubs competing in the group stage. learning from the excessive travel budgets and other travel-related issues from previous competitions, the champions league divided teams into regional groups. with these changes, more teams and leagues were willing to join the competition, which grew in and again in when it moved to its current format with teams participating in group play. one final change that was made in , when the competition moved away from a home-and-away final, and moved the finals to a single championship game played at a neutral venue. between and , the neutral venue finals were hosted twice in japan and twice in south korea. both matches in japan featured teams from south korea and the middle east, and the interest by japanese fans in these matches was low, with attendance around , for both finals matches. the and finals had a greater boost in attendance, with around , attending both matches that again featured south korean and middle eastern clubs. the single venue final was clearly not a format that would guarantee large crowds and media success. the key to large crowds at the afc champions league matches was teams from the host nation being present in the match. in , the afc champions league returned to a home-and-away finals format, with guangzhou defeating seoul to decide the winner of the ultimate title in asian club football. although the growth and success of the afc champions league can be attributed to the explosion of professional football in east asia starting in the mid- s, it is important to note that west asia has also played an important role in the growth of the competition, and the afc as a whole. there has always been a difficult coexistence between east and west asian leagues in the afc. regional differences in terms of culture and race have generated a rivalry between east and west asian league champions that has spilled over to the management, organization, and leadership of the afc. winning the afc champions league has become important not only for club and country, but also in signaling the football and business prowess of the region. in terms of sponsorship, emirates airline based in west asia and nikon and asahi in east asia have played heavy roles in funding the competition. competition for business and prominence in asian markets has helped to drive organizations to bid higher to be official partners and sponsors of the afc. the asian based marketing company world sport group (wsg) has also invested heavily into both the afc and the afc champions league, understanding the potential importance the competition has with audiences across asia. the wsg reportedly signed an eightyear sponsorship agreement with the afc that was valued around $ billion. as the afc champions league has continued to grow, the competition and the federation overseeing all football in asia has profited. the payoffs for the afc champions league are structured like those in the uefa champions league. teams are paid based on how far they progress in the competition. while the pot of money for the uefa champions league continues to grow, there has been fluctuation of the total sum of money paid out in the competition. the amount offered in was valued around $ million; five times the amount that was offered the previous year. this jump in the total tournament payout was partly due to increased sponsorship deals. however, the afc reduced the total payout in subsequent years, with the total amount given out to clubs each year estimated at around $ -$ million. more than $ million is awarded to the winner of the tournament. while the afc has awarded larger sums of money to the winning club over time, many early rounds still have more money given out in travel subsidies for teams than for actually winning or advancing in the tournament. the addition of australia to the afc in increased travel costs for east asian clubs. possibly, the most interesting development in the afc champions league (and potentially the afc as well) has been the emergence of powerful chinese professional clubs backed by domestic billionaires. the emergence of strong east asian football began at the national team level with south korea, followed by japan who would develop a professional league which set the standard for professional football in east asia. now that chinese teams have the backing of deep-pocket owners who want to field winning teams, it appears that china would be poised to dominate professional club football in east asia. there have been winners of the afc champions league and the earlier pan-asian club competitions. though professional leagues are relatively recent developments in east asia, the clubs from this region have won of titles, with another teams having been runner-up. in the afc champions league, south korean and japanese clubs have won six of the ten titles, and have been runners up three times. with the final to be played between a chinese and korean club, it appears that the poser in the afc has shifted from the western asia to the eastern asia. a boom occurred in professional sports leagues in east asia over the past three decades. not only did the number of leagues and franchises continued to grow, but the revenues, financial sponsorship and backing of professional sport organizations has continued to grow as well. however, this growth has not generated stability, as a significant amount of reorganization of franchises, leagues, and pan-asian championships have also taken place. although it is natural for some upheaval to take place when an industry undergoes rapid growth and expansion, there are some reasons to believe that east asian professional sports leagues and teams may experience some problems in the future. there has already been evidence of some teams having difficult times being able to stay financially solvent, especially for teams in smaller markets or with relatively small corporations backing them. the city of fukuoka, japan has experienced several cases of financial difficulties with sports teams. the npb team in fukuoka, the softbank hawks, was purchased by the softbank corporation after their previous owner; the daiei corporation underwent financial distress. as noted earlier in this chapter, professional teams in japan have not always focused on proper business operations. by treating teams as part of a corporation's marketing or public relations department, it is only natural to expect sport organizations to experience financial loses. thus, when the parent corporations experience financial problems, the sports franchise is usually one of the first things sold, or in some cases, abandoned. in the fall of , it was discovered that the professional football club in fukuoka (avispa fukuoka) had been struggling financially and had lost about half a million us dollars in the previous year. avispa fukuoka is a club with no corporate ownership and is a publicly traded stand-alone corporation. because avispa lack the power of a parent corporation to fully sustain them, the losses have hit the club hard, and there is question about whether the club will be able to remain in j-league. to date, there has not been any other j-league club which has gone bankrupt and been removed from the j-league, and avispa fukuoka are the closest team in the league has come to such a dire financial situation. the presence of teams owned and operated by industrial groups or large corporations is an interesting feature of east asian professional sports leagues. other leagues around the world have not adopted this ownership form, although corporations owned some early teams in the national football league; for example, the green bay packers were started by a large meat-packing firm. this ownership form either disappeared early or was not present in most of the professional sports leagues in the us and europe. it remains to be seen if this ownership model can support significant growth in leagues to make east asian football and baseball leagues competitive with the us and european leagues they modeled their competitions on. however, the emergence of billionaire team owners in china may signal increased problems there, and in other east asian leagues if chinese billionaires turn their attention to buying teams in other nearby countries. the presence of deeppocket owners willing to spend large amounts on start players and sustain large operating losses has led to problems in many european football leagues, in terms of on-pitch outcomes and increased player costs across all leagues as demand for star players drives up salaries all salaries. if a sufficiently large number of east asian teams are bought by "sportsmen owners" like russian billionaire roman abramovich, owner of the chelsea football club in england and united arab emirates billionaire sheikh mansour bin zayed al nahyan, owner of manchester city football club in england, these leagues could experience financial problems like those currently taking place in europe. the prevalence of corporate-owned teams could exacerbate this problem, if the corporate owners sell teams or refuse to compete with billionaire owners of other teams. east asian leagues have also experienced a large number of match fixing scandals over the past years. in part, these scandals can be attributed to the relatively low salaries paid to players and referees in these leagues. forrest and simmons ( ) showed that low salaries were key factors in explaining match fixing incidents, as players and officials with lower salaries have a lower cost of fixing matches, and a higher expected net payoff. as salaries in these leagues rise, match fixing should decline as players are less likely to risk loss of these high salaries if they are caught fixing matches. npb commissioner ryozo kat resigns over juiced ball controversy hope and pay soar in china's soccer league k-league in crisis as corruption probe widens sport and gambling sumo-rigging born of necessity? japan times business and finance of international sport leagues does salary discrimination by nationality exist? the case of nippon professional baseball league incheon united goalkeeper dies rule changes and competitive balance in japanese professional baseball take me out to the yakyushiai: determinants of attendance at nippon professional baseball games eagles win rights to coveted lefty matsui the impact of the flat world on player transfers in major league baseball you gotta have wa the influence of a leader and social interaction on attendance: the case of the japanese professional baseball league convergence, clustering, and their effects on attendance in the japanese professional baseball league key: cord- -d s akd authors: akabayashi, akira title: a brief history of bioethics in japan date: - - journal: bioethics across the globe doi: . / - - - - _ sha: doc_id: cord_uid: d s akd in this chapter i look back at the history of bioethics in japan, which can be divided into three phases: phase i, introduction ( – ); phase ii, development ( – ); and phase iii, the recent past ( –present). phase i marks the period when the concept of bioethics that originated in the west came to japan. it was also when japanese society faced its first difficult bioethical issues: namely brain-death and organ transplantation. other issues emerged during this period, particularly pertaining to death, such as end-of-life medical care and euthanasia. in phase ii, the problems shifted to those pertaining to the beginning of life, such as the moral status of the human embryo. as well, during this period the government implemented ethical guidelines for research ethics. during this period, social awareness of bioethics increased, and bioethics education began to appear not only medical education, but also within high school curricula. in phase iii, japan began to tackle its own ethical issues, such as enhancement, regenerative medicine, neuroethics, public health ethics, and precision medicine. some of my thoughts concerning projections for the future are discussed at the end of this chapter. in this introductory chapter, i consider the current period (ca. ), look back on the history of bioethics in japan over the past years, and finally, look briefly toward the future. first, i present an overview of the types of problems that have been handled in the fields of bioethics and medical ethics in japan since the s. i begin my discussion in this era because modern bioethics began its development in japan in the early s. i divide this time period (beginning in the early s until around ) into three parts, corresponding to phase i, introduction: - ; phase ii, development: - ; and phase iii: the recent past ( -present) . no significant distinction is intended between use of the terms "bioethics" and "medical ethics." for full description, refer to references [ ] [ ] [ ] . this chapter is a brief summary of those three papers and some further considerations. in japan, the issue of brain-death and organ transplantation was highly influential to the development of bioethics (see chap. ) . beginning in the s, nationwide debates began as to whether brain-death constituted human death, and whether organ transplantation from a brain-dead body should be permissible. these issues were discussed in medical circles, religious groups, political groups, the media, and among the general population. with the establishment of a commission on braindeath, and the enactment of the organ transplantation law, organ transplantation from a brain-dead body was finally deemed permissible, with strict conditions. specifically, the clear and written expression of the intent to donate from the organ donor ( years or older) as well as the family's consent were both required. politically, this marked a major milestone. several positive outcomes were achieved, including discussion on the definition of death and japanese views on life and death; this set a healthy tone for how bioethical arguments should be handled in japan from that point forward. at about the same time, the idea of informed consent began to emerge as an issue in clinical settings (see chap. ) . informed consent in the clinical context entails an act in which "medical caregivers provide sufficient explanation to those with sound judgment capacity and ensure that the patient understands, while the patient then offers consent of their own volition." today this is considered common protocol, but such was not always the case in japan. there was even extensive debate on how to translate the english term "informed consent" into japanese; today, the term "infomudo konsento" is used, but is presented in katakana, the phonetic alphabet used for foreign terms and names. in the course of this discussion, those arguing to protect patient autonomy, basing their arguments on self-determination and the right to know, were in a dominant position. others countered that argument, stating that prioritizing individual autonomy so heavily is unsuited for japan. however, an explanation of the patient's condition has now been systematically added to the treatment plan at the time of hospital admission and discharge, such that it is covered by health insurance [ ] . informed consent is therefore a common theme that has been integrated into medical care. this flow of events necessitated a change in paternalism among physicians. it also led to the medical record disclosure system based on the private information protection law in . one other major event during phase i was the tokai university euthanasia incident. in this case, a physician, in response to a clear request from the patient's family, administered potassium chloride (kcl) to a terminal cancer patient [ ] . he was found guilty of murder and given a suspended sentence by the yokohama district court in . the trial was an unprecedented case in which a doctor performed euthanasia. as the court decision presented the conditions that would allow for active euthanasia in the obiter dictum, it was erroneously relayed to other countries that active euthanasia was legally permissible in japan. reports of physicianassisted suicide emerged from abroad, and terms such as euthanasia, mercy killing, and dying with dignity were used without clarity. currently in japan, the act of a medical caregiver administering muscle relaxants or kcl to a patient, leading to the latter's death, carries with it a high probability that the medical caregiver will be placed on trial for murder. notably, unlike several other countries, japan has not legalized active voluntary euthanasia. in addition, with the increased popularity of hospice or palliative care, fewer cases of "withholding treatment" requested ahead of time by patients are legally problematic. however, the issue of "treatment withdrawal" (removal of an artificial respirator) from a terminal patient has yet to be subject to legal resolution (see chap. ). there are three characteristics of phase ii. first, the topics of discussion shifted from issues pertaining to the end of life (brain-death, end-of-life medical care, and euthanasia) to those dealing with the beginning of life, particularly the moral status of the embryo and problems related to reproductive medicine. second, numerous policy guidelines and legislation were produced in the fields of life sciences and . phase ii: development ( - medical care, paving the way for the establishment of a framework for policy decisions. third, bioethics came to be socially acknowledged in the fields of medical education and research. in the second phase, topics in medical ethics shifted to issues pertaining to the beginning of life. among them, the heightened discussion surrounding the moral status of the human embryo became particularly significant. at this time, research on embryonic stem cells (hereafter, escs) had become widespread. human escs have the capacity to differentiate into all kind of tissues and all cell types, and there was great hope for these cells in the field of regenerative medicine. however, in order to create human escs, one must destroy a fertilized egg (embryo) that has the capacity to become a human individual in the future. if one perceives that human life is present in an embryo, then this type of use of embryos cannot be approved. in , the council for science and technology policy and the experts panel on bioethics compiled the document "basic principles concerning the handling of human embryos." this document stated that the human embryo is not a person per se, but it is also not an object. the document instead uses the phrase, "sprout of human life." the writers conclude that a human embryo is worthy of respect and requires careful handling (see chap. ). fundamentally, "the guidelines for derivation and utilization of human embryonic stem cells" and "the act on regulation of human cloning techniques" take the same stance as "the basic principles concerning the handling of human embryos." given that the moral status of the human embryo is a topic of deep religious and political debate in other countries, it seems that japan has actually handled this issue with relative ease. however, it remains a mystery as to why abortion-another debate that should be begging many questions about the moral status of an individual prior to birth-did not gain as much momentum as that surrounding the human embryo. at the end of the twentieth century, numerous governmental ethical guidelines (ethical guidelines for human genome/gene analysis research, ethical guidelines for epidemiological research, and ethical guidelines for clinical research) as well as a number of laws (the organ transplant law, the act on regulation of human cloning techniques) came into the fields of life science and medical care. many of these were drafted by governmental review boards or committees. these drafting sessions came to include medical and legal specialists as well as ethicists, and representatives from the general public such as journalists. by this time, a new framework had been put in place for policy-making in the fields of life sciences and medical care. this was a linear process that began with forming a public committee comprising specialists from many disciplines, drafting the guidelines, implementing the public comment system, and publishing the final results on the internet, followed by media reporting and revisions over several years. this framework reflected the nature and process of the debate surrounding organ transplantation from brain-dead donors. by the year , "medical ethics" had been added to the curriculum of medical schools nationwide, and faculty members were tasked with teaching these classes. in the early stages, these classes were taught by faculty in forensic medicine, public health, and philosophy/ethics departments as part of general education courses. however, in , the first graduate level department of biomedical ethics was established at the kyoto university graduate school of medicine, and several full-time faculty members were hired. in , the university of tokyo graduate school of medicine also created a department of biomedical ethics. as this process continued to unfold, the "ethics of medicine" education for students in healthcare-related areas became standard. meanwhile, the elsi (ethical, legal, and social implications) programs in the life sciences and medicine were offered a substantial amount of public and private research funding. by the end of phase ii, bioethics had earned a well-respected place in education and research. in what follows i introduce several topics that have emerged or are just emerging in japan. these are not unique to japan, but i would like to emphasize those which have become prominent in the japanese context. "enhancement" refers to a specific use of medical technology, that is not been used to treat or prevent disease. it has been defined as an "intervention aimed to improve the human form and function more than is necessary for maintaining health or recovery" [ ] . a more straightforward definition is set forth by kato, a japanese philosopher, who defines enhancement as "employing medical technology for a purpose other than treatment." examples of enhancement include the administration of growth hormones for children who are not suffering from growth restriction, "doping" or the use of steroids to enhance muscle strength in sports, and the use of mind-altering drugs such as ritalin (methylphenidate) and prozac (fluoxetine hydrochloride) by healthy individuals to enhance their attention (learning capacity) or mood ("smart pills" or "happy pills"). in the future, we may be dealing with designer babies due to genome editing, increased longevity, and enhancement using a brain-machine interface that can operate machinery by connecting a brain to a computer. ideas to improve humans are not new, but developments in medical technology now present the possibility of altering the human body, which has led to many debates surrounding the ethical issues involved. those opposed to enhancement are concerned about its widespread use, advocating that it is unnatural for human beings to "play god," that there are unknown dangers, and that we would lose sacred values such as the importance of weakness and being interlinked with one another. meanwhile, those whose stance is one of the passive promotion, that is "so long as each individual's freedom to choose is respected we cannot go so far as to prohibit this," are in favor of enhancement, as are those who actively promote it based on utilitarian principles, arguing that enhancement promotes the happiness and enjoyment of human beings, and thus we are obligated to pursue it. in japan, rather than either approving or prohibiting all forms of enhancement, each issue is considered individually. some also feel that as long as society respects individual self-determination and the multidimensionality of values, blanket prohibition of enhancement cannot be enforced, but some restrictions may be warranted. these discussions will help us to grapple with some important issues related to the nature of human beings and society. in recent years, a new and rapidly developing field of study has emerged that deals with ethical problems related to neurology or neuroscience and applicable technologies. this field has come to be known as "neuroethics." safire defines neuroethics as "the examination of what is right and wrong, good and bad about the treatment of, perfection of, or unwelcome invasion of and worrisome manipulation of the human brain" [ ] . two major developments in neuroscience aided in creating momentum for neuroethics. first, the development of brain functional imaging technology such as pet and fmri made it possible to observe the brain functions of a living human being. this new technology presents the possibility of obtaining an even more diverse array of information, such as lie detection and clarifying an individual's level of consciousness. however, it also opens up possibilities for mind-reading, or the ability to read the condition of another person's spirit. the second major technological development involves selective pharmacological and anatomical intervention/manipulation of neurological processes. for example, it is now possible to control tremors caused by parkinson's disease with deep brain stimulation. this technology could potentially be applied to patients with other diseases. moreover, it could also be used for brain enhancement in healthy people. brainwashing and mind control for military purposes have also come into the debate. the thought of a brain-machine interface and chimeras of humans and animals being created evokes science fiction-like images of cyborg production, generating fear among people. some attempts have been made to resolve this fear through twoway communication between scientists and non-scientists, for example, and by improving scientific literacy among the general population and performing risk assessments and encouraging participation from ordinary citizens. the japanese government is proactive about neuroscience research and has a national brain science project; the strategic research program for brain science. while there are no specific topics unique to japan, elsi studies of techniques developed in japan such as decoded neurofeedback are ongoing. in , dr. shinya yamanaka created the nobel prize-winning human induced pluripotent (ips) cells at kyoto university, using technology that allows for the creation of cells with the same differentiation function as human escs by incorporating multiple genes into an adult somatic cell. this technology did not require the destruction of a human embryo, and thus generated much hope for cell transplantation medicine that does not involve ethical issues or rejection responses. it was a breakthrough in the field of life sciences as well as the ethical arena. currently, ips cell research has moved from the basic research stage to drug development and even clinical applications such as cell transplantation. the japanese government is actively promoting ips cell research. however, were the ethical problems truly and completely eliminated by ips cell technology? here, i discuss some of the remaining issues. the first of these is safety. the fact that these cells can differentiate into many cell types means that the possibility of cancerization cannot be ruled out. in this regard, attempts are being made to create ips cells (using drugs, for example) that lower the number of, if not eliminate the need for, gene recombinants. this is something that future technological advancements will likely be able to overcome. next, whether or not it is permissible to create reproductive cells (sperm or egg cells) from ips cells is an important ethical issue. in research using human cloned embryos, there was some concern that if the cloned embryo was returned to the uterus, then an individual (a cloned human being) would develop. as such, returning a cloned embryo to the uterus was prohibited by the act on regulation of human cloning techniques. for ips cell research, at least in theory, if it is possible to induce differentiation into sperm and egg cells, then it may also become possible to fertilize these. as was the case with the argument surrounding cloning, problems arise concerning the uniqueness of a human individual. another developing discussion in this field concerns differentiation into neurons. for example, if a human neuron is grown for research in an animal brain, there is some concern that this animal will develop a human personality. this may present to us a problem related to fusion between humans and animals at the cellular or genomic level. another challenge is that of stem cell research, for which various uses can be imagined (for example, controlling the direction of differentiation, genetic recombination, and fusion with cells from other species). the problem for researchers then becomes how much consent to obtain from cell donors, and for cell donors, how much they should understand about the future destinations of their cells. the course of medical ethics in the second half of the twentieth century can be summed up by the phrase "from paternalism to individual self-determination." part of the background for this was the change in disease patterns. namely, from infectious diseases to lifestyle diseases, which led to a change in thinking about disease as something that should be prevented at a population level, to that which should be prevented and treated individually. however, in recent years, the dangers of new and re-emerging infectious diseases such as hiv/aids, malaria, sars, and novel forms of influenza have increased. in addition, concern has mounted for bioterrorismled smallpox outbreaks, or a recurrence of polio, leading us to refocus our attention on the importance of mass prevention. in japan, the worldwide spread of the h n influenza virus in is a recent collective memory. this engendered social debate, especially with regard to who would be the first to receive the vaccine. it is clear that situations are emerging that cannot be well addressed by the kind of bioethics that prioritizes individual self-determination. the opposition and tension between public welfare and individual freedom can be seen in the debates about quarantines and vaccinations for infectious diseases, discussion on handling vaccine distribution, and individual lives being affected by governmental interventions aimed to promote health. meanwhile, they also create more difficulties than ever before for equal distribution (justice) of benefit and cost. simply stated, the central arguments concerning these issues pose the following questions: in what situations should individual autonomy and self-determination be restricted, and what degree of intervention for the sake of the common good and paternalistic interventions should be permitted? a new discussion framework for medical ethicsone that is not limited to the conventional patient-caregiver relationship but rather forms the basis for healthcare policy for the various issues plaguing modern-day public health-is in the process of development. discussion concerning this topic is just beginning in japan. due to groundbreaking developments in genomic medicine, the time will come when at the time of our birth (or indeed at conception), we will know our genetic destiny. this raises many serious ethical issues. for example, if it is discovered at birth that a child would develop a severe disease by the age of , what sort of care should that child receive? what would we do if we found out that we would develop dementia by the age of ? would we be tested early on and take preventive drugs? adults may have the capacity to make these sorts of decisions, but the ethics of administering these tests to underage individuals is not as clear. is testing justifiable in children if the intent is to prevent disease or slow its progression? should genome editing be conducted, ensuring that the genes causing disease are eliminated prior to birth? alternatively, is this an area into which humans should not intervene? this would change the nature of medicine entirely. similar issues are raised by the development of artificial intelligence, as implementation of this technology may change the physician's role markedly. currently, in an age in which progress has been realized in genomic medicine, precision medicine will likely become one of the most difficult ethical dilemmas for mid-twentyfirst century medical care. having touched on the history of bioethics in japan, i will turn to some issues for the future. first, i anticipate that the life sciences and medical technology will continue to progress further, giving hope to many suffering from currently incurable diseases. some of the developments, such as immune-checkpoint inhibitors, ips cells, and escs, are technological innovations that may help to overcome conventional ethical problems. however, as is evident in issues related to enhancement and neuroethics, forward progress in medical technology that hides within itself great possibilities can, on the one hand, give us great hope for the development of new treatments, while also creating friction between conventional values and ways of living, causing anxiety in society. moreover, when technology is first introduced, it is difficult to envision sufficiently the influence it will have. how should society handle the "uncertainties" that inevitably and constantly accompany these new technologies? constructing a framework to ensure that these are addressed effectively is one major challenge for the future of medical ethics in the japanese context. in addition, even if the arguments in medical ethics shift over to those concerning the beginning of life against the backdrop of new technological developments, issues with terminal medical care and end of life will continue to cause worry among patients and their families, as well as among medical caregivers involved in treatment. with regard to the many problems that will inevitably emerge, as well as for those that already exist, attempts to address these will likely occur by establishing guidelines or legislation through japanese governmental committees, as described above. thus, the framework required to address these issues, while befitting the japanese context, also comprises an effective method that listens to external voices and adjusts to create harmony in society in order to resolve ethical issues in life sciences and medical care. yet, i cannot help but assume that the excessive use of . the future of bioethics in japan guidelines and laws creates a dependency among medical caregivers and researchers, such that they lose the motivation to face problems head-on and think deeply about them, or when necessary, communicate to the outside world about issues. in questioning whether a hospital ethics consultation (a system in which an ethics committee or its equivalent would offer advice for individual cases) would even function well in this particular climate, i can imagine numerous medical caregivers who would be baffled by having to find answers to difficult ethical issues in the clinical setting. indeed, the demand not only to accomplish all they do in their busy workday but also to tackle these ethical issues is a tall order. however, we cannot assume simply that because laws and guidelines are in place, all problems will be resolved quickly. if we hope to resolve these ethical issues, we must take a multifaceted approach that is both policy-based (guidelines are issued, ethics committees are established, and healthcare systems are improved) and education-based (awareness and problem-solving capacities are increased among medical caregivers and researchers in those fields as a result of medical ethics education). this is what i envision for bioethics in japan. in the chapters that follow, there are many passages in which i question the japanese government's policies in regard to bioethics and research ethics. however, my intent is not to criticize the government, but rather to bring the issues to light and describe them in their rawness through the lens of japanese culture. if i do not do this, my concern is that international trust and confidence in japan's research and medical care will deteriorate over time, and the value quality of japan as a nation will decrease. this is not a lack of patriotism. as noted in the introduction, the present book was written to achieve a global scale of bioethical dialogue; to this end, i cannot shy away from criticism of my own country's policies or cultures in this process when this is necessary. the late japanese scholar donald keene, in dialogue with jakucho setouchi in the book "nihon no bitoku [the virtues of japan]," said, "since obtaining japanese citizenship, i have come to think that i should …express my opinions as a japanese citizen," and, "now that i have become a japanese citizen, i intend to freely speak out against japan in ways that i have refrained from in the past." there can be no deep-rooted democracy in a society where the act of criticism itself comes under critique even before validation of the substance of the criticism. in japanese society today, however, many people internalize an obedient spirit of servility toward current systems, without so much as questioning authority. i too have been quite hesitant to criticize japan up to this point, but like keene, i must say what should be said before i reach the final years of my life. in this book, i speak of what i call "the dark side" of japan, as seen through the lens of bioethics, so that non-japanese readers can truly understand japan. there are already enough publications which describe japan's advantages. open access this chapter is licensed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. the images or other third party material in this chapter are included in the chapter's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the chapter's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. ): importation, development, and the future biomedical ethics in japan: the second stage nurturing the future of medical ethics in japan paying for informed consent euthanasia, assisted suicide, and cessation of life support: japan's policy, law, and an analysis of whistle blowing in two recent mercy killing cases enhancement uses of medical technology neuroethics: mapping the field. new york: dana foundation key: cord- -u dola authors: morimoto, konosuke; suzuki, motoi; ishifuji, tomoko; yaegashi, makito; asoh, norichika; hamashige, naohisa; abe, masahiko; aoshima, masahiro; ariyoshi, koya title: the burden and etiology of community-onset pneumonia in the aging japanese population: a multicenter prospective study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: u dola background: the increasing burden of pneumonia in adults is an emerging health issue in the era of global population aging. this study was conducted to elucidate the burden of community-onset pneumonia (cop) and its etiologic fractions in japan, the world’s most aged society. methods: a multicenter prospective surveillance for cop was conducted from september to january in japan. all pneumonia patients aged ≥ years, including those with community-acquired pneumonia (cap) and health care-associated pneumonia (hcap), were enrolled at four community hospitals on four major islands. the cop burden was estimated based on the surveillance data and national statistics. results: a total of , cop episodes out of , hospital visits were enrolled during the surveillance. the estimated overall incidence rates of adult cop, hospitalization, and in-hospital death were . ( % confidence interval, . to . ), . ( . to . ), and . ( . to . ) per , person-years (py), respectively. the incidence rates sharply increased with age; the incidence in people aged ≥ years was -fold higher than that in people aged - years. the estimated annual number of adult cop cases in the entire japanese population was , , , and . % were aged ≥ years. aspiration-associated pneumonia ( , ) was the leading etiologic category, followed by streptococcus pneumoniae-associated pneumonia ( , ), haemophilus influenzae-associated pneumonia ( , ), and respiratory virus-associated pneumonia ( , ), including influenza-associated pneumonia ( , ). conclusions: a substantial portion of the cop burden occurs among elderly members of the japanese adult population. in addition to the introduction of effective vaccines for s. pneumoniae and influenza, multidimensional approaches are needed to reduce the pneumonia burden in an aging society. guardians. the requirement for obtaining written consent from all participants was waived by all irbs because of the study's observational nature without any deviation from the current medical practice. hospital doctors verbally described the study objectives and methods to eligible patients and their guardians during their consultations. we also provided the necessary information to patients and their guardians using a standardized questionnaire sheet and a poster presentation at the outpatient department. anonymized data were used for the analysis. according to the national statistics, the total population in japan was million in [ ] . of this population, . % were ! years of age, and . % were ! years of age [ ] . although no national recommendation for the -valent polysaccharide pneumococcal vaccine (ppv ) existed at the time of study, the cost of ppv was partially or fully subsidized by the local government. the estimated coverage rate of ppv for adults aged ! years was approximately % in [ ] . the study was conducted at four community-based hospitals in four prefectures (hokkaido, chiba, kochi, and nagasaki) in japan from september through january . one hospital is located on each of the four main islands (ebetsu city hospital in hokkaido, kameda medical center in honshu, chikamori hospital in shikoku, and juzenkai hospital in kyusyu). because of japan's universal health insurance system, % of the medical costs for people aged < years and - % of the medical costs for people aged ! years are covered, regardless of whether the individual is treated in the private or public sector [ ] . therefore, we assume that the characteristics of the pneumonia patients visiting these hospitals do not significantly differ from those visiting neighboring hospitals. all the outpatients were screened by hospital physicians, and eligible patients were identified using a standardized case definition. patients who fulfilled all the following criteria were enrolled in the study: ) age ! years, ) with symptoms compatible with pneumonia (e.g., fever, cough, sputum, pleuritic chest pain, and dyspnea), and ) with new pulmonary infiltrates on chest x-ray (cxr) or computed tomography (ct) scan films that were consistent with pneumonia. to ensure that all eligible cases were enrolled, the study investigators screened the hospital database for international classification of diseases, th revision (icd- ) codes and reviewed hospital medical records. all the enrolled cases were classified into cap and hcap groups according to the definitions in the ats/idsa guideline [ , ] . if a patient developed the disease hours after admission, he or she was classified as having hap and was excluded. repeated episodes of pneumonia in the same patient within a -week period were regarded as a single episode. demographic and clinical information were collected from patients and medical charts using a standardized data collection form. sputum and blood samples were collected from the participants on admission; sputum was induced with the inhalation of hypertonic saline solution if the patients were unable to cough up sputum. cxrs were taken from all patients within hours of admission, and ct scans were ordered by clinicians based on their judgment. clinical specimens were immediately transported to the laboratory at each hospital. gram staining was performed on each sputum specimen, and the specimen quality was evaluated by trained laboratory technicians according to miller and jones' classification [ ] . all sputum samples were examined using semi-quantitative or quantitative culture methods. sputum samples were further tested at the institute of tropical medicine, nagasaki university, using in-house multiplex polymerase chain reaction (pcr) assays to identify bacterial and viral pathogens. three typical bacterial pathogens (s. pneumoniae, h. influenzae, and m. catarrhalis), three atypical bacterial pathogens (mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila), and thirteen viral pathogens ( [ , ] . we also performed urinary antigen tests for s. pneumoniae and l. pneumophila using commercial kits (binax now streptococcus pneumoniae, binax now legionella; alere inc., waltham, ma, usa). all pneumococcal isolates were serotyped using the capsular quelling method. the etiological category of pneumonia was defined according to the microbiological findings of sputum, blood, and urine samples. the causative pathogens were determined by hospital clinicians and study investigators. because no culture, pcr or rapid urine test is perfect, we estimated the prevalences of s. pneumoniae and h. influenzae using two different methods: ) positivity was defined if either a sputum culture or a urinary antigen test showed a positive result (standard estimation), and ) positivity was defined if a sputum culture, sputum pcr, or urinary antigen test showed a positive result (maximum estimation). to estimate the etiology-specific incidence of pneumonia, aspiration-associated pneumonia was defined independent of microbiological profiles. cases were classified as aspirationassociated pneumonia when the patients had any of the following known risk factors: episodes of aspiration, the presence of dysphagia, consciousness disturbances, neuromuscular diseases, cerebrovascular diseases, tube feeding, and bedridden status [ ] . the prevalence of aspiration-associated pneumonia was calculated separately from those of pathogenspecific category. the age-group specific incidence rates of pneumonia, hospitalization and death in the four prefectures were estimated using the surveillance data and the national statistics. we used the pneumonia-outpatient ratio (por)-based estimation model that was used in our previous study [ ] . in this model, the number of age group-specific pneumonia cases was estimated using the age group-specific number of outpatients and the age group-specific por. pors were calculated for hospitals and clinics separately. the model used to estimate the number of pneumonia outpatient visits was as follows: c ijk : the annual number of outpatients reported to the patient survey in age group i, prefecture j, and facility type k (k = , hospitals; k = , clinics) α ijk : the ratio of confirmed pneumonia patients to the number of total outpatients in age group i, prefecture j, and facility type k p ij : the population in age group i and prefecture j c ijk was obtained from the patient survey conducted by the mhlw in [ ] . the survey was conducted at hospitals and clinics on one designated date set for each hospital on one of three days in october. p ij was obtained from the national demographic survey in [ ] . the proportion of pneumonia cases among outpatients in hospitals (α ij ) was calculated from the surveillance data. to estimate the proportion of pneumonia cases among outpatients in clinics (α ij ), we obtained the clinic databases from two clinics in nagasaki city. pneumonia diagnosis was confirmed using the case definition identical to that used for the hospital-based surveillance. α was estimated for each age group using curve fitting. age-standardized rates were calculated using the who world standard population [ ] . the number of pneumonia cases in the entire japanese population was estimated assuming that the incidence rates (i ij ) were constant across all prefectures. during the study period, a total of , patients visited the study hospitals; , of these patients were enrolled in the study. after excluding cases that did not meet the criteria, , patients were eligible for our analysis (see supplementary material, s fig) . ct scans were administered to , patients ( . %), including ( . %) who did not demonstrate any infiltrates on their cxrs. the demographic and clinical characteristics of the enrolled cases are shown in table . fifty-nine percent of all patients were male, and the median age was years (range: to ). seventy-five percent of the patients were elderly people aged ! years, and % were aged ! years. the case fatality rate was %. seventy-two percent of our cases were hospitalized, and these cases were more likely to be male, older, smokers, and classified as having hcap; more likely to have underlying conditions, aspiration-associated conditions, and severe conditions; more likely to visit the hospital early; and more likely to have a fatal outcome compared with outpatients. among , sputum samples tested using conventional cultures, causative bacterial pathogens were isolated from ( %) samples; ( %) were monoclonal and ( %) were polyclonal (table ). h. influenzae was the most common bacterial pathogen isolated ( %), followed by s. pneumoniae ( %). h. influenzae was more frequently isolated from outpatient cases, while staphylococcus aureus, klebsiella pneumoniae, and escherichia coli were more frequently isolated from hospitalized cases. among the sputum samples tested using multiplex pcr, ( %) were positive for any bacterial pathogens; % were positive for s. pneumoniae, and % were positive for h. influenzae. h. influenzae and m. pneumoniae were more frequently detected in samples from outpatient cases than those from hospitalized cases. s. pneumoniae was isolated by blood culture from . % (n = / , ) of cases, and s. pneumoniae urinary antigen was detected in % (n = / ). taken together, . % of samples were positive for s. pneumoniae either by culture, pcr, or urinary antigen tests. among s. pneumoniae isolates, were serotyped; serotype was the most dominant (n = , %), followed by serotypes a (n = , %) and f (n = , %). ppv covered % of all serotypes, and % were covered by -valent pneumococcal conjugate vaccine (pcv ) (see supplementary material, s fig) . multiplex pcr was used to test , sputum samples for rvs, and % of the samples were positive for at least one rv (table ) . hrv was the leading virus identified, followed by infa and rsv. the positivity rates for rvs were similar between the outpatient cases and the hospitalized cases. among sputum samples tested for viral and bacterial pcrs, ( %) were both positive. among all , patients, ( . %) were with aspiration-associated conditions (ie, aspiration-associated pneumonia): ( . %) had episodes of aspiration, ( . %) dysphagia, ( . %) consciousness disturbances, ( . %) neuromuscular diseases, ( . %) cerebrovascular diseases, ( . %) tube feeding, and ( . %) bedridden status. among sputum samples available from aspiration-associated pneumonia, staphylococcus aureus was the most common bacterial pathogen isolated by culture ( %), followed by s. pneumoniae ( %) and klebsiella pneumoniae ( %) (see supplementary material, s table) . s. pneumoniae and h. influenzae were less frequently detected in samples from aspiration-associated pneumonia than those from non-aspiration-associated pneumonia by culture and pcr. the burden of cop the overall annual incidence rate of adult pneumonia in the four prefectures was . per , py ( % ci, . to . per , py) in japan, and the asr was . ( . to . ; table ). the incidence rate was highest in kochi ( . per , py) and lowest in chiba ( ) ( ) ( ) . m. pneumoniae ( ) ( ) ( ) < . ( . per , py), while the asr did not differ significantly by prefecture, ranging from to . per , py (see supplementary material, s table) . the rate was higher in males than in females ( . vs . per , py in all age groups; . vs . per , py in people aged ! years). it was lowest in those aged - years ( . per , py), increased sharply with age, and became highest in the population aged ! years ( . per , py; %ci, . to . ; fig ) . this age-associated increase in the rate was more apparent in males than in females (the point estimates of the rate ratios comparing males and females in people aged - years, - years, - years, and years were . , , . , and . , respectively). this increasing trend was also observed for pneumonia-related hospitalizations and in-hospital death. for the etiology-specific incidence estimates, the etiology-associated incidence rate was calculated according to age group (table ) . although aspiration-associated pneumonia had the highest incidence, there were substantial numbers of infection-associated pneumonia cases among older people; s. pneumoniae-, h. influenzae-, and rv-associated pneumonia followed. the exception was atypical pneumonia; the incidence of atypical bacteria-associated pneumonia was highest among younger people (aged - years). in our study population, only out of , cases had positive blood cultures, indicating that the incidence of bacteremic pneumococcal pneumonia was per , py ( % ci, . to . per , py). assuming that these proportions of pneumonia etiologies were constant across all prefectures, the estimated annual number of cop in the entire japanese adult population was , , ; of these, , , cases ( %) occurred in people aged ! years (fig ) . seventy percent were hospitalized cases, and % of all pneumonia cases were cap. among cop cases, , died in hospitals. , cases were aspiration-associated, and % of patients with this condition were aged ! years. , cases were s. pneumoniae-associated, , were h. influenzae-associated, and , were rv-associated pneumonia. to our knowledge, this study is the first to estimate the burden of pneumonia in the entire japanese adult population. in , the overall incidence of community-onset pneumonia, including cap and hcap, among people aged ! years was . per , py; the incidence sharply increased with age and reached up to . per , py among people aged > years. the estimated annual number of adult cop patients in japan was , , , % of which were elderly people aged ! years. aspiration was the leading etiologic category of pneumonia, though a substantial number of cases were still associated with infections, such as s. pneumoniae. our findings clearly indicate that pneumonia is an age-related disease that causes an enormous burden in this aging population. the incidence of pneumonia in japanese elderly people was higher than the incidences observed in large-scale population-based studies in both the united states and european countries. in japan, the incidence of cop among people aged ! years was . per , py; in contrast, cop incidences were . per , py in the united states [ ] , per , py in spain [ ] , and per , py in the united kingdom [ ] . the high pneumonia incidence in japan may be partially explained by its high proportion of extremely elderly people aged ! years ( % of the elderly population in [ ] ). however, the trend did not fundamentally change after the incidence was standardized using the who world standard population: the asrs of cop among the elderly in japan, the united states, spain, and the united kingdom were . , . , . , and . per , py, respectively. the in-hospital mortality rate for cop in japan ( . %) was lower than that reported for other countries ( . % in the united states [ ] , % in spain [ ] , and > % in the united kingdom [ ] ). these findings suggest that patients with mild pneumonia cases that may be overlooked in other countries are visiting clinics and being diagnosed in japan. in fact, % of our pneumonia cases were examined with ct scans to diagnose pneumonia. the good access to healthcare facilities resulting from universal health coverage and the wide use of sensitive diagnostic tools may explain this high incidence. however, it does not indicate that the hospitalized cases in japan are milder than those in other countries; the proportion of severe cases (curb score ! ) in the current study was %, compared with % in the united kingdom [ ] and % in spain [ ] . the pneumonia incidence and in-hospital morality were higher among males than among females, especially among the older age group, confirming previous works [ , , ] . considering the higher incidence of childhood pneumonia among males, they may be genetically vulnerable to pneumonia; however, there is no evidence to support this hypothesis. according to our estimates, aspiration was the leading cause of pneumonia, and the burden of pneumonia associated with aspiration was higher than that associated with any single pathogen, including s. pneumoniae. the burden was particularly high among the elderly population; . % of aspiration-associated pneumonia cases occurred in patients aged ! years. the inhospital mortality for aspiration-associated pneumonia ( . %) was higher than that for other pneumonia categories ( %). aspiration-associated pneumonia has been overlooked in current pneumonia control programs. although previous studies have shown that this condition is common among hospitalized pneumonia patients [ , ] , its burden has never been evaluated at the population level in the past. aspiration-associated pneumonia is a multi-factorial condition observed in older people. impaired swallowing and an abnormal cough reflex increase the risk of oropharyngeal aspiration; the aspiration of colonized pathogens and gastric acid causes lower respiratory tract infection and/or lung injury [ ] . compromised immunity, comorbidity and changes in lung function in this age group underlie this condition and are associated with the high mortality. nursing home residents are at high risk for aspiration, but hcap and aspiration-associated pneumonia are not identical conditions. in fact, in our study, . % of cap and . % of hcap cases were associated with aspiration. effective clinical management and preventive measures targeting aspiration-associated pneumonia remain underdeveloped. ats guidelines recommend using β-lactam/β-lactamase inhibitors for this condition [ , ] , but the management of recurrent and refractory cases is challenging. for prevention, oral hygiene care and dysphagia rehabilitation have been suggested for reducing the risk of aspiration pneumonia, but with limited supporting evidence [ ] . the burden of aspiration-associated pneumonia may further increase as the number of elderly people who require long-term care increases. effective clinical and public health intervention measures are urgently needed. in the current study, s. pneumoniae was the leading single etiological pathogen and was associated with - % of pneumonia, confirming previous reports [ ] . recent studies in japan have shown that the positivity of s. pneumoniae among cap cases was % [ ] to % [ ] . according to a recent meta-analysis, the proportion of pneumococcal pneumonia among cap cases was - % [ ] . the proportion of pneumococcal pneumonia among all pneumonia cases is declining in high-income countries, reflecting the wide use of antibiotics and pneumococcal vaccines [ ] . in our study, the positivity of s. pneumoniae by sputum culture was only %. considering the low sensitivity of sputum culture, we included urinary antigen test-positive cases for the standard estimation and further included pcr-positive cases for the maximum estimation. the true value must lie between these values (i.e., to %). the proportion of bacteremia among pneumococcal pneumonia cases was % in our study. a meta-analysis showed that approximately % of pneumococcal pneumonia is bacteremic [ ] ; our figure was lower than this estimate. however, our results showed that the incidence of bacteremic pneumococcal pneumonia among japanese adults was per , py, a figure that was comparable with those reported for other countries, such as the united states [ ] and australia [ ] . the findings suggest that pneumococcal pneumonia, either bacteremic or non-bacteremic, remains the leading target for pneumonia control programs in japan. ppv reduces the risk of invasive pneumococcal diseases (ipds) among adults; however, its effectiveness against pneumococcal pneumonia is still controversial, particularly for the elderly [ ] . the recently approved pcv is expected to prevent almost half of the pneumococcal pneumonia cases in the elderly [ , ] ; however, the vaccine covers only serotypes of pneumococcus, and its long-term effects remain unknown. in japan, before the introduction of pcv for children in , % of ipd isolates were ppv serotypes, and % were pcv serotypes [ ] . in the current study, % of the isolates were ppv serotypes, and % were pcv serotypes. the vaccination policy for pneumococcus has been dramatically changing in japan. pcv for children was replaced by pcv in late , and ppv was also included in the ministry of health, labour and welfare recommended vaccines for elderly people in late . the proportion of vaccine-covered serotypes is known to decline after widespread use of pcv [ ] ; thus, these figures will decrease in coming years. the true efficacy of pcv for adult pneumonia among the japanese population must be evaluated along with cost-effectiveness analyses before it is introduced into the national immunization program. a substantial proportion of pneumonia was associated with rvs ( % of all pneumonia cases). recent studies suggest that rvs play crucial roles in the development of pneumonia, including severe cases [ , , [ ] [ ] [ ] ; however, their biological mechanisms remain largely unknown. rvs such as influenza, rsv, and human metapneumovirus (hmpv) cause outbreaks among the elderly in nursing homes [ , ] , and these rvs are potential targets for vaccination. currently, only seasonal influenza vaccines are available for adults, but their effects on pneumonia prevention have not yet been established [ ] . further investigations are needed to clarify the public health impact of rv-associated pneumonia in aging societies. our findings have important implications for effective pneumonia control programs in the aging society. the burden of pneumonia is higher in older people, and the pneumonia etiology largely varies by age group: the incidences of aspiration-, s. pneumoniae-, h. influenzae-, rv-, and pdr pathogen-associated pneumonia increase with age, while the incidence of atypical bacteria-associated pneumonia decreases. it must be noted that the proportion of pneumonia caused by unknown pathogens is higher among elderly people. this category most likely represents multifactorial conditions. therefore, in coming decades, the pneumonia burden will likely increase, and its etiology will become more diverse. in this situation, the current etiologyspecific approach (i.e., vaccinations for pneumococcus and influenza, guidelines for appropriate antibiotics use) must have only a limited impact. a multidimensional approach integrating vaccination programs, clinical management guidelines, training for health care workers, and education for people must be needed; further studies are warranted. this study is the first to estimate the national burden of cop in japan. although pneumonia is a common disease, its true burden remains unclear, even in high-income countries. a number of studies have reported the incidence of adult pneumonia, but their estimates substantially varied from setting to setting [ , , - , , , ] . several factors explain this variation. first, the definition of pneumonia differs among studies. some studies have reported incidences of cap that include outpatients and hospitalized patients [ , , ] , while other studies have reported hospitalized cases only [ , ] . it was not clear whether these studies included hcap cases. additionally, the diagnosis of pneumonia is not standardized in clinical settings; thus, the burden estimates based on existing database are unreliable. second, study designs vary. pneumonia is a common disease, and it is not included in national surveillance. cohort studies may not represent the entire population of a country, while hospital-based studies do not capture all the cases in the community. different designs may produce different estimates in an identical population. third, the health care-seeking pattern affects the incidence estimates. mild cases must be overlooked in countries in which access to health care is limited. in the current study, we enrolled pneumonia cases prospectively, and all were confirmed by study clinicians using the standardized case definition. considering the high reliability of national statistics in japan, our estimates can be reasonably assumed to reflect the true pneumonia burden among japanese adults. our study has limitations. for the incidence estimation, we assumed that the pneumonia-outpatient ratios in the study hospitals were constant across all hospitals in the four prefectures. additionally, to calculate the national burden, we assumed that the incidence of cop in the four prefectures was constant across all prefectures. our hospitals were community-based general hospitals that provided primary, secondary, and tertiary care for residents; thus, the patients visiting these hospitals reflected the general population. in fact, the asrs in the four prefectures were almost identical in our study. furthermore, according to the national patient survey, the proportion of reported acute respiratory infections among all outpatients in the four prefectures was identical to the national average (see supplementary material). we believe that these assumptions are reasonable. in contrast, we did not consider seasonal differences in the pneumonia etiology. our etiology-specific burden estimates must be confirmed using multi-year surveillance data. in this study, aspiration-associated pneumonia was defined based on the presence of known risk factors; thus, we could not distinguish between "aspiration pneumonia" caused by aspiration of colonized oropharyngeal pathogen or "aspiration pneumonitis" caused by aspiration of gastric contents [ ] . however, there is no reliable marker to identify aspiration [ ] . further studies are needed to better define this pneumonia category. a substantial portion of the cop burden in the japanese adult population occurs in the elderly. aspiration was the leading etiology of pneumonia, followed by s. pneumoniae. in addition to the introduction of vaccines for s. pneumoniae and influenza, multidimensional approaches are urgently needed to reduce the pneumonia burden in this aging society. supporting information s database. database of community-onset pneumonia cases. table. microbiological profiles of patients with community-onset pneumonia with and without aspiration-associated conditions. 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and critical care medicine etiology and factors contributing to the severity and mortality of community-acquired pneumonia changing disparities in invasive pneumococcal disease by socioeconomic status and race/ethnicity in connecticut the epidemiology of community acquired bacteremic pneumonia, due to streptococcus pneumoniae, in the top end of the northern territory, australia-over years vaccines for preventing pneumococcal infection in adults. the cochrane database of systematic reviews pneumococcal conjugate vaccines for adults: reasons for optimism and for caution rationale and design of capita: a rct of -valent conjugated pneumococcal vaccine efficacy among older adults. the netherlands journal of medicine serotype and antibiotic resistance of isolates from patients with invasive pneumococcal disease in japan. epidemiology and infection pneumococcal serotype diversity among adults in various countries, influenced by pediatric pneumococcal vaccination uptake incidence and characteristics of viral community-acquired pneumonia in adults etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods viral infection in patients with severe pneumonia requiring intensive care unit admission. american journal of respiratory and critical care medicine outbreaks of human metapneumovirus in two skilled nursing facilities-west virginia and idaho respiratory syncytial virus outbreak in a long-term care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods vaccines for preventing influenza in the elderly. the cochrane database of systematic reviews new perspectives on communityacquired pneumonia in patients. results from a nationwide mandatory performance measurement programme in healthcare quality the influence of age and gender on the population-based incidence of community-acquired pneumonia caused by different microbial pathogens we are grateful to all the laboratory staff at the participating hospitals. we would like to thank rina shiramizu and kyoko uchibori for performing pcr and yumi yamasaki for administrative work. we also wish to thank hidefumi yamamoto and masatoshi ide for sharing their data. adult pneumonia study group-japan (apsg-j) are: masahiko abe , takao wakabayashi , masahiro aoshima , naoto hosokawa , norihiro kaneko , naoko katsurada , kei nakashima , yoshihito otsuka , eiichiro sando , kaori shibui , daisuke suzuki , kenzo tanaka , kentaro tochitani , makito yaegashi , masayuki chikamori , naohisa hamashige , masayuki ishida , hiroshi nakaoka , norichika aso , hiroyuki ito , kei matsuki , yoshiko tsuchihashi , koya ariyoshi , bhim g dhoubhadel , akitsugu furumoto , sugihiro hamaguchi , , tomoko ishifuji , shungo katoh , , satoshi kakiuchi , emi kitashoji , takaharu shimazaki , motoi suzuki , masahiro takaki , konosuke morimoto à , kiwao watanabe , lay-myint yoshida key: cord- - ih jdpe authors: shibuya, kazuhiko title: identity health date: - - journal: digital transformation of identity in the age of artificial intelligence doi: . / - - - - _ sha: doc_id: cord_uid: ih jdpe identity health has especially specific meanings for social relationships in contemporary digital age. first, computerized digital communication makes many citizens in severe maladaptation. the who often warns mental addictions of internet usages and online gaming among the youth. the advent of social media and online networking has endangered them in ambiguous situations which are not stabilizing in those basic grounds for human relationships. further, because social networking sites and social gaming frequently enforce each member to interconnect with the others, many of participating members often hold harder mental debts to respond and maintain their interconnections. in this situation, in other words, it can say that all of users simultaneously might share common conditions under mental illness. who (the world health organization) has already published their warning reports on gaming disorder and its mental addiction (who ) . and their site also says: "gaming disorder is defined in the th revision of the international classification of diseases (icd- ) as a pattern of gaming behavior ("digital-gaming" or "videogaming") characterized by impaired control over gaming, increasing priority given to gaming over other activities to the extent that gaming takes precedence over other interests and daily activities, and continuation or escalation of gaming despite the occurrence of negative consequences". those who can be identified as icd- case are not globally well known, but suspicious cases are roughly estimated as adults (approximately . million) and youths (approximately a million) in japan (data at ). since the beginning of the internet revolution, internet communication and online activities among the youth had been frequently accused by serious concerns in mental health. the first is violent behavior induced by playing violence game. until recently, video game has been argued by media psychologists (xu ) . they often verified violent behavior of adolescents related to experiences and the extent of game playing. the causal relationships across those patterns have not been clarified enough yet. some reasons endorsed by experimental designs are still difficult to interpret clearly (bruner and bruner ) . secondly, there are still controversial issues on online addiction cases (griffiths ) . colleagues ( , ) have been publishing serial reports on online addictions. the number of people diagnosed with the condition of addiction for internet usage has been increasing in the era of social media. especially, adolescents and young adults are eager to immerse in online cyber-world activities. they consume their time through browsing web, watching online video, participating in online games, and internet communication. in japan, mic (ministry of internal affairs and communications) at , reported the latest statistics that the total average of internet usage time of young citizens (ages vary from to ) was h and min every day. their motivations for internet usages diversified categories such as watching online movie ( %), playing games ( %), and communicating by sns and emails ( %) (multiple answers). the most important is how we think about such online additions and youth's sound development. these are a kind of mental illnesses and conditions as a maladaptation of gaming and social withdrawals from actual society, or they are overadaptation in somewhat online communities rather than physical environment. the former is to step further grounding in social living, and the latter may suggest that they extraordinarily prefer to online human relationships. it should be clinically observed in each case. thus, online gaming and social networking sites are indeed based on somewhat human relations and such online communities organized by providers often offer to share some comforts, cooperative achievements, and entertainments among active participants. and simultaneously those services require much engagement among participants, and then such mental obligations enforce each participant to keep playing online game and committing with the other online partners during longer times (oberst et al. ) . here, it experientially indicates that group commitments reduce anxiety of members and enhance comfort and mental bonding among members (leary and baumeister ; baumeister et al. ) . their group life intends to maintain such conditions in in-group memberships, and commitment belonged in group whether offline or not has crucially important meaning for them. to date, advanced data analysis on our phr (personal health record) and personality dispositions has been conducting in medics (pol and thomas ) . further, the advancement of big data and the ai driven medical services is to rush into the daily life contexts (marin et al. ; king et al. ) . namely these services can offer to assess and promote both mental and physical health of each individual. first, as mentioned valuation by the ai at chap. , those services already contain some questionnaires on psychological assessments related to personality characteristics, health attitudes, and social adaptation in daily life. those assessed data might intend to statistically reveal our strength of mental health and degree of adaptation in social relations, and then automatic prediction for those who answered personality tests enables to trustfully measure financial limitations for loans and transactions in actual contexts. therefore, our mental conditions and its social adaptations, to date, have been unveiled by such ways, and those applied services could be built for somewhat vigilance system on mutual trust among citizens. mental disorder and maladaptation of each individual have possibilities to further pervade unsound influences among the others, and vice versa. financial bad-debt by personality dysfunctions of individual will also engender chain bankruptcy among stakeholders, but those services would intend to predict such consequences in advance through checking personality maladaptation in daily life. hence, our digital life has been already founded in those mechanisms, and the ai and big-data operations indeed interlude into our mentality. secondly, wearable devices and sensing tools for human behavior can help monitoring and analyzing latent patterns of physical and mental conditions in daily life (morahan-martin and schumacher ; clifton ; zhu et al. ) . and telephone communication patterns using smart phone can be interacted by identifiable chronotype of each user in daily activities (aledavood et al. ) . medical cares in each country has the demands to organize national health information systems, and it includes big data in the relations to national assurances for health cares, medical quality, demographic statistics, financial investment, quality of life (qol), quantifications for modeling (shibuya ) , and other social welfares (brady et al. ) . those information systems may be governing own centered database, but it will be replaced by distributed blockchain database in the future. during the previous era, there were some troubles yet to bridge between clinical psychology and sociological studies as well as computational models and experimental cases. sympathy interpretations for client's latent mental process and their needs should be taken carefully. but case studies often mean that there are no effective ways to explore future patterns and expectations based on past clinical cases in the daily interactions (leary ; kircher and leube ) . traditionally, the clinical psychological way is usually beneficial to manage mental dynamics that are impossible to be generalized and formalized. clinical psychology and its fundamental assumptions usually hesitate to do generalizations from clinical case studies. that is because each personal condition and mental distress may be too individualized, and rather researchers in this field recommend qualitative and intensive caring ways for understanding each personal experiences embedded in actual conditions. historically, emerging diseases have been suffering us ever since our civilization (roeser et al. ) . human history could be said as somehow survival process from lethal diseases. for example, there were smallpox, pest, dysentery, tuberculosis, and other diseases. otherwise recent outbreaks of emerging diseases such as hiv, ebola, sars (severe acute respiratory syndrome : shibuya ) , zika, and others are still ongoing matters, and those medical ways such as drugs and examination tools have not completed enough yet. in contrary, smallpox can be exemplified as a success case. the humankind had finally achieved the extinction of this disease threat in natural conditions using vaccine as a land-breaking medical way. parts of above diseases could be cured by specific medicines, but known well, there is another problem on resistant bacteria against those medicines. to date, in those fields, immunological and medical investigations have been accelerated by biotechnological and gene-technological advancements. those who obtained the nobel prize in physiology or medicine, and nobel prize in chemistry contributed toward enhancing medical progress for wellbeing of the humankind. in fact, there were contributions by japanese scientists in the medical field (e.g., tonegawa, s, yamanaka, s, ohmura, s, ohsumi, y, etc.). but, the humankind cannot completely repel both disease and death. according to the who report "top causes of death globally ," infectious diseases such as lower respiratory infections (over million deaths), diarrheal diseases (nearly million deaths), and tuberculosis (nearly million deaths) still remain within list of top . otherwise, the worst three cases were ischemic heart disease (nearly million deaths), stroke (nearly million deaths), and chronic obstructive pulmonary disease (approximately million deaths). additionally, total deaths caused by alzheimer disease and other dementias can be estimated to approximately million per year globally. in those areas, as mentioned before, computational searches by the ai driven system and big-data analysis will boost enhancing diagnosis of subtle symptoms, image processing on medical data, pharmacologic utility discovery, and statistical precisions for future risks of each patient. precision of diagnosis by the ai's pattern recognition systems on medical images has outperformed more accurately than human doctors (zhang et al. ) . as larger growing size of knowledgebase on medical science increasingly requires much experience for them, and it will be impossible to operate any clinical cases unless the ai's supports can be provided. and other computational contributions to epidemics can enumerate such as computer simulations of mathematical models on spreading infectious diseases (e.g., sir (susceptible-infected-recovered), small-world networking model (moore and newman ; newman ) ), gene analysis of virus in bioinformatics (ksiazek et al. ) , and risk management on health data of patients and medical policies for future controls of emerging diseases (shibuya ) . in terms of medical cares, epidemiological actions should lay weights on governmental policy, because there are great needs to control against secondary contagions and predict precisely dynamic trends on diseases. traditionally, those fields must be accumulated from onsite clinical data on diagnosis of patients and analyze statistical trends which localized in each region. regarding these concerns, to date, online query results mostly reflect citizens' intentions and latent needs for specific actual events in society. recently, ginsberg and his colleagues ( ) had unveiled such facts by their big data, and their findings by statistical analyzations on logistic positive correlations between actual trends of data from cdc (centers for disease control and prevention, usa) and web-query data among citizens had become a pioneer for big-data age. namely this study clearly suggested that citizens were usually apt to seek more accurate and necessary information in uncertainty conditions such as disaster (see chap. ), unwelcome infectious disease, terrorism, and other fascinated events. their study seemed to be a first breakthrough for researches using web data analysis. as an implication of this finding, many researchers realized significant meanings on synchronizing and corresponding evidence between web trends and offline events. namely "big data" can be analyzed by computational engineering methodologies such as artificial intelligence, statistical machine learning techniques, and natural languages processing, and it can open the gate to investigate novel findings automatically. let me exemplify an actual case. according to data from the who, , from the spring of to , global pandemic caused by new type of influenza (h n ) had suffered global citizens. the total amount of the death was estimated as , in globally (the present data at ). at the peak of this pandemic, the author investigated those trends using google insights for search services. japanese patients were roughly estimated as totally . million (it finally includes at least death cases), nevertheless many citizens have traditional customs encouraged to treat and keep their hygiene in daily living. below fig. . indicates a trend of google query result inputted into keyword "influenza" in japanese. at , it was certainly that there were mostly three peaks during this year. and fig. . , in contrary, shows only seasonal trends of ordinary influenza (except for data of pandemic patients), and both peaks (earlier weeks of this year and the late of year) can be identified during year. seasonal trends on influenza can be also recognized in each year, and only bizarre peak around the middle of this year can be specified. namely, because the pandemic caused by new type of influenza virus occurred around the beginning of may , it can understand that the middle peak in fig. . was underlying in above pandemic influences. and then, in this japan case, google trends could entirely indicate correlational patterns between information needs among citizens and actual influenza trends, and each peak corresponded with seasonal or pandemic ones. as an alternative of google query, using twitter as one of microblogging tools, signorini et al. ( ) revealed synchronizing phenomena on online tweets about influenza corresponded with actual trends of influenza given from cdc data, and https://www.who.int/csr/disease/swineflu/en/ https://www.who.int/wer/ /wer .pdf?ua= implementation of the international health regulations ( ) http://apps.who.int/gb/ebwha/ pdf_files/wha /a _ -en.pdf?ua= fig. . trends of google query result which inputted into a keyword "influenza" in japanese. y axis means frequencies of a query on a specific keyword and x axis shows each year in this case they could find efficient results. those metrics have stronger merits being qualified for online and real-time analyzation than trend data obtained from google query. similarity, broniatowski et al. ( ) reported significant correlation between normalized prevalence data on influenza filtered from twitter's real-time tweets data and cdc actual trends of influenza. they further attempted to forecast influenza trends using data from twitter (paul et al. ) , and then social media consequently enables to do real-time sensing among citizens. as succeeding previous chap. , in the digitized society, disaster, environment, and climate data have also become a target for big-data analyzation. larger natural disasters and human-made hazards have globally potentials to corner to the crisis of humanity. because our global society has been endlessly threatened by various disasters (unisdr ), more than million citizens have been globally harming by natural disasters every year. and this data contains more than , deaths per year. natural disasters are almost interrelated to numerous factors such as climate, demography, environment, and anthropogenic events. further, it seemed obvious that complicated factors related to climate changes in global level have . y axis denotes reported new patients, and x axis periodically shows serial weeks. each line (from to ) means each site given data from medical hospital. this figure was cited from idsc (infectious disease surveillance center, japan) (http://idsc.nih.go.jp/idwr/kanja/weeklygraph/ flu.html) been influencing those meteorological disasters (e.g., hurricanes, drought, flood, etc.). of course, the anthropogenic factors (e.g., industrial damages to the environment, carbon gas emissions) should be occupied in system models in order to examine detail mechanisms (meadows et al. ) . especially, at , after serial tragedies of the tohoku quakes and fukushima nuclear disasters, the unisdr as a part of the united nations (at , unisdr was renamed to undrr : the un office for disaster risk reduction) held the global conference on disaster management at both tokyo and sendai city of japan. as consequences of much discussion, the committee finally proposed following the four priorities for global actions which entitled "sendai framework for disaster reduction ." . priority : understanding disaster risk . priority : strengthening disaster risk governance to manage disaster risk . priority : investing in disaster risk reduction for resilience . priority : enhancing disaster preparedness for effective response and to "build back better" in recovery, rehabilitation, and reconstruction disaster management means just our preparedness against disasters. certainly, the digitized global world can offer artificial space satellites, wireless internet, mobile computing, social networking services, and other mechanical relief robots. and the ai driven systems and big-data analyzations on the disaster can be useful for us. recently, noaa (the national oceanic and atmospheric administration, usa) has released their online services, which is named as the coastal inundation dashboard. it visually enables people to know and prepare for floods. in this way, nevertheless digitized systems for global monitoring the earth, collaboration with each nation and analyzation of vast necessary data had been pervasively equipped, but our acquired technologies for forecasting and preventing disasters have not been achieved enough yet. as above four priorities said, the undrr as a part of the united nations lays heavier weights on rather mitigations and resilience from disasters. because of such inevitable reasons why our survival efforts from the tremendous disasters are never diminishing, there are still greater needs to consider the human existential issues in disaster management. here, as introduced a bit at chap. , the author had a chance to conduct own researches on the tohoku quake and nuclear disasters in fukushima. and this time, a part of findings and evidences can be exhibited in health topic (shibuya (shibuya , (shibuya , (shibuya , (shibuya , ). after , the fukushima case of nuclear power plant accident had incubated another problem (oecd/nea ines (international nuclear event scale) ranked level (the worst)). this fukushima case can be called as a nuclear power plants' crisis (nrc ; roeser et al. ; oecd/nea ; shibuya ) . at the time, many of fukushima citizens lost not only hometown, but background of their identities. in this point, they forced to be laid in identity crisis. according to theoretical sociologists berger et al. ( ) and giddens ( ) , they commonly argued that western post-modernizations could reconstruct mindsets on reality and social identification ways among citizens during achieving industrial progresses, if above severe incidents of nuclear power plants and those systems failures could be regarded as malfunctions as a symbol of modernity, above consequences of nuclear crisis on the fukushima case (and other human-made disasters) might be contextualized to reexamine social adaptation and consciousness among fukushima citizens by sociological verifications. how did daily belief systems among fukushima naïve citizens against the safety surrounding in nuclear power plants deal with? their attitudes had been rather steadily stabilizing among many of them before the crisis. however, risk cognition, social constructive senses of reality, and meaningful understanding against nuclear disasters among citizens would be collapsed in those conditions. they were betrayed by advanced technologies, government, and optimistic beliefs shared among them. it is probably that their theoretical discussions have few suggestions for any policies on fukushima case more than awkward theoretical bases, and rather there are quite needs to tackle grounding in actuality, resilience of identification, purifying environment, and rebuilding community for the fukushima citizens (science council of japan (scj) ; shibuya (in press)). actually, this fukushima case was a reluctantly controversial issue on an accident of nuclear power plant by academic researchers in japan. since the tohoku quake, many natural scientists in japan were very criticized by ordinary citizens. especially these were academic scholars such as nuclear physicists, governmentside natural science researchers and engineers at nuclear power plant, and of course politicians must be also confronted with such serious criticisms (funabashi and kitazawa ) . with deep reflections, the science council of japan (scj) ( ) published globally their investigated documents by belonging scientists and researchers in various academic fields. this report laid stress on the fact description of the fukushima nuclear power plant accidents and the statement of actual conditions for researchers in foreign countries. here, the author conducted to investigate this scj's statement ( ) in depth using text mining. table . shows a result by text mining analyzation on the whole contents of scj's statement, and it depicted a part of frequent and important words (it extracted around top words among the most frequent words) and its total counts within above scj's statement text. at a glance, it appeared that their motivations were implicated by some words such as nuclear, radioactive, cooling, accident, safety, emergency, evacuation, and so on. tepco means an abbreviated name of administrative company for electric power plants in fukushima. figure . shows an example of network structure of words' co-occurrence. in this case, it configured mathematically to color each separated subgraph structure that limited to important co-occurrence words. the author found some clusters of words on radioactive materials, fukushima nuclear plant, accident inside-out and others. these patterns were weighted and frequently articulated by document writers. otherwise, fig. . depicts a result of three-dimensional visualization which analyzed by mds (multi-dimensional scaling: this time was configured by kruskal and jaccard models). this method located statistically each word in cubic dimensions, and it appeared some clusters such as power plants (e.g., power, plant, nuclear, and fukushima), quake (e.g., tsunami, earthquake, situation, and operation) and others. as result of these malfunctions, confidence for ruling party critically had been fallen down. what text mining analysis made clear was that this report concluded the negative consensus against the nuclear hazard among scientific community in japan, and their statements explicitly described that mythical beliefs among stakeholders were no avail in the case of fukushima nuclear power plant accident. obviously, they intended to publish the truths for globally foreign academicians and citizens in terms of mainly nuclear physics and energy engineering after the fukushima crisis. as mentioned at chap. , without doubts, many of global citizens were eager to know more accurate and immediate information in detail at that time of moment. those results by text mining could exhibit japanese governance of risk management against nuclear power plants and energy policies before the fukushima case. there were no rational reasons for excuses that risk communication and consensual discussion had not been openly organized among stakeholders in fukushima, as it was differently the canada's case (johnson ) . rather, tepco and governmental ministry had oppressed to scientifically contemplate and examine nuclear risks and their published data by ordinary citizens and external professionals. with these backgrounds, the focal point of disputes on the compensations has been accused by plaintiff (i.e., citizens, evacuees, and victims) under trials in courts (oecd/ nea ). for the digitized society, this fukushima case indicated further unneglectable facts. iot and xaas will be deployed anywhere (geng ) . many of those systems such as computational controls and sensing networking related to power plants and sensitive artifacts will be impossible to keep under controls unless electric fig. . it depicts a part of network structure of co-occurrence words by text mining. this configuration was to separately color each subgraph structure which limited to important co-occurrence words power can be continually provided. nuclear power plants and its control systems per se always also require independent electric power for controlling those mechanisms. serial incidents of the fukushima nuclear power plants can be determined by the serious factors on both the loss of external electric power supply and vent malfunctions for refrigerating systems caused by tremendous tsunami attacks. besides, until now, the ai-driven robots sensing inner-damaged power plants cannot be activated over the physical limitations because inner-damaged power plants still remain with higher radiation dose (i.e., the human dies immediately and computational mechanisms will be disabled by radiations sooner). then, the lessons for the digitized future must be intensively deduced and learned by convincible investigations. in the usa, after this disaster, a taskforce team was immediately assembled for investigations to report the cause of the nuclear power plants, reconstruction of the nuclear power plants for safety, and future policy on energy management in the after that, it should turn eyes to persevering purification from the radiation damage caused by the nuclear accidents, its environmental restoration, health monitoring, and socioeconomic reconstruction in community (nrc ; iaea ) . consequently, the fukushima case definitely needs to solve nuclear accidents and future design for long-term reconstruction on their devastated communities and hometown. at least, the following points should be tackled. . decommissioning work for the wrecked nuclear power plants in fukushima . removal of nuclear fuel and substances . chemical management on nuclear substances . purifications in polluted places . temporary storage and final disposal of contaminated soil, water, and garbage . medical health survey and care for victims . risk assessment against environment and people exposed by nuclear substances . city and socioeconomic reconstructions as consequences of serial incidents of the nuclear power plants in fukushima, nuclear pollution provoked the severe disputes on human rights, health, radiation contaminations to foods and environmental restorations. both environments (soil, waters and air) as well as ordinary peoples who lived in fukushima were polluted and exposed by both nuclear substance and radiation (gibney ; merz et al. ; oecd/nea ) . sampling data accumulated by agricultural scientists endorsed that many of crops absorbed radioactive substances such as sr, cs, cs, and others (takahashi ) . and excessive intake and exposure of radioactive pollutants will endanger citizens' and workers' health (hiraoka et al. ) . consequentially, enacted provisions often reflect social actualities. for conquests against those hardships of citizens and evacuees, the "basic act on reconstruction in response to the great east japan earthquake ( th, june )" enacted the basic policies for reconstructions. for example, a part of provisions clearly stipulated as follows. • article . the reconstruction in response to great east japan earthquake will be implemented based on the following -the unprecedented disaster resulted in enormous damage, where countless lives were lost, numerous people were deprived of their basic living infrastructures and have been forced to evacuate in and out of the disaster-affected regions. also, the disaster's influence extends over the entire nation; the economic stagnation in the disaster-afflicted areas is affecting business activities and peoples' lives nationwide…(hereafter omitted) at the fy, the total budget of fukushima prefecture for reconstruction after the disaster was approximately . billion yen (including both quakes and nuclear disaster countermeasure portion of . billion yen). and it includes population declining and aging countermeasures as well as restoration of birth number ( . billion yen). in addition, other items were . billion yen for environmental restorations, . billion yen for living reconstruction assistance, billion yen for expenses to protect medical health of the citizens, . billion yen for expenses for children and youths who will be responsible for the future, and other expenses. further, in addition to above costs, the fukushima case requires unprovoked compensations and its litigation disputes are ongoing matters in courts (shibuya ) . namely, there are still requirements to solve future designing for reconstruction from devastation in their hometown. next, the total amount of casualties in japan was more than , at the time of . moreover, as aftermath of the fukushima disaster, one of the hardest matters was collective immigration of evacuees from their hometowns to other places in japan (akabayashi and hayashi ; library of congress ) . at the peak (may ), gross migrants from fukushima (e.g., total population of evacuees) were estimated over , . and including this, gross migrants (e.g., total evacuees in japan) were estimated over , at the time of . this estimation was not too low. please recall similar past cases, for example, the case of chernobyl in reported that total population of evacuees was approximately , around km (ines level ). and, in , the case of the three mile island accident was estimated over , around km (ines level ). table . shows a part of outflow data on migrants across major cities (it includes mobility data within same city). it queried into big data of mic (e.g., demographic data of migration and population) and the geospatial information authority of japan (e.g., geospatial data and distance information). the numbers of citizens lived in fukushima prefecture has been notably decreasing from . million ( ) to . million ( ) . and this area is statistically , km (the third widest area in japan). and it compares fukushima with people lived in major metropolis such as tokyo area (total population is approximately million people and within million people in special districts), nagoya area (approximately . million people within central city), and osaka area (approximately . million people within central city) in japan. it namely denotes moving flows within fukushima cities, moving toward one of metropolises from fukushima and moving patterns between metropolises. actually, there is still another problem in residential data. in year , a national census every years was carried out in japan, and it has achieved to unveil many data discrepancies and inconsistencies of population in each local area of fukushima. comparing with statistics on resident data holding municipal government office and actual population from census, the latter cases were almost too lower than estimated populations in many cases of fukushima. to date, these missing populations have not been traced properly and many evacuees did not intentionally apply immigration cards to municipal government office. it means that many of them still have strong intentions to go back to fukushima in the near future. however, the stumbling blocks still remain against their returns, even though the government purifies radioactive substances and pollutions around their towns. according to the general surveys by tokyo capital government for evacuees and interviews for evacuees by the author (shibuya ), they found that evacuees' motivations which choose the destination and refuge were relying on following critical factors: ( ) to tie with any kindred relationships (a factor of human relationship), ( ) rich opportunities for jobs in the new address (a factor of new job opportunity), ( ) conveniences to manage their own real estates, farms, livestock, and factories in their hometown (a factor of holding estates). first factor implies mutual cooperation and helping among local acquaintances, and second answer clearly reflects their needs for jobs in new dwelling. and third factor relates to geospatial location, and parts of them have been living in two places of both fukushima and refuges. namely, their conditions were back and forth between hometown and refuge. thus they could not leave to far refuges, and geographical area around km within fukushima and refuges satisfied their above motivations using transportations such as the bullet train, highways, and other land transportations (it drives toward the destination within h). for those who required living needs, tokyo and nearby area of tokyo could properly offer new job opportunities and dwelling availabilities for them. our identifications are often determined by not only own cognitive factors but human relational and spatiotemporal factors, and their daily conditions of mental health would be interlinked with those internal and external surroundings. the fukushima case similarly indicated those mental malfunctions of evacuees caused by the human-made disaster. when such individuals lose all (or a part) of the linkages with both human relationships and living place, their mental foundation for identification will be seriously damaged in severe situations. such accidents and events caused by both natural and human-made disasters will be easy to engender secondary damages against social adaptation and subjective well-being of each individual. it is too important to care for each, but there are resilient needs to wholly repair and revive the community among them and their human relationships such as social capital (putnum ; kawachi and berkman ; kawachi et al. ; oecd/nea ) and family bondages. even though digitized communication styles renewed our daily commitment for online community, physical-contact based commitments in onsite community have still special meanings for their well-being. frey and osborne ( ) reported how will contemporary industries and jobs be changed and replaced by computerization and the ai-driven robotics, and they simulated socioeconomic trend patterns of jobs fitted by gaussian stochastic model. their estimations have indeed shown that the ai society will engender emerging job market and require other skills for citizens. but it will be clearly understood whether correct or not in the future. but, will a meaning on working life be steeply altered by such innovation? some theorists said that both living and working are indispensable relations each other. as one of the renown episodes, psychoanalyst freud answered that it requires "lieben und arbeiten" for becoming sound and independent adult. the former "lieben" means the accepting and loving for the others as a partner. and the latter "arbeiten" devotes to achieving the goals for maintaining daily living by own works and pursuing enhancement of own intellectual abilities and skills. both are still certainly the fundamental necessary factors for people. and a life-course approach in developmental psychology has much suggestion to wholly understand our mental development and health promotion during the life process (erikson (erikson , (erikson , . in each development stage of identity, each individual closely faces the problems and what should be conquered by each of them. such tasks can be furnished for own rich experiences of each, and each can be reorganized to adapt in own life process (e.g., self-actualization). working, learning, and other daily activities will be achieved by undertaking self-development and adaptation in social surroundings. it motivates to enhance each quality of life (qol) through own working experiment. it is certainly that the ai and big-data-based society enables to change our qol and working life. such innovation progress in our working styles has already become the cascading to collapse larger barriers by the big wave of digital transformation. working environment has been crucially invested in the contexts of either employees or employer. but quality in working environment cannot be determined by factors of material and physical surroundings, and it should be cared about human factors such as enhancement of human relationships and well-being (oecd ; strack et al. ; buunk and gibbons ) . for example, oecd proposed total framework ("measuring well-being and progress: well-being research") , and they enumerated necessary factors related to measuring both economic and well-being value in working and daily life. the digital transforming society will enlarge our working from actual physical space to virtual online space through tele-existence and online collaboration tools. and our working skills and abilities will be required conquering the harder roads of uphill progress of the ai and data sciences (boyd and holton ) . further, traditional stressful working environments can be attempted to quantify and coordinate with each parameter of employees such as personality characteristics, demanded skill levels, abilities, chemistry among members, and other necessary factors. now, such hr (human resource) technology (i.e., a case using business microscope ) enhances our working styles and improves productivities in various situations (khartri and samuel ) . analytics teams have vividly rushed to dive into the ocean of big data, but matching between the needs and their analyzed solutions can be improved by further efforts. active workers are usually facing issues at the marriage and family in their life courses. in some developed countries, the reasons, due to which unmarried rate during the lifespan of the youth generation has been increasing, may be understandable in work-life balance context. in japan, statistical data of ipss (national institute of population and social security research, japan) show such facts: men's case of unmarried rate excessed %, and women's case was . % at . many adolescents frequently hesitate to lose their free time and conformity, and it simultaneously means that they hate any interruptions by others and physical contacts. as necessary, they can choose tentative friends online (su and hu ) . using smartphone, many matching service applications for marriage among future partners have been launched in japan. and then, the matching needs can be bridged with the youths for their marriages. those matching services might be applied by stable matching problem in economics of mechanism design (roth ) . such algorithm can be formalized for matching pairs of stable marriage. otherwise, daily living enriches its big data (ganchev et al. ) . especially, there are strong requirements for children and elder people in their local community and living environment. first, smart sensing and ubiquitous technologies aim to enhancing our daily life (shibuya ) , and smart cities and smart house have cutting-edges for improving health services for us (lee ) . for example, the digital human research center in japan proposed an autonomous caring system and simulators for toddlers and little infants. due to their sudden and unpredictable manner of behaviors, serious accidents such as injuries and death at home often happen. this system intends to monitor and analyze daily patterns for improving their safety. of course, those systems which are equipped in smart houses are also applicable for elder people to watch their daily cares and health monitoring. on the other hands, secondly, airbnb and similar sharing house services have been launched in many nations, and big data on those paring patterns between house-owners and visitors will be arranged to analyze such trip purposes, sharing durations, other preferences by the ai-driven services (koh et al. ) . in these regards, digitization has already reshaped our quality of living in those contexts. using smartphone, mental health monitoring can be possible recently (ben-zeev et al. ; bakker et al. ) . especially, using social media data, there were innumerable examinations to analyze the relations with mental health and diagnosis of discourses on twitter and sns services. for example, there were enumerable cases on depression trends in community corresponding with data of geospatial location (yang and mu ) , depression detection on twitter (guntuku et al. ) , adhd (attention deficit hyperactivity disorder) diagnosis using discourses on twitter (coppersmith et al. ) , and other borderline cases in clinical psychology (e.g., hopeless, loneliness, social withdrawal). as social networking services clearly indicate a part of human relationships online (lazakidou ) , it can consider that their relations itself still have sharing illness personalities and depressed mental health. namely, there is a possibility that latent patients were apt to be participating in such social media, and some of them flocked online each other. inclusive cares within communal and relationship level can be also very effective for each person. traditionally, in the studies of communication and communal health, the concept on illness identity in interactive communication has potentials to recognize identification process for caring illness and health (hecht et al. ) . illness identity could be regarded as interactive processes from personal to communal layers in terms of the communication theory of identity. these mechanisms should be inclusively cared by various viewpoints from personal to social communal level. that is because, for example, social anxiety, depression, and other mental (and physical) illness could not be easily emancipated from not only individuals but also more diverse interactions and social groups. according to wegner et al., they discussed mental control and relationships with the others. and their findings can be understood only by including perspectives of social contexts. that is, it should pay attention to not only individual experiences of depression and mental distress but social relationship and interaction process with standpoints from the others (aneshensel et al. ). as implied earlier, rapport interaction between clinical psychologists and clients has been better focused in empathy oriented understanding as client-centered therapy (rogers ) . clinical psychological cases such as autism, psychopathy and other diagnoses usually display specific patterns of behaviors and assertions. especially, those who have specific disabilities against soundly interactions with the others are managed by tom (theory of mind), and this study intends to reveal our mental manners to recognize and coordinate with the others in dyad models (semeijn ; freitas et al. ) . such client cannot understand any intentions asserted by the others, and they often confuse meanings pretending and deceptions by the others. namely, understanding for the others flexibly requires more imaginable coordination in social context. and the loss of such basic intellectual skills becomes difficult for them to behave appropriately against troublesomeness with the others. in other words, some evolutionary psychologists and neuroscientists told that the humankind could be evolved both to lie against the others and detect . therapy for human by ai deceptive intentions. they further said that acquisitions for those neural mechanisms of social intelligence took advantages of beating against other wild animals during surviving history of ancient peoples. according to attachment theory by developmental psychologist bowlby, it authorized that physical attachment between mother and children offers strong and comfort foundation during child development (bretherton ) . suggestively, serial experiments also shown that an alternative of "mother" could be sufficiently replaced for children. for example, a fluffy doll as alternative of mothership could fulfill having comfort emotions for child (in their experiments, they tried to use a child of monkey). in this concern, personal intelligent robots have potentials to assist our daily life (coeckelbergh ) . as an example case, paro already achieved improving many patients of mental illness and alzheimer diseases. it calls robot therapy assisted by ai and adorable doll-like robots. such new services can be adapted in the social welfare institutes, daily cares in home and hospitals (wada et al. ; yu et al. ) . their attachments with physically autonomous entities may offer them some reliefs and comforts. in such ways, as mentioned earlier, there are certainty to automatically diagnose clients by the ai using telecommunications and smartphone. sensing data accumulated by wearable devices in 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bayesian inference to fuse biosignal sensory estimates for personalizing care key: cord- -cbbtrksq authors: foxton, richard m. title: current concepts for operatively managing carious lesions extending into dentine using bioactive and adhesive direct restorative materials date: - - journal: jpn dent sci rev doi: . /j.jdsr. . . sha: doc_id: cord_uid: cbbtrksq this article reviews the current concepts for operatively managing carious lesions extending into dentine with minimal removal of tooth structure and restoring with dentine adhesives and direct composite resin. it looks sequentially at the operative steps involved including remineralisation with bioactive cements, bonding to caries-affected dentine including smear-layer modification using hocl, creation of an acid-base resistance zone,steps to optimise the performance of all-in-one adhesives, and incremental placement of direct composite resin to improve bond strengths to cavity floor dentine. it explores how understanding the phenomenon of colour-shifting at the composite resin-enamel/dentine restoration borders can help in creating near “invisible” restoration margins through a “chameleon” effect. with the increased risks of transmission of covid- when aerosol generating procedures are carried out in dentistry, following a minimally invasive approach to managing dental caries should be given serious consideration. this article reviews the current concepts for operatively managing and restoring carious lesions extending over one-third into dentine with adhesively-retaine ddirect composite resin. the following topics will be discussed: operative management of caries extending into dentine, remineralisation with bioactive cements, bonding to caries-affected dentine including smear-layer modification using hocl, creation of an acid-base resistance zone, steps to optimise the performance of all-in-one adhesives and incremental placement of direct composite resin to improve bond strengths to cavity floor dentine. an understanding of how the phenomenon of colour-shifting at the composite resin-enamel/dentine restoration borders can help in creating near "invisible" restoration margins through a "chameleon" effect will also be addressed. with the increased risks of transmission of covid- when aerosol generating procedures are carried out in dentistry, following a minimally invasive approach to managing dental caries should be given serious consideration with a heavy emphasis on prevention of caries by providing patients with appropriate dietary advice and oral hygiene instruction [ ] . should operative treatment be required, then reducing the amount of tooth structure that is removed, carrying out appropriate caries removaland use of a rubber dam will help lessen the quantity of aerosols generated, which might contain microorganisms that could be inhaled by dental staff involved with treatment [ ] . advances have taken place in understanding how dentine can be remineralised, bonding to caries-affected dentine can be achieved and how the strength and acid resistance of the adhesive interface created by dental adhesives can be improved. these factors can aid the clinical success of adhesive direct composite restorations, which when combined with the adoption of a minimally invasive approach now allow us to minimise the intervention required and these will be discussed in more detail. carious lesions extending beyond one third from the enameldentine junction (edj) into dentine will require operative management [ ] . operative management should aim to preserve as much tooth structure as possible. this can be achieved by carefully accessing the dentine caries with as little removal of tooth structure as possible and understanding the pathology of the carious process together with the published research on the clinical management. fusayama's classification of carious dentine into the two zones of "caries-infected" dentine and "caries-affected" dentine has https://doi.org/ . /j.jdsr. . . - /© the author. published by elsevier ltd on behalf of the japanese association for dental science. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). provided important guidance on how the histology of the dentine carious lesion can be related to the clinical situation [ ] . fusayama advocated removing caries-infected dentine, which is substantially demineralised and is physiologically un-remineralisable [ ] . this dentine consists of irreversibly denatured collagen fibrils and to leave behind the caries-affected dentine, which in contrast is minimally infected with bacteria, partially demineralised and physiologically remineralisable [ ] . it is not easy clinically to differentiate between caries-infected dentine and caries-affected dentine and it may not matter so much if the floor of the cavity is still caries-infected dentine rather than caries-affected dentine if a seal can be created after restoration. there is no solid evidence that it is necessary to remove all the caries-infected dentine and if present, even all the soft, wet carious dentine. if there is enamel present at the cavity margins then the deeper caries can be "sealed" after removal of superficial soft cariesif an adhesive restorative material is used [ ] . the classic study by mertz-fairhurst's group adhesively bonded composite and amalgam over soft carious dentine that was present even adjacent to the edj and although only % of the patients presented at recall years later, this approach led to a similar outcome as conventional caries removal [ ] . it is accepted that removal of "softened" carious dentine using hand excavators, initially adjacent to the enamel-dentine junction, keeping away from the deeper caries, will lessen the likelihood of a pulp exposure [ ] . however, it is important to understand that it is not necessary for the cavity to be completely caries-free as this will result in sound dentine being present on the cavity floor, which will lead to the exposure of dentinal tubules that will cause pain for the patient. what has been sometimes forgotten is that fusayama as well as advocating that complete caries removal is not necessary, also introduced the concept of "painless dentistry" [ ] . he advocated leaving the cavity floor consisting of caries-affected dentine, where the tubules are filled with whitlockite crystals formed by reprecipitation of calcium and phosphate ions lost during the carious process that can re-mineralise and forms a so-called "barrier" to the underlying pulp. he wrote in , "although dentists have heretofore believed that dental caries treatment must surely be accompanied by pain, our new concept of dental caries pathology reveals that the pain is caused by removing tissue that should not be removed", thereby introducing the concept of "painless" dentistry [ ] . removing "soft" caries carefully with an excavator that will be caries-infected dentine can be often accomplished without causing pain to the patient and this is all the operative intervention that is required on the carious dentine. this will also minimise the risk of post-operative sensitivity. it is not recommended that caries removal should be taken any further from a biological point of view [ ] . we should be thinking biologically and aiming to preserve pulp vitality at all costs ( fig. ) . with the increased risks of virus diffusion when carrying out aerosol generating procedures on patients who may have been infected with covid- [ ] , access cavities for caries ought to be kept as small as possible if using an air turbine, followed by careful excavation of only superficial, soft, wet, "infected" carious dentine.it could be argued that a clinician managing caries for the first time on a tooth carries a great responsibility to preserve as much healthy tooth tissue as possible. opting if possible, for a restorative material such as an adhesive direct composite resin restoration in the first instance after accessing and managing the caries. this often only requires minor subsequent cavity modifications and there have been several papers published showing its long-term clinical success over a period of years or more [ , ] . on the other-hand, if instead an indirect restoration is chosen, tooth tissue will need to be removed to create a cavity with diverging cavity walls, which is likely to give rise to additional loss of peripheral enamel and most likely sound dentine. cutting of sound dentine exposes denti-nal tubules causing the patient pain and must be sealed to prevent post-operative sensitivity. this could create an additional clinical problem that might otherwise be avoided. this approach therefore does not make sense from a biological point of view where only "diseased" tooth tissue is removed and as much healthy tooth tissue as possible is retained thus ensuring the tooth has the best possible chance of long-term survival. caries that has extended into dentine most likely leads to the pulp becoming "stressed". in , abou-rass introduced the "stressed" pulp as a clinical concept in which the pulp should be considered to be "stressed" if it has been subjected to repeated challenges to pathological changes such as caries [ ] . nowadays, if we believe this to be the case, it would make sense to consider placing a "bioactive" restorative material that may promote repair and remineralisation of the demineralised carious dentine left on the cavity floor, such as a glass-ionomer based or calcium-silicate based material [ ] . the setting reaction of glass-ionomer based materials and calcium-silicate materials are unlikely to stress the pulp further and the calcium-silicate cement, biodentine tm (septodont, saint-maur-des-fossé, france) in particular may have a therapeutic effect of 'pulp-healing" [ ] (fig. ). biodentine tm (septodont, saint-maur-des-fossé, france) or glass-ionomer provisional restorations should be cut back and layered with direct composite resin to provide a durable occluding surface [ ] . this could be as little as . - . mm, which could be pain-free for the patient if sound dentine is not cut. the cut-back restorative material will act as a "dentine" replacement and the overlying direct composite resin material as the enamel replacement (fig. ) . clinically, a decision has to be made therefore after managing a tooth with deep caries into dentine. the options are to proceed straightaway with a directly bonded resin composite restoration, which will create a "seal" and a durable, aesthetic restoration, or to adopt a two visit technique in which an attempt is made to manage the remaining carious dentine and potentially "stressed" pulp biologically. partial caries removal and cavity preparation using excavators and burs will leave a smear layer and caries-affected dentine as the substrates at the base of the cavity, which should be managed appropriately prior to applying a dentine adhesive. nakajima's group have reported that caries-affected dentine compared to sound dentine is softer even though the dentinal tubules are occluded with mineral deposits [ ] . moreover, nakajima described the smear layer created on caries-affected dentine as being thicker and more enriched with organic components than the smear layer created on sound dentine because the caries-affected dentine is partially demineralised compared to normal dentine [ ] . the remaining caries-affected dentine on the cavity floor is a challenging substrate to bond to. in the era of evidence-based dentistry it is poignant to appreciate that the majority of research on dentine adhesives has been carried out on extracted molars or bovine teeth with sound dentine. however, research on bonding to caries-affected dentine is limited. the clinician has various choices with regards to dentine adhesives and there are two broad groups: etch and rinse adhesives and self-etching adhesives. with regards to the "etch-and-rinse" adhesives, the phosphoric acid etchant will dissolve the smear layer and demineralise the surface dentine. it can be difficult for the resin monomers to infiltrate to the full depth of demineralised dentine and when a "wet bonding" technique is used, water will also compete with the resin monomers to penetrate to the full depth of demineralisation [ ] . the occluded dentinal tubules in the caries affected dentine cannot be completely dissolved by phosphoric acid and this makes them acid resistant [ ] . however, because the caries-affected dentine is less mineralised compared to normal dentine, a deeper zone of demineralised dentine is created after phosphoric acid etching, which will result in a thicker hybrid layer after resin infiltration [ ] . on the other hand, beneath the hybrid zone there will be a porous region of dentine that has been created because of the greater depth of demineralisation and the mineral deposits present in the tubules [ ] . the net result of this is a reduction in resin-dentin bond strengths to caries-affected dentine compared to sound dentine. this porous zone of weakness below the hybrid layer can be potentially strengthened by the addition of chemical cross linkers, which mechanically strengthen this incompletely resin-infiltrated zone [ ] . self-etching adhesives, in particular the two-step self-etching systems, offer the advantage of not requiring dentine to be etched by phosphoric acid and the acidic self-etching primer will dissolve the smear layer and demineralise a thin zone of underlying dentine. the zone of demineralisation is not as deep in normal dentine as that created by phosphoric acid, which makes it easier for the subsequently applied bonding resin to infiltrate the demineralised dentine resulting in a thin but "high quality" hybrid layer [ ] . however, in caries-affected dentine, the hybrid layer created by a self-etching adhesive is thicker, though not as thick when an "etch and rinse" adhesive is used, and a porous zone has been observed beneath the hybrid layer created by a self-etching adhesive [ ] . a deeper zone of demineralisation is created in caries-affected dentine and resin infiltration is also hampered by the thicker smear layer, which the mildly acidic self-etching primer is not able to fully dissolve. this results in the self-etching adhesives having lower bond strengths to caries-affected dentine compared to sound dentine [ ] . in order to improve the bonding of self-etching adhesives to caries-affected dentine, researchers have looked at modifying the thicker smear layer present on caries-affected dentine [ ] . initially, sodium hypochlorite solution was investigated as this solution can dissolve organic substances and when applied to smear-layer covered caries-affected dentine at a concentration of % for s, it was shown that the smear layer was thinned down and dissolved away resulting in significant improvements of the bond strengths of one and two-step self-etching adhesives [ ] . further research by the same research group has investigated the application of a mild acidic solution of hocl (comfosy, haccpperadvantec corp., tokyo, japan), which has antimicrobial, deproteinising and low cytotoxicity properties on caries-affected dentine and observed that a better quality of hybrid layer was created as well as more stable bond strengths [ ] . it has been mentioned earlier that phosphoric acid etching of dentine may result in the phenomenon of "over-etched" dentine, whereby the resin is unable to fully penetrate to the full depth of demineralisation resulting in a zone of potential weakness beneath the hybrid layer [ ] . this zone will be demineralised and not be fully infiltrated with resin and could influence the longevity of a composite resin restoration particularly if placed in a tooth subjected to high occlusal stress. on the otherhand, self-etching adhesive systems do not require prior phosphoric acid etching of dentine and therefore, "aggressive" etching of dentine is avoided [ ] . nikaido previously discovered an "acid-base resistant" zone beneath the hybrid layer, when bonded dentine-composite resin discs were subjected to an artificial secondary caries attack [ ] . his research group investigated a two-step, self-etching adhesive, clearfil se bond (kuraray noritake dental inc., japan), and observed that the hybrid layerwas approximately m thick after argon ion etching in sound dentine and was slightly thicker in caries-affected dentine [ ] . beneath the hybrid layer of this adhesive, an acid-base resistant zone was observed in both sound and caries-affected dentine bonded specimens, which was observed to be m thick in the sound dentine and . m thick in the caries-affected dentine [ ] . this zone was not observed to be present for adhesive systems that required prior etching with phosphoric acid. it was reasoned that this was because phosphoric acid aggressively etched the dentine and created a zone of demineralisation that was too deep for resin to fully infiltrate [ ] . this is potentially significant in that secondary caries will occur at the margins of restorations between the dentine and resin restoration and therefore the presence of an acid-base resistant zone may help prevent secondary caries around a restoration. it is relevant at this point to mention that in , nakabayashi described the resin infiltration of tooth structure as follows; "the resin reinforced dentine and enamel is a hybrid of natural tissue and artificial material and is valuable in the prevention of secondary caries after restoration" [ ] . and so, even though it was thirty-five years ago when prevention of secondary caries by hybrid layer formation was first proposed, it is interesting that discussion on the effectiveness of the hybrid layer to prevent secondary caries is sometimes omitted in the scientific discussions when adhesive performance is discussed. nikaido described the acid-base resistant zone as a combination of dentine and the adjacent hybrid layer which, because it is effectively reinforced, has suggested this region of the adhesive interface should be called "super dentine", which possesses an ability to prevent primary and secondary caries [ ] . this is because it is superior to sound dentine mechanically, chemically and biologically. nikaido's proposal is arguably, a further development and extrapolation of the concept of that the hybrid layer can prevent secondary caries formation, as was first described by nakabayashi in [ , ] . having now discussed how we could provide "painless" dentistry by careful removal of soft, caries-infected dentine, seal the cavity and prevent caries progression by adhesive enamel bonding and how the hybrid layer and an acid-base resistant zone offer the ability to prevent primary and secondary caries, it is clear that carefully applied adhesive direct composite resin restorations can offer the patient additional benefits by their ability to fill minimally prepared cavities, offer aesthetically pleasing results and good long-term durability [ ] . while the words "minimally invasive dentistry" are mentioned nowadays as if it was a new treatment modality, it's origins are surely in the mid s with the work of fusayama and nakabayashi who suggested that caries can be prevented by hybrid layer formation at the adhesive interface of direct composite resin restorations. the remainder of this article will discuss some key aspects of direct composite resin placement to obtain good adhesion and colour matching. there are multiple choices for the clinician when selecting which direct composite resin materials should be used as a restorative material. an initial question might be whether a thin layer of a "flowable" composite resin should be placed initially in the cavity? as with many clinical techniques it is often difficult to provide a definitive answer. the rationale of placing a low-viscosity or flowable composite resin as an initial thin layer after curing of the adhesive is that this may dissipate contraction stresses that are created when the composite resin is light-cured by the creating of a stress-absorbing layer that helps preserve the integrity of the bonded adhesive interface [ ] . in , reis and colleagues looked at the effect of filling techniques and a low-viscosity composite liner on bond strength to class ii cavities prepared in extracted human third-molar teeth and found that bond strengths were not improved when a low-viscosity composite resin was used but an incremental filling technique improved bond strengths [ ] . research was then focussed on whether a particular type of adhesive might benefit from a subsequent application of a thin layer of flowable composite resin and work by de goes' group tried to answer this question by looking at adhesives representing the four classes of adhesives at that time [ ] . the occlusal surfaces of extracted third molars were ground to expose dentine and were treated with one of four adhesives: -step etch-andrinse (scotchbond multi-purpose, m, usa); -step etch-and-rinse (singlebond, m, usa); all-in-one self-etching adhesive (adper prompt, m, usa); -step self-etching adhesive (clearfil se bond, kuraray noritake dental inc., japan) [ ] . their results showed that application of an intermediate flowable composite layer improved the bond strength of all four adhesives and this was significant for clearfil se bond (kuraray noritake dental inc., japan) [ ] . early all-one-adhesives such as adper promptl-pop ( m, usa) contained a functional monomer that dissociated into a hydrolytically unstable monomer because of the presence of water at a concentration of %- %. this allowed fluid penetration through the adhesive and led to the hybrid layer being susceptible to cracking after polymerisation of the overlying composite resin [ , ] (fig. ) . the simplicity of all-in-one adhesives led many researchers to look at ways of improving the durability of the adhesive bond and the mechanical properties of the adhesive interface to withstand the contraction stresses of the overlying composite resin as it polymerised and manufacturers to look at new compositions. the expiry of the patent in for mdp, which was held by kuraray noritake dental inc. japan, has led to several manufacturers, developing new adhesives that contain mdp as the functional adhesive monomer and their terminology has also evolved to "universal adhesives" [ ] . mdp has been shown to exhibit ionic bonding to hydroxyapatite more effectively than other adhesive monomers such as -met through the creation of an mdp-ca salt deposition and nanolayering [ ] . applying the all-in-one adhesive as a double application before curing has been investigated using four, one-step self-etching adhesives (ibond se, kulzer gmbh, germany; clearfil s bond,(kuraray noritake dental inc., japan; xenov, dentsply sirona, usa; and scotchbond universal, m, usa) with extracted human third molars as the bonding substrate under different storage conditions [ ] . it was found that ibond se, clearfil s bond and xenov exhibited significantly higher bond strengths when they were applied as a double layer before light-curing [ ] . another adhesive application technique, "scrubbing"the dentin surface with a micro-sponge, has been suggested for certain onestep self-etching adhesives to increase the bond strength [ ] . the effect of scrubbing dentine during application was investigated using two, one-step self-etching adhesives on extracted third molar teeth (se one, kuraray noritake dental inc., japan; and scotchbond universal, m, usa) and it was found that scrubbing improved etching ability and significantly improved the bond strength of scotchbond universal ( m, usa) [ ] . one more technique proposed to improve the bond strength of universal adhesives is to apply a subsequent coating of a thin layer of a hydrophobic resin, thereby converting the adhesive strategy to a two-step self-etching one [ ] . extracted third molar teeth were used and when a layer of heliobond (ivoclarvivadent ag, liechtenstein) was applied to three universal adhesives (all-bond universal, bisco, usa; g-bond plus, gc corporation, japan; and scotchbond universal, m, usa) in their self-etching mode, adhesive performance was improved [ ] . what this means for clinical practice is that while the abovementioned studies using universal adhesives all used extracted third molars, their results do indicate that if a universal adhesive has been chosen by the clinician, then thought should be given not only to its application technique, be it by "scrubbing" or as a double-coat, but whether consideration should be given to applying an additional thin layer of hydrophobic resin before applying the composite resin or a flowable composite resin material. it has been established that shrinkage stresses develop in composite resin during polymerisation and if the cavities increase in size then volumetric shrinkage may also increase [ ] . polymerisation shrinkage cannot be avoided, and the adhesive system must possess sufficient mechanical strength to avoid being weakened [ ] . research using simulated occlusal cavities in resin and occlusal cavities cut in extracted human molarsshowed that an incremental filling technique was more effective in improving adhesion to the cavity floor than a bulk filling technique [ , ] . for patients, a good aesthetic outcome is often important and direct composite resin can give excellent results if attention is paid to shade matching with the surrounding tooth structure. the colour of the resin composite is influenced and perceived by various factors; colour elements of hue value and chroma; and optical properties. the optical properties include light transmission characteristics (straight-line and diffusion), translucency, fluorescence, and opalescence, along with the surface characteristics of the composite resin [ ] . composite resin and enamel are translucent materials, which can reflect colours from adjoining substrates, leading to the colour shifting between each other [ ] . obtaining a near "invisible" restoration margin through good colour matching with the adjacent tooth structure can help ensure a pleasing result for the patient (fig. ) . several factors come into play when considering how best to get a good colour match with a synthetic restorative material to natural living tooth structure. extracted human premolar teeth and four different composite resin restorative materials (clearfil ap-x, kuraray noritake dental inc., japan; clearfil majesty, kuraray noritake dental inc., japan; tetric n ceram, ivoclarvivadent ag, liechtenstein; and ceram x mono, dentsply sirona, usa) have been used to look at the colour differences in four regions: the resin composite restorations, within the tooth, at the composite border area and at the tooth border area [ ] . it was found that colour changes occurred at the borders of the composite resin restoration with the adjacent tooth structure and on both sides of the restoration border, within the tooth structure and the composite restoration [ ] . colour changes in the tooth structure adjacent to the restoration border were therefore due to colour reflection from the composite restoration. colour changes within the border of the composite restoration were due to colour reflection from the tooth structure. colour changes in the tooth structure adjacent to the restoration border, were larger than the colour changes in the composite restoration adjacent to the border [ ] . a goniophotometer was also used to investigate how light was transmitted through the four resin composite materials [ ] . ceram-x and clearfil majesty showed that light diffused more within the composite material than within clearfil ap-x and tetric n ceram. different depths of cavity preparation ( . mm and mm) were also investigated [ ] . for the mm-deep cavities, the values for light diffusivity were compared with the colour differences measured for each material at the different border regions and it was concluded that the materials exhibiting good light diffusivity within the composite resin are able to create a chameleon effect as result of reflection of the surrounding tooth colour and projection of its own colour into the surrounding tooth structure, which would result in good colour matching [ ] . on the otherhand, in the cavities that were . mm deep, it was found that light diffusivity had less influence on the colour changes and the colour rendition of the composite restorations was more influenced by the colour of the cavity floor. in the shallower cavity, the materials which exhibited better straight-line light transmission through the resin in this case, tetric n ceram, reflected the background colour better [ ] . the clinical relevance of these findings is that in order to successfully colour match a composite restoration with the surrounding tooth structure, consideration must be given to the surrounding tooth structure, which might have to be masked. moreover, the straight-line and diffused light transmission properties of the composite resin materials must be considered.further work has been carried out on how the colour of the surrounding tooth structure might influence the colour of the composite restoration border [ ] . how the age of the tooth, in particular colour changes that occur in dentine with aging, through factors such as physiological aging and the creation of transparent dentine as a result of mineral deposition in the dentinal tubules, might influence the colour of the restoration at the borders has been investigated [ ] . human premolars extracted for orthodontic reasons (younger teeth, age - years) and premolar teeth extracted for periodontal reasons (older teeth, age - years) were used along with four direct composite resins, kalore (gc corporation, japan), solare (gc corporation, japan), clearfil majesty (kuraray noritake dental inc., japan) and beautiful ii (shofu, japan)) all of shade a [ ] . all four materials exhibited colour differences between the restoration borders and the dentine and composite resin in both the "younger" and "older" teeth, indicating "colour shifting" had occurred at the restoration borders [ ] . however, when the "younger" and "older" teeth were looked at, colour differences in the older teeth at the restoration borders were greater than those for the "younger" teeth, indicating that potential for colour adjustment in older teeth would be less than it would be for younger teeth [ ] . the straight-line and diffusivity light transmission properties of the young and old dentine were also investigated. the older dentine exhibited higher straightline light transmission values than younger dentine, indicating better light transmission, whereas the younger dentine exhibited better light diffusion values, indicating less light transmission [ ] . clinically, this would mean that better colour matching would be required when restoring older teeth compared to younger teeth. the effect of the enamel margin on colour adjustment has also been investigated [ ] . three types of enamel margin (non-bevel, -degree bevel and -degree reverse-bevel) and four composite resin materials (estelite asteria a b, tokuyama dental corp., japan; estelite pro a e, tokuyama dental corp., japan; kalore e , gc corporation, japan; and clearfil majesty es- premium a e, kuraray noritake dental inc., japan) were studied in bovine enamel [ ] . the study found that the enamel margin configuration affected colour adjustment at the borders of the restoration with the bevel preparation improving colour adjustment at the borders [ ] . with regards to the four composite resin materials, they had different values for straight-line light transmission and light diffusivity within the materials and the degree of colour adjustment at the borders of the restoration was material dependent [ ] . if a cavity involved the labial aspect of the tooth, which is likely to be highly visible to the patient, then the orientation of the enamel prisms would differ depending on whether the cavity margin was in coronal or cervical regions of the enamel. enamel prisms are the most important structure in enamel for influencing light scattering, an important optical property.the effect of enamel prism orientation on colour shifting at the border of composite resin restorations was investigated using bovine enameland four composite resin materials (estelite asteria a b, estelite pro a e, kalore e and clearfil majesty es- premium a e) bonded using a one -step self-etching adhesive (clearfil se one) [ ] . in the coronal enamel, the prisms would be cut diagonally, whereas in the cervical region, they would have been cut longitudinally [ ] . two sizes of composite restoration in enamel were prepared; mm wide and mm wide, both mm thick. the colour shift in the l* value was greater in the enamel side of the restoration [ ] . the colour shift in the l* value was also significantly greater at the cervical border than the coronal border and the difference in value was also larger in the small cavity [ ] . clinically this would indicate that the potential for colour adjustment, particularly lightness, is greater in the coronal border region of a composite restoration because the gentle colour shifting and larger area of colour shifting would make the restoration border less obvious. with regards to placement of the composite resin, as mentioned previously, placing the composite resin in increments can aid colour matching, ensure adequate polymerisation of the increment and minimize the effect of contraction stress on adhesion to cavity walls [ ] . the effect of layering composite resin using both conventional and bulk-filling techniques on translucency and colour appearance has been investigated [ ] . three resin composites (estelite sigma, tokuyama dental corp., japan; beautiful ii, shofu, japan; clearfil majesty, kuraray noritake dental inc., japan) in both a and oa shades were evaluated [ ] . the universal (a ) shade specimens exhibited higher translucency than the opaque-shade (oa ) specimens, which would indicate that the oa shades would be better for masking colour [ ] . in addition, layering in mm-thick incre-ments resulted in less transparency than a mm-thick increment indicating light diffusion occurring at the composite-composite joint [ ] . clinically this means that after a shade of composite resin is chosen, the final colour might change depending on how thick the increments of composite resin were when they were placed. finally, a restoration will be viewed by the patient from many directions and the effect of the direction of the light illuminating a direct composite restoration has been investigated [ ] . it was found that the colour appearance at the border of composite resin restorations is influenced by the light illuminating direction in conjunction with the enamel rod orientation [ ] . in other words, the appearance of a patient's restoration around the cavity margin, particularly towards the coronal aspect, will differ depending on the angle at which it is viewed by another person. none. covid- transmission in dental practice: brief review of preventive measures in italy when to intervene in the caries process? an expert delphi concensus statement two layers of carious dentin: diagnosis and treatment how "clean" must a cavity be before restoration? ultraconservative and cariostatic sealed restorations: results at year treatment of deep caries lesions by complete excavation or partial removal a simple pain-free adhesive restorative system by minimal reduction and total etching a randomized controlled years follow up of three resin composites in class ii restorations a randomized controlled years follow up of three conventional resin composites in class ii restorations the stressed pulp condition: an endodontic-restorative diagnostic concept present and future of glassionomers and calcium-silicate cements as bioactive materials in dentistry: biophotonics-based interfacial analyses in health and disease clinical and radiographic assessment of the efficacy of calcium silicate indirect pulp capping. a randomized controlled clinical trial clinical evaluation of the performance and safety of a new dentine substitute, biodentine, in the restoration of posterior teeth -a prospective study bonding to cariesaffected dentin tensile bond strength and sem evaluation of caries-affected dentin using dentin adhesives bonding of selfetch and total-etch adhesives to carious dentin improving the effect of naocl pretreatment on bonding to caries-affected dentin using self-etch adhesives pretreatment effect of mild acidic hocl solution on adhesive to caries-affected dentin using self-etch adhesive new strategy to create "super dentin" using adhesive technology: reinforcement of adhesive-dentin interface and protection of tooth structures morphological and mechanical characterization of the acid-base resistance zone at the adhesive-dentin interface of intact and caries-affected dentin bonding of restorative materials to dentine: the present status in japan assessment of the nanostructure of acid-base resistant zone by the application of all-in-one adhesive systems: super dentin formation a morphological and tensile bond strength evaluation of an unfilled adhesive with low-viscosity composites and a filled adhesive in one and two coats the effects of filling techniques and a low-viscosity composite liner on bond strength to class ii cavities microtensile bond strength of adhesive systems to dentin with or without application of an intermediate flowable resin layer origin of interfacial droplets with one-step adhesives long-term durability of one-step adhesive composite systems to enamel and dentin universal adhesives: the next evolution in adhesive dentistry? compend selfassembled nano-layering at the adhesive interface effect of double-layer application on dentin bond durability of one-step selfetch adhesives effect of scrubbing technique with mild self-etching adhesives on dentin bond strengths and nanoleakage expression in vitro longevity of bonding properties of universal adhesives to dentin increased wall-to-wall curing contraction in thin bonded resin layers effect of incremental filling technique on adhesion of light-cured resin composite to cavity floor bond strength and ultimate tensile strength of resin composite filled into dentine cavity; effect of bulk and incremental filling technique the combined effect of light-illuminating direction and enamel rod orientation on color adjustment at the enamel borders of composite restorations color shifting at the border of resin composite restorations in human tooth cavity the effect of tooth age on colour adjustment of resin composite restorations effect of enamel margin configuration on color change of resin composite restoration influence of enamel prism orientations on color shifting at the border of resin composite restorations influences of composite-composite join on light transmission characteristics of layered resin composites key: cord- - v sz xm authors: lee, k.; iwata, t.; nakadai, a.; kato, t.; hayama, s.; taniguchi, t.; hayashidani, h. title: prevalence of salmonella, yersinia and campylobacter spp. in feral raccoons (procyon lotor) and masked palm civets (paguma larvata) in japan date: - - journal: zoonoses public health doi: . /j. - . . .x sha: doc_id: cord_uid: v sz xm to estimate the public and animal health risk that alien species pose, the prevalence of salmonella, yersinia, and campylobacter spp. in feral raccoons (procyon lotor, n = ) and masked palm civets (paguma larvata, n = ), which are abundant alien species in japan, was investigated in urban and suburban areas of japan. salmonella enterica was detected from samples [ raccoons, . %, % confidence interval (ci) . – . %; three masked palm civets, . %, % ci . – %]. many of the isolates belonged to serovars that are commonly isolated from human gastroenteritis patients (e.g. s. infantis, s. typhimurium, and s. thompson). the antimicrobial susceptibility test showed that . % of the isolates from raccoons were resistant to at least one antimicrobial agent, whereas none of the isolates from masked palm civets were resistant. yersinia sp. was detected from samples ( raccoons, . %, % ci . – . %; masked palm civets, . %, % ci . – . %). all virulent yersinia strains belonged to yersinia pseudotuberculosis, which was isolated from seven ( . %, % ci . – . %) raccoons and six ( . %, % ci . – . %) masked palm civets. according to the detection of virulence factors, all the y. pseudotuberculosis isolates belonged to the far eastern systemic pathogenicity type. campylobacter spp. was detected from samples (six raccoons, . %, % ci . – . %; masked palm civets, . %, % ci . – . %). among these, three isolates from raccoons were identified as c. jejuni. these results showed that these pathogens can be transmitted by human activities, other wild animals, and the environment to feral raccoons and masked palm civets, and vice versa. as these animals have omnivorous behaviour and a wide range of habitats, they can play an important role in the transmission of the enteric pathogens. the raccoon (procyon lotor) is a medium-sized mammal that is widely distributed in north america. raccoons were introduced into japan in the s and have become naturalized in at least of prefectures (ikeda et al. , to estimate the public and animal health risk that alien species pose, the prevalence of salmonella, yersinia, and campylobacter spp. in feral raccoons (procyon lotor, n = ) and masked palm civets (paguma larvata, n = ), which are abundant alien species in japan, was investigated in urban and suburban areas of japan. salmonella enterica was detected from samples [ raccoons, . %, % confidence interval (ci) . - . %; three masked palm civets, . %, % ci . - %]. many of the isolates belonged to serovars that are commonly isolated from human gastroenteritis patients (e.g. s. infantis, s. typhimurium, and s. thompson). the antimicrobial susceptibility test showed that . % of the isolates from raccoons were resistant to at least one antimicrobial agent, whereas none of the isolates from masked palm civets were resistant. yersinia sp. was detected from samples ( raccoons, . %, % ci . - . %; masked palm civets, . %, % ci . - . %). all virulent yersinia strains belonged to yersinia pseudotuberculosis, which was isolated from seven ( . %, % ci . - . %) raccoons and six ( . %, % ci . - . %) masked palm civets. according to the detection of virulence factors, all the y. pseudotuberculosis isolates belonged to the far eastern systemic pathogenicity type. campylobacter spp. was detected from samples (six raccoons, . %, % ci . - . %; masked palm civets, . %, % ci . - . %). among these, three isolates from raccoons were identified as c. jejuni. these results showed that these pathogens can be transmitted by human activities, other wild animals, and the environment to feral raccoons and masked palm civets, and vice versa. as these animals have omnivorous behaviour and a wide range of habitats, they can play an important role in the transmission of the enteric pathogens. roundworm (baylisascaris procyonis) (gavin et al., ) , rabies virus (finnegan et al., ) , leptospira spp. (hamir et al., ) , and francisella tularensis (berrada et al., ) . bigler et al. ( ) found that raccoons are so adaptive that they can bridge the gaps among avian, terrestrial, and aquatic environments, and thus they are appropriate as an indicator of the prevalence of various infectious diseases and pollutants. the masked palm civet (paguma larvata) is also a medium-sized carnivore and is distributed widely in asia. masked palm civets are thought to have been introduced into japan, although this contention is controversial (abe, ) . masked palm civets often live in similar places as raccoons and can be carriers of human and animal pathogens, such as severe acute respiratory syndrome (sars) virus (tu et al., ) and canine distemper virus (machida et al., ) . in japan, attention has been focused on the role of these highly adaptive mammals in the transmission of zoonotic pathogens, including trichinella t (kobayashi et al., ) , babesia microti-like parasite (kawabuchi et al., ) , ehrlichia spp., anaplasma phagocytophilum (inokuma et al., ) , strongyloides procyonis (sato and suzuki, ) , and canine distemper virus (machida et al., ) . pathogens carried by these animals can present serious public and animal health problems in the habitats to which they have been introduced. however, in spite of the fact that these two species often inhabit urban and suburban areas, there have been few reports on the prevalence of enteric pathogens among them. the objective of this study was to determine the prevalence of the causal agents of enteric diseases including salmonella spp., yersinia spp., and campylobacter spp. in feral raccoons and masked palm civets. we then assessed antimicrobial resistance in the isolates and analysed them using polymerase chain reaction (pcr) to elucidate their virulence factors and transmission routes. from march to may , feral raccoons and feral masked palm civets were captured in kanagawa, gunma, and tokyo prefectures by each municipality as a part of the local governmental control and eradication programmes. the animals were caught using box traps and were killed by humanitarian methods (japan veterinay medical association, ). of feral raccoons, ( . %) were males, ( . %) were females, and ( . %) were of unknown sex. of feral masked palm civets, ( . %) were males, ( . %) were females, and two ( . %) were of unknown sex. the age of the feral raccoons and masked palm civets was determined on the basis of tooth eruption and cranial suture obliteration by the method of montgomery ( ) and junge and hoffmeister ( ) . of feral raccoons, ( . %) were juveniles (< months), ( . %) were sub-adults or adults, and ( . %) were of unknown age. of feral masked palm civets, ( . %) were juveniles (< months), ( . %) were sub-adults or adults, and ( . %) were of unknown age. faecal samples were collected and preserved in cary and blair transport medium (eiken chemical co. ltd., tokyo, japan) or sterile centrifuge tubes. the samples were then transported to the laboratory. the samples were suspended in ml of sterile phosphate-buffered saline (pbs; ph . ) and tested within a week after collection. one millilitre of each specimen was inoculated into ml of buffered peptone water (bpw; becton dickinson, franklin lakes, nj, usa). after incubation at ºc for h, ml of bpw culture was transferred to ml of hajna-tetrathionate broth (eiken chemical). the broth was incubated at ºc for h, then one loopful of each tube was inoculated onto a plate of desoxycholate hydrogen sulfide lactose agar (dhl; nissui pharmaceutical co. ltd., tokyo, japan) and mannitol lysine crystal violet brilliant green agar (mlcb; nissui). these plates were incubated at ºc for h, and at least two suspicious colonies morphologically similar to salmonella spp. from each plate were subcultured for biochemical examinations. biochemical characteristics were examined on triple sugar iron medium (nissui) and lysine indole motility medium (nissui). the subspecies of salmonella isolates were confirmed by biochemical examinations and the multiplex pcr assay (popoff and le minor, ; lee et al., ) . serotyping for salmonella isolates was accomplished with commercial o and h antisera (denka seiken co. ltd., tokyo, japan) according to the method of popoff and le minor ( ) . specimens were incubated at ºc for weeks. after alkali treatment (aulisio et al., ) , a loopful of sample suspension was spread on virulent yersinia enterocolitica (vye) agar (fukushima, ) and irgasan-novobiocin (in) agar containing . mg/l of irgasan and novobiocin in yersinia selective agar base (difco laboratories, detroit, mi, usa) (schiemann, ) . all plates were incubated at ºc for h. colonies morphologically similar to yersinia spp. were subcultured for biochemical examination. the identification of yersiniae was performed by the methods of wauters et al. ( ) . all isolates identified as yersiniae were subjected to autoagglutination tests to evaluate their potential pathogenicity (laird and cavanaugh, ) . then, virulent isolates were subjected to further analysis. serotyping of y. pseudotuberculosis was accomplished by slide agglutination with commercial antisera (denka seiken). isolates identified as y. pseudotuberculosis were genotyped by the presence patterns of the gene encoding y. pseudotuberculosis-derived mitogen typea (ypma) and high-pathogenicity island (hpi) so as to analyse their geographical origin using pcr assay (fukushima et al., ) . one millilitre of the specimen was inoculated into ml of preston enrichment broth (oxoid cm + sr e + sr e) containing % defibrinated horse blood. after incubation at ºc for h, one loopful of each tube was inoculated onto a skirrow blood agar plate (oxoid cm + sr e + sr e) containing % defibrinated horse blood. all skirrow blood agar plates were incubated for h at ºc under microaerobic conditions and examined for the presence of characteristic colonies of campylobacter. confirmation and characterization of the isolates were performed on the basis of microscopic morphology, an oxidase test, a catalase test, growth at ºc and ºc, and a multiplex pcr assay for c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus (wang et al., ; vandamme et al., ) antimicrobial susceptibility test antimicrobial susceptibility testing was performed according to the disc diffusion method (national com-mittee for clinical laboratory standards, ; luangtongkum et al., ) . the following antimicrobial paper discs (sensi-disc; becton dickinson) were used: ampicillin ( lg/disk), cefazolin ( lg/disk), ceftriaxone ( lg/disk), ciprofloxacin ( lg/disk), chloramphenicol ( lg/disk), gentamicin ( lg/disk), kanamycin ( lg/disk), nalidixic acid ( lg/disk), oxytetracycline ( lg/disk), and streptomycin ( lg/disk) for salmonella and y. pseudotuberculosis; and ampicillin, ciprofloxacin, clindamycin ( lg/disk), erythromycin ( lg/disk), gentamicin, kanamycin, nalidixic acid, norfloxacin ( lg/disk), oxytetracycline, and streptomycin for campylobacter. differences of the prevalence were analysed by the chi-squared test in spss software (spss inc., chicago, il, usa). the raccoons and masked palm civets were essentially normal except for various external parasites. no discernible pathological evidence or signs of disease were observed. there were no significant sex-and age-specific differences in salmonella spp., yersinia spp., and campylobacter spp prevalence in both animals (table ) . there was no sample which was positive for more than two bacterial species or serotypes tested in this study, except nonpathogenic yersinia spp. enteric pathogens in raccoons and masked palm civets k. lee et al. salmonella enterica was isolated from the faecal samples of of raccoons ( . %, % ci . - . %) and three of masked palm civets ( . %, % ci . - %). table shows the serovars and antimicrobial resistance patterns of the isolates. nine and three serovars were identified in the isolates from raccoons and masked palm civets, respectively. those serovars included common serovars in gastroenteritis patients and domestic animals (e.g. s. infantis, s. typhimurium, s. thompson, and s. enteritidis) (esaki et al., ; ishihara et al., ; national institute of infectious disease, ). seven of isolates ( . %) from raccoons showed resistance to at least one antimicrobial agent used in this study, whereas all isolates from masked palm civets were susceptible to all of the antimicrobial agents. yersinia spp. were isolated from the faecal samples of of raccoons ( . %, % ci . - . %) and of masked palm civets ( . %, % ci . - . %). among the isolates, seven strains from raccoons and six strains from masked palm civets showed positive reactions in autoagglutination tests and were subsequently identified as y. pseudotuberculosis. therefore, the prevalence of y. pseudotuberculosis in raccoons and masked palm civets was . % ( % ci . - . %) and . % ( % ci . - . %), respectively. four, one, and two y. pseudotuberculosis isolates from raccoons belonged to serotypes b, , and b, respectively. three, two, and one y. pseudo-tuberculosis isolates from masked palm civets belonged to serotypes b, , and b, respectively. all y. pseudotuberculosis isolates were susceptible to all of the antimicrobial agents used in this study. in all of the y. pseudotuberculosis isolates, the gene of ypma was detected and the gene of hpi was not detected by pcr assay. campylobacter spp. were isolated from the faecal samples of six of raccoons ( . %, % ci . - . %) and of masked palm civets ( . %, % ci . - . %). three isolates from raccoons were identified as c. jejuni by multiplex pcr assay. the other campylobacter isolates from raccoons and masked palm civets exhibited three different phenotypic patterns (table ). all the campylobacter isolates were susceptible to all of the antimicrobial agents used in this study. in this study, the prevalence of s. enterica in raccoons ( . %) was almost concordant with the prevalence in western pennsylvania, usa ( . %) (compton et al., ) . investigations in other wild mammals also have revealed similar prevalence rates in uk ( . %) and spain ( . %) (euden, ; millan et al., ) . however, morse et al. ( ) reported that s. enterica was isolated from . % of feral raccoons. this finding may suggest that raccoons have the potential to harbor s. enterica at a high rate. the prevalence of s. enterica in masked palm civets was somewhat lower than that in raccoons. differences in their behaviour could be responsible for the difference in the prevalence. further analysis of their food habits and habitat choice may verify this hypothesis. some of the isolates were indicated to have originated from human activities, because many of the serovars isolated have been common in human gastroenteritis and in domestic animals, and the isolates from raccoons showed a relatively high resistance rate ( . %). interestingly, six strains isolated from raccoons were identified as s. enterica subsp. diarizonae. because this salmonella subspecies is not common in warm-blooded animals but is common in cold-blooded animals and the environment, it may be associated with their omnivorous behaviour and proclivity for wet habitats (zeveloff, ; bopp et al., ; haley et al., ) . yersinia pseudotuberculosis isolates belonged to serotypes b, , and b, which are predominant serotypes in human patients and wild animals in japan (hamasaki et al., ; fukushima and gomyoda, ; hayashidani et al., ) . the prevalence of y. pseudotuberculosis in raccoons and masked palm civets was comparable with that of those studies. it is difficult to compare the prevalence with that in its place of origin, because there are few reports on isolation of pathogenic yersinia from raccoons and masked palm civets (hacking and sileo, ) . all of the y. pseudotuberculosis strains showed the same genotypic pattern with the ypma + hpi ) far eastern systemicpathogenicity type. fukushima et al. ( ) reported that most of the strains isolated in far east asia showed such a pattern and differed from the strains isolated in european countries. in addition to the information regarding geographical origin, this virulence characteristic has a clinical implication, because ypma encodes ypma, which contributes to the virulence of y. pseudotuberculosis in systemic infection (carnoy et al., ) . these results lead to the conclusion that raccoons and masked palm civets probably have acquired their infections in japan and play a similar role to other indigenous animals on the ecology of y. pseudotuberculosis. in previous studies, natural reservoirs of y. pseudotuberculosis in japan have been suggested to be wild mammals and birds, especially rodents and raccoon dogs (nyctereutes procyonoides) (hamasaki et al., ; fukushima and gomyoda, ; hayashidani et al., ) . the major transmission routes of the pathogen in wildlife are suggested as preying upon infected animals or ingesting environmental substances contaminated with y. pseudotuberculosis rather than contact with human activities (fukushima and gomyoda, ) . it is likely that raccoons and masked palm civets acquired their infection, as a result of sharing habitats with other reservoirs. the principal reservoirs of campylobacter in the environment are wild mammals and birds (mörner, ) , with the occurrence of enteric campylobacter higher in birds than in wild mammals. this tendency is consistent with our results. the prevalence rates in raccoons and masked palm civets were . % and . %, respectively, whereas the rate is often more than % in wild birds (kapperud and rosef, ; matsusaki et al., ; ito et al., ) . however, wild mammals, especially species that have direct or indirect contact with human activities, are still important reservoirs of the pathogen. workman et al. ( ) demonstrated that dogs were one of the most likely sources of human campylobacteriosis. domestic animals, including dogs, are more likely to have opportunities to acquire infections from excretory substances or from environments contaminated by raccoons and masked palm civets. among the isolates, three strains were identified as c. jejuni. however, the other isolates from both raccoons and masked palm civets could not be identified by the multiplex pcr assay. additional biochemical tests suggested that these strains belonged to uncommon species of campylobacter (table ) . as such species (e.g. c. hyointestinalis, c. larienae or c. rectus) are isolated from healthy animals and enteritis patients, their pathogenicity and epidemiology have not been well known (vandamme et al., ) . because there is little information about the carriage of uncommon campylobacter species in wild mammals, further investigations are required to elucidate the real impact of these species in wild animals on public and animal health. from these results, we concluded that raccoons and masked palm civets could be potential reservoirs of enteropathogenic campylobacter (c. jejuni) and are more likely to possess uncommon campylobacter species. to assess the risk and potential source, wild birds in the same habitat and the environment where the animals live should be investigated. in the present study, we investigated the prevalence of three important enteric pathogens, including salmonella, yersinia, and campylobacter spp. in feral raccoons and masked palm civets. these results lead to the conclusion that these animals are potential reservoirs of the pathogens. the characteristics of the isolates showed that these animals probably acquired the pathogens from human activities, other wild animals, and the environment. the presence of human-associated serovars and the antimicrobial resistance of the salmonella isolates revealed the effect of human activities on these animals. this represents a typical spill-over of pathogens from human activities to wildlife (daszak et al., ) . meanwhile, the carrying of pathogens which are usually isolated from wildlife or from the environment (e.g. s. enterica subsp. diarizonae and y. pseudotuberculosis) indicated that these animals could play an important role in the life cycles of those bacteria in their habitats. these findings are in concordance with their omnivore behaviour and their wide range of habitats from forests to urban areas (zeveloff, ; abe, ) . our results revealed that raccoons and masked palm civets play an important role on the spreading of humanrelated pathogens. moreover, carriage of wildlife-and environment-related pathogens in these animals showed the possibility of the transmission of these pathogens to humans and domestic animals. raccoons and masked palm civets live near areas of human habitation and often nest in attics or in the feed stores of livestock. the enteric pathogens that we investigated can be transmitted to humans and domestic animals via feces, contaminated water and soil (fukushima et al., ; humphrey and bygrave, ; handeland et al., ) . thus, not only the ecological threats but also the public and animal health risks presented by these animals should be assessed in detail. a guide to the mammals of japan alkali method for rapid recovery of yersinia enterocolitica and yersinia pseudotuberculosis from foods raccoons and skunks as sentinels for enzootic tularemia wildlife and environmental health: raccoons as indicators of zoonoses and pollutants in southeastern united states manual of clinical microbiology superantigen ypma exacerbates the virulence of yersinia pseudotuberculosis in mice salmonella infections in the common raccoon (procyon lotor) in western pennsylvania wildlife ecology -emerging infectious diseases of wildlife -threats to biodiversity and human health antimicrobial susceptibility of salmonella isolated from cattle, swine and poultry salmonella isolates 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of japan correlation of autoagglutination and virulence of yersiniae a novel multiplex pcr assay for salmonella subspecies identification comparison of antimicrobial susceptibility testing of campylobacter spp. by the agar dilution and the agar disk diffusion methods canine distemper virus infection in a masked palm civet (paguma larvata) prevalence of campylobacter jejuni and campylobacter coli among wild and domestic animals in yamaguchi prefecture salmonella isolates from wild birds and mammals in the basque country (spain) tooth eruption in preweaned raccoons campylobacter infection raccoons (procyon lotor) as carriers of salmonella performance standards for antimicrobial disk susceptibility tests m -s . nccls, villanova, pa. national institute of infectious disease antigenic formulas of the salmonella serovars bergey's manual of systematic bacteriology: the enteric pathogens in raccoons and masked palm civets k gastrointestinal helminths of feral raccoons (procyon lotor) in wakayama prefecture synthesis of a selective agar medium for yersinia enterocolitica bergey's manual of systematic bacteriology: the proteobacteria; the alpha-, beta-, delta-, and epsilonproteobacteria colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus yersinia mollaretii sp. nov and yersinia bercovieri sp. nov, formerly called yersinia enterocolitica biogroups a and b pet dogs and chicken meat as reservoirs of campylobacter spp. in barbados raccoons: a natural history this work was partially supported by health sciences grant for research on emerging and re-emerging infectious disease from the ministry of health, labour, and welfare of japan. we are grateful to local government offices, m. kaneda, cacodaemon network and s. kato, strain ltd. for providing fecal samples in this study. key: cord- -rnia mbq authors: gai, ruoyan; tobe, makoto title: managing healthcare delivery system to fight the covid- epidemic: experience in japan date: - - journal: glob health res policy doi: . /s - - - sha: doc_id: cord_uid: rnia mbq amid the global pandemic of a novel coronavirus disease (covid- ), healthcare delivery system is being stretched. in japan, rapid spread of the epidemic brings hospitals to the brink of exhaustion. this commentary aims to briefly review related policies of japan in managing healthcare delivery system. among the relevant actions, strengthening the hospitalized care is emphasized to save lives. despite of limitations, the policies show a success in preventing a collapse of healthcare delivery system and skyrocketing mortality from happening so far. on the other hand, huge concerns remain if the infections continue to rapidly increase. the experience in japan indicates the urgency of planning of healthcare delivery system, mobilizing all relevant social sectors by consensus, and guiding people with calm manner based on the best shared knowledge and evidences. the pandemic of a novel coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov ) has posed a severe global crisis. in japan, the incidence is escalating, causing a large number of community transmission. as of april , , the time point that the statement of emergency is initiated, the number of domestic confirmed infection cases reaches up to , with deaths (fig. ) . the policies in japan highlight the planning of healthcare delivery system, in particular the prevention of an "overshoot" in hospitals. to this end, a crucial lesson has been learned from wuhan, china, where hospitals once melted down at the beginning as the result that patients were overwhelmingly seeking healthcare, and consequently those severe cases urgently needing inpatient care cannot be hospitalized. the exhaustion of healthcare delivery system is one of the reasons for the incredibly high mortality rate in that period [ ] . the overcrowded and contaminated environment of hospitals exposed a large population to high risk of the infection, further exacerbating the epidemic. similar conditions now also occur in seriously hit countries such as italy and spain, where the crude mortality rate due to covid- largely exceeds the global average level of . % [ ] . confronting the epidemic, it should be noted that the capacity of healthcare delivery system is not unlimited and consequently it entails a series of policies to avoid a surge of the infected. this commentary focuses on adaptation of healthcare delivery system for the containment of covid- in japan, for knowledge sharing with the global society. the major related measures adopted in japan are outlined. as covid- is listed to be a designated infectious disease in the infectious diseases control law, the patients are rendered to the hospitalized care in the designated facility in general. on the other hand, the overwhelming healthcare seeking may lead to a collapse of healthcare delivery system as mentioned above. in this regard, the basic policies for covid- control emphasized the hospitalized care for the severe / critical case based on previous experiences [ ] , and recommended people with mild flu-like symptoms to stay at home first and seek medical care after consulting the call center or a family doctor [ ] . after the declaration of state of emergency, tokyo metropolitan government decides to move patients with mild or no symptom (except the elderly, those with pre-existing health conditions and pregnant women) from hospitals to the assigned facilities to increase bed capacities with serious cases for covid- . to mitigate mortality and morbidity, it is worthy to note that the elderly, those with pre-existing diseases and pregnant women are specially considered for healthcare delivery, including detection and inpatient-based treatment, because of their vulnerability [ , ] . in japan, because of a limited laboratory capacity, priorities are given to determining cases requiring an inpatient care and tracing the cluster transmission, rather than to exploring the infection in the overall population through massive screening. meanwhile, the test method based on polymerase chain reaction (pcr) has a limited accuracy [ ] , especially when the test is used for a large cohort of population. a relevant proportion of false positive is prone to cause spread of infection from these tested negative but truly positive, whereas a large number of false negative leads to unnecessary occupation of limited healthcare resources that could be otherwise for severe cases. with regard to this, the implementation of detection test based on pcr has been scheduled by the call center for consultation, which was established within each health center of prefectures and government-designated cities right in early february . the criteria of seeking consultation are stipulated and updated, with consideration of high-risk population, such as the elderly, those with pre-existing health conditions and pregnant women. the call-center-based consultation system plays a key role of guidance of healthcare seeking and referral. it also provides a clue to capture the overall epidemic scale in the country. as the laboratory capacity has gradually improved, the pcr test has been included into benefit package of the social health insurance schemes and offered under physician's discretion since march . the capacity of healthcare delivery system is challenged by rapidly growing severe / critical cares [ ] . in japan, it is largely calling for wide negotiations and collaborations among the national and local governments, manufacturers and healthcare providers. since emerging of covid- , the national government has urged local authorities to prepare to secure hospital beds, intensive care unit and medical equipment in hospitals, especially for those not originally designated and specialized for infectious diseases. guidelines for infection control measures developed by the ministry of health, labour and welfare help to inform hospitals and health professionals. the government has also mobilized the manufacturing to ensure the supply chain of medical equipment and materials. on the other hand, it is reported that difficulties nevertheless remain in local authorities to offer the number of beds equivalent to the estimates of the expert panel assigned by the health ministry during the peak period of the epidemic [ ] . besides limited hospital beds and materials, a shortage of doctors, especially infectious disease specialists remain a huge concern [ , ] . the high risk of iatrogenic infection and lack of support to child care during school closure may further deteriorate the situation. the limitation in the capacity of healthcare delivery system suggest an urgency to suppress the scale of the epidemic. in japan, routine hygiene behaviors and health literacy of self-motivated and well-informed public compose the first line of defeating covid- . effective measures such as mask wearing [ ] , hand washing and health management are well penetrated in daily life. since the outbreak, clusters and contacts are closely traced by the public health officers to generate the firsthand data source for evaluation by the expert panel of mhlw. then a number of measures have been implemented with engagement of various stakeholders, including different governmental sectors, expert panels, local authorities, schools, industries and civil society, such as a nationwide school closure, promotion of telecommuting, staggered commuting time and voluntary restraints. knowledge on pathological, epidemiological and clinical characteristics of the virus and disease and public health measures are constantly updated to the public. social distancing is being reinforced. on the other hand, a lockdown as the most radical measure of social distancing may lag behind because of lack of legal force, suggesting the containment in japan is largely relying on consensus, self-motivation and empowerment of the public. as mentioned, underpinning the promotion of these policies are public understanding and consolidated trusts. as public health measures are not compulsory but voluntary in the country, the government is striving to get consensus and to consolidate the trusts from relevant stakeholders including local authorities and the public by providing information that is accurate, prompt and easy to understand and practice. the current policies also emphasize a thoughtful consideration of the growing concerns and fears in public. especially at this moment when public anxiety and fear are spreading together with the growing number of infections, effective communicating channels to the public are necessary to promote public understanding and encourage calmness. in japan, preventing the exhaustion of healthcare delivery system and softening the spike of the epidemic are regarded to be of paramount importance. these policies have limitations in testing, resource mobilizing and social distancing. however, the number of death cases since the emerging of covid- suggests these prioritybased measures have partly contributed to deferring the occurrence of the spike, adapting healthcare delivery system and saving lives so far. it should be recognized that, on the other hand, concerns remain in the capacity of healthcare delivery system if the epidemic continues to rapidly progress. to this end, dealing with the crisis further requires an agile and coordinative leadership to consolidate consensus and trusts. the experiences in japan to date indicates the importance of ( ) preparing healthcare delivery system for the epidemic, ( ) mobilizing all relevant stakeholders and social sectors by the consensus, and ( ) guiding people with calm manner based on the best shared knowledge and evidence, and all that are attributable to contingency planning and preparedness at early stage. estimating risk for death from novel coronavirus disease, china world health organization. who-audio emergencies coronavirus press conference estimates of the severity of coronavirus disease : a model-based analysis basic policies for novel coronavirus disease control (released on analysis of the pregnancy outcomes in pregnant women with covid- in hubei province (in chinese) the way to reduce the "false negative results" of novel coronavirus nucleic acid detection (in chinese) are high-performing health systems resilient against the covid- pandemic? organization for economic cooperation and development (oecd) rational use of face masks in the covid- pandemic the opinions presented in this commentary are based on individual view and do not reflect that of national institute of population and social security research, japan and that of japan international cooperation agency. authors' contributions dr. ruoyan gai concepted and wrote the manuscript. dr. makoto tobe then added essential materials for interpretation and revised the manuscript. the authors have approved the submitted version.authors' information not applicable. not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. not applicable. key: cord- -p vqpazu authors: tsai, theodore f.; rao, raman d.s.v.; xu, zhi yi title: immunization in the asia-pacific region date: - - journal: plotkin's vaccines doi: . /b - - - - . - sha: doc_id: cord_uid: p vqpazu nan and inactivated vero cell-derived je vaccines from china, japan, and korea, regionally; live attenuated hepatitis a vaccine from china, regionally; live attenuated and inactivated pandemic and seasonal influenza vaccines from india and china, internationally; and oral cholera vaccine from vietnam, internationally). previously, asian manufacturers did not themselves market novel vaccines in europe or the united states, choosing to distribute their innovative products, such as acellular pertussis and live attenuated varicella vaccines, through multinational companies. however, an increasing global integration is taking place, as multinational companies acquire asian manufacturers (e.g., sanofi-aventis, france, acquired shantha biotechnics, india); asian companies acquire or obtain technologies and distribution rights from european countries (e.g., inactivated polio vaccine by serum institute of india ltd. acquiring bilthoven biologicals, netherlands; astellas, japan, acquiring recombinant influenza hemagglutinin from protein sciences, u.s.; thai government pharmaceutical organization acquiring chimeric je vaccine from sanofi-pasteur, france; and biological evans, india, acquiring je vaccine from intercell ag, austria); and vaccine codevelopment is agreed between entities in developed and asian countries (e.g., genetically modified, inactivated hiv vaccine codeveloped by sumagen, korea, and the university of western ontario, canada; mycobacterial proteinag a candidate tuberculosis vaccine codeveloped by tianjin cansino biotechnology, china, and mcmaster university, canada; universal influenza vaccine codeveloped by xiamen wantai and sanofi-pasteur, france; and novel pneumococcal conjugate vaccine codeveloped by sk chemicals, korea and sanofi-pasteur, france). , , the role of asian companies as developers and providers of neglected and improved vaccines for the region and, for developing countries more generally, is an emerging trend as illustrated by the joint research activity agreement between the national research council, canada, and the chinese national biotec group that covers development of h. influenzae type a and hib bivalent conjugate vaccine, novel mucosal adjuvants and therapeutic vaccines against helicobacter pylori infection, and cell culture manufacturing platforms for viral and vectored vaccines. a korean-manufactured biosimilar (generic) biological, infliximab, now is licensed in europe, a step toward commercial expansion of asian region-manufactured biologicals to developed countries. the emergence of asia as the base of new multinational vaccine companies with broad development, production, and distribution capabilities is on the horizon, even as consolidation of existing companies occurs elsewhere. [ ] [ ] [ ] the broad income range within countries in the region results in large population segments that have sufficient means to pay for vaccines out-of-pocket. even among countries that otherwise qualify economically for gavi funding (e.g., india), substantial numbers of families can avail themselves of vaccines not covered by the national epi, resulting in a two-tiered system of vaccination, paralleling the public-private dichotomy of healthcare delivery in general. practitioners serving these and expatriate families generally follow current u.s., european, or australian vaccine recommendations, or some modification of those schedules. five je vaccines have been developed and licensed in asian countries. the widely used first-generation inactivated suckling mouse brain (smb)-derived vaccine is being replaced rapidly in economically disadvantaged countries by the chinese developed and manufactured live attenuated or inactivated vaccine (sa - - strain) grown in primary baby hamster kidney (phk) cells and in higher-income countries with vero cell-derived inactivated vaccines (licensed in the united states, australia, canada, and europe, as well as several asian countries) or a replicating chimeric yellow fever-je virus recombinant vaccine (manufactured in thailand). details are provided in chapter . to control cases and occasional outbreaks of the far eastern subtype of tickborne encephalitis virus in northeastern china, the changchun biologicals institute developed a formalininactivated vaccine, derived from a human isolate, senzhang strain, and grown in phk cell cultures. related vaccines prepared from central european strains and distributed in europe are described in chapter . the kyasanur forest disease virus (kfdv) is a highly pathogenic member of the family flaviviridae causing a zoonosis, kfd, that is transmitted by the bite of infective ticks (haemaphysalis spinigera) primarily in its nymphal stage, and characterized by acute febrile illness with severe hemorrhagic manifestations. it was first described from outbreaks centered in karnataka state, india, among herders and villagers with forest exposure and was considered to be localized in the shimoga district area of the state. however, since first being reported in , the virus has been found in other areas of india including the kutch and saurashtra parts of gujarat state, andaman islands and west bengal. it is estimated that close to cases of kfd occur in india every year and, from to , among confirmed cases, were fatal. following the outbreak in india various vaccines including a formalin inactivated russian spring summer encephalitis virus, a russian spring-summer encephalitis virus-based mouse-brain vaccine, and a live attenuated vaccine that was serially passaged in tissue culture were tried but with limited success. finally, a formalin inactivated vaccine with the kfd virus grown in chicken embryo fibroblasts was tested in a large field trial from to among inhabitants of affected villages. the disease attack rates reported were . % ( / ) among persons receiving one dose and . % ( / , ) among recipients of two doses, respectively, compared to an attack rate of . % ( / , ) in unvaccinated persons, for vaccine efficacies of . % and . %, respectively. the vaccine was subsequently commercialized and is produced by the state institute of animal health and veterinary biologicals, hebbal, bangalore, and has been central to kfd prevention efforts in the state of karnataka. annual vaccinations have been done since in the shimoga and adjacent districts wherein two doses of the vaccine were administered in individuals to years of age at an interval of month. periodic boosters were also administered after to months. however, recent observations suggest a lower field effectiveness than had been reported previously, especially following a single dose, while overall coverage has also been low. between and , effectiveness among individuals perceptions of the value of vaccines and their risks also range widely, regionally and within individual countries, from largely enthusiastic acceptance and even demand for additional routine vaccinations (e.g., for je vaccine in southern and southeast asia) to a degree of skepticism equal to, if not more deeply and widely held, than vaccine hesitancy in europe and the united states. within the last years, japan discontinued routine childhood vaccine programs for combination measles-mumps-rubella, influenza, and je, and withdrew recommendations for the human papillomavirus (hpv) vaccine for adolescents, owing in several of instances, to incorrectly thinking that coincidental adverse events were causally related. the requirement for subcutaneous, as opposed to im administration for all vaccines, exemplifies the misattribution of adverse reactions, arising in this case from an extrapolation of muscle contractures resulting from repeated im administration of antibiotics, to other intramuscularly administered products, including vaccines. , the extrapolation has had unintended consequences of impeding the licensure of vaccines with newer adjuvants with mechanisms of action that require im administration. with the global spread of information, concerns over the thimerosal content of childhood vaccines and vaccine-associated autism have been as active a parental concern among middle-class families in developing countries as elsewhere. parental refusal of routine je vaccination in korea and significant declines in vaccine coverage occurred in a different context after seven cases of temporally related cases of anaphylactic shock and neurological disease, including five deaths, occurred in . the cases could not be excluded as causally related to administration of the mousebrain-derived vaccine, prompting a national debate and establishment of a vaccine adverse events reporting scheme, a national vaccine injury compensation system, and introduction of a live attenuated je vaccine derived from a nonneural tissue substrate. from this mosaic, we describe some common themes, highlighting representative approaches and unique issues that hold a wider interest. because they are covered elsewhere in this volume, we have not reviewed specific vaccines of regional concern (e.g., pandemic influenza and je vaccines) or vaccination topics common to developing countries (e.g., initiatives surrounding injection safety, measles and neonatal tetanus elimination, and polio eradication, nor financing mechanisms). we concentrate, instead, on other aspects of vaccine development and implementation, organized by the steps of vaccine development, approval, production, recommendation, and delivery. we also focus on childhood vaccines and vaccination and on selected countries in the region. japan is acknowledged as the innovator of several vaccines now used internationally, including acellular pertussis and live attenuated varicella vaccines, but other novel vaccines have been developed by japan, china, india, australia, and vietnam for region-specific needs (table . ). these include vaccines for je, hantaan (htn)-and seoul (seo) virus-related hfrs, russian spring-summer encephalitis, kfd, cholera, severe acute respiratory syndrome, and q fever. in addition, novel attenuated strains of measles, mumps, hepatitis a, rotavirus, and intranasally delivered pandemic h n virus have been derived for products distributed principally within the region. additional novel vaccines for hepatitis e and ev-a have potential for broader use internationally, an indicator of the region's transition from a provider of fill-finish and manufacturing capacity to a full-fledged participant in biotechnology research and clinical development. text continued on p. the reasons for low vaccine efficacy and coverage rates need to be investigated and the appropriate vaccine regimen for effective control requires further definition. newer vaccine approaches (e.g.., chimeric or virus protein subunit vaccines) are being investigated to potentially replace the current vaccine. elsewhere, a nearly identical strain to the kfdv was isolated from a patient suffering from acute febrile illness from yunnan province, china in . seroprevalence studies indicate that kfdv (or the nanjianyin virus or a related tickborne flavivirus) may be present in various parts of southwestern china. in a virus similar to kfdv called alkhurma hemorrhagic fever virus was isolated from patients with febrile illness in saudi arabia. overall, cases with two deaths occurred in sheep and camel handlers exposed to a tick ornithodoros savignyi. the disease has now been confirmed to be more widespread in the country than previously considered. as tickborne diseases are "diseases of place," kfd virus itself, if it spreads, is likely to disseminate locally. nevertheless, the discovery of antigenically related viruses elsewhere, such as alkhurma hemorrhagic fever virus, suggests a potential for more widespread use of kfd vaccine, depending on public health needs. hfrs, a widespread rodent-borne bunyaviral zoonosis in asia, is a pantropic infection with prominent capillary hemorrhages, interstitial nephritis, and a % to % case-fatality ratio that, until the last decade, caused more than annual cases in the republic of korea and more than , cases in china. although the disease had been well known in parts of russia and asia as a sporadic and occasionally epidemic disease among farmers, soldiers, and others exposed to campestral and sylvatic habitats, it was largely unknown in the west until thousands of military cases and deaths occurred during the korean war, when the disease was described as korean hemorrhagic fever. the etiologic agent eluded investigators until , when a novel bunyavirus, htn virus was isolated from the striped field mouse, apodemus agrarius, which proved to be the principal viral reservoir in most areas of asia. later, antigenically related seo virus was isolated from rattus rattus and rattus norvegicus, explaining the occurrence of sporadic hfrs cases and outbreaks in urban areas. subsequently, sin nombre and related hantaviruses were discovered in the western hemisphere, where rare encounters with infected rodents lead to small numbers of cases that feature prominent pulmonary involvement. a multitude of hantaviruses now have been described globally. the widespread impact of hfrs in china led public health authorities in the s and s to pronounce the disease second only to hepb as a public health menace, and, beginning in , several chinese vaccine manufacturers used smb, primary baby gerbil kidney cells (gkcs) or phk cells to produce inactivated, monovalent vaccines against htn or seo viruses. the gkc vaccine was inactivated by β-propiolactone and the other two by formalin. subsequently, vero cell linederived vaccines have been developed. these vaccines were evaluated in nine chinese provinces hyperendemic for hfrs during to . the gkc-derived vaccine against htn virus produced seroconversions to putatively protective titers of neutralizing antibody in . % of subjects after three primary doses at , , and days, the proportion rising to . % after a booster at year, and declining to . % at years and . % at years. similar immunogenicity results were reported for the phkderived vaccine and the purified smb vaccine. in a randomized, controlled, three-arm trial of gkc vaccine in which vaccinated subjects received three primary doses and a booster at year, hfrs cases were observed in the age-, sex-, and residence-matched controls, and cases in the , unvaccinated subjects of similar age ( to years), compared with none in vaccinees during months of follow-up, for a protective efficacy of % ( % lower confidence limit of . %, p = . , cumulative binomial probability). efficacy of the three primary doses alone was shown in the year between administration of the three primary dose series and the booster dose: with zero cases in the vaccinated, and nine and cases in the unvaccinated and control groups respectively. among cases in the control and unvaccinated groups, were caused by htn virus, were caused by seo virus, and four by a virus of indeterminate serotype. thus, the monovalent gkc-derived htn virus vaccine was protective not only against the homologous virus, but also cross-protective against seo virus. no vaccine-related serious adverse event was reported during the trial, and mild local and systematic reactions were reported in . % of vaccinees. the efficacies of the phk vaccine and the purified smb vaccine were similar: in nonrandomized trials, one hfrs case was found in , recipients of phk vaccine, compared with in , unvaccinated subjects, a reduction of . %; for the purified smb vaccine, the rates were . per , ( / , ) versus . per , ( / , ) for vaccinees and unvaccinated subjects, respectively, a reduction of . %. the observed reductions were maintained through years of follow-up. nonsevere reactions were found in . % of phk vaccine recipients and in . % of smb vaccine recipients. , bivalent htn and seo gkc-and phk-derived vaccines were developed and improved by purification procedures through gradient density ultracentrifugation or chromatography to be more immunogenic and less reactogenic. the purified bivalent gkc vaccine induced neutralizing antibody seroconversion against htn virus and seo viruses in . % ( / ) and . % ( / ) of volunteers, respectively, after two doses with an interval of days, and . % ( / ) and . % ( / ), respectively, after a booster dose at months. only mild reactions were observed; local reactions in . % ( / ) and systemic reactions in . % ( / ) of the vaccinees. the purified bivalent phk vaccine induced neutralizing antibody seroconversion against htn virus and seo virus in . % ( / ) and . % ( / ) of subjects, respectively, after two doses separated by days, and . % ( / ) and . % ( / ), respectively, after a booster dose at months. no systemic reaction was found among vaccinees and mild local reactions were observed in two ( . %). the purified, bivalent gkc vaccine was tested for protective efficacy in a nonrandomized trial among , subjects, to years of age; , persons received the two primary doses with an interval of days and a booster dose at months; , persons were unvaccinated. the two groups were similar in age distribution. during years of follow-up, hfrs cases were found in , person-years among the unvaccinated, a rate of . per , , compared with none in the vaccinated , person-years, a reduction of %. several manufacturers have adapted their processes from primary gerbil or hamster cells to continuous vero cells. the purified, bivalent vero cell-derived vaccine administered in two doses separated by days, induced neutralizing antibody against htn virus and seo viruses in . % ( / ) and . % ( / ) adult volunteers, respectively. mild systemic reactions were observed in . % ( / ) and mild local reactions in . % ( / ) of vaccine recipients. the immunogenicity and safety profiles of the vero cell-derived, purified bivalent vaccine were similar in children and older adults. based on the above data, a schedule of two primary doses with an interval of days, plus a booster at months, has been recommended for the purified bivalent gkc-, phk-, and vero-cell-derived vaccines. a postlicensure, retrospective study was conducted to measure the long-term effectiveness of the gkc vaccine among , adults to years of age, in villages located in a hyperendemic area of shaanxi province. hfrs incidence rates were compared between the vaccinated and the unvaccinated adults: . % ( / ) versus . % ( / ), respectively, for the first years after vaccination; . % ( / ) versus . % ( / ) in years to ; . % ( / ) versus . % ( / ) for years to ; and . % ( / ) versus . % ( / ) at to years. the vaccine's effectiveness was thus estimated at . %, . %, . %, and . %, respectively, for the four study periods. the effectiveness was underestimated because the year of onset of hfrs was unknown for cases, all of whom belonged to the unvaccinated group and were not included for analysis. the overall hfrs attack rate was . % ( / ) in the vaccinees and . % ( / ) in the unvaccinated subjects, a reduction of . %. a long-term study of the monovalent phk-derived seo virus vaccine also was conducted among adults to years old in a seo virus-predominating area, from through . only three primary doses were given at , , and days without a booster. seven hfrs cases were found in , , subjects in the vaccine group, a rate of . per , , and cases were found in , , controls, a rate of . per , , with an overall reduction of . % ( % ci, . % to . %) during the years of the study. the vaccine's effectiveness was estimated at % for the first year, . % for the second year, and . % for the th year. the rate reductions in other years were approximately %. a smb-derived htn virus vaccine also was developed in the republic of korea and is available for at-risk individuals. the incidence of hfrs in china and korea has declined in the last years with the introduction of vaccination and probably, more importantly, because of urbanization, rural economic development leading to improved (cement) houses, and grain harvesting and storage practices, resulting in reduced exposures to the rodent reservoir. this trend has been most evident in rapidly developing areas of southeastern china and likely will continue in other regions, leading to a diminution of disease incidence and, potentially, discontinuation of routine vaccination in endemic provinces. see table . . two similar live attenuated hepatitis a vaccines, based on the h and la- strains, and measles vaccines, based on the shanghai s- and changchun- strains, have been licensed in china and are used domestically and exported. the national institute of hygiene and epidemiology in vietnam developed an oral bivalent o -o killed whole-cell cholera vaccine that now is produced and distributed by vabiotech, company for vaccine and biological production no. , in hanoi; another oral bivalent o -o vaccine based on the vabiotec vaccine but with improved production design is produced in india by shantha (sanofi, france). both vaccines are used domestically and also exported. similarly, live rotavirus vaccines based on local strains have been developed in india and china for local use. ev-a and related enteroviruses have emerged in major seasonal epidemics in asia and australia, leading to millions of cases and extensive social disruption as daycares and schools are closed. the extent and impact of seasonal outbreaks stimulated vaccine development in china, taiwan, malaysia, singapore, and japan, with government prioritization and support in some countries, analogous to mechanisms that facilitated pandemic influenza vaccine development. an escherichia coli-expressed capsid peptide virus-like particle hepatitis e vaccine, approved by the china food and drug administration, is the first novel recombinant vaccine developed and licensed in asia. its potential use in africa, south asia and, possibly, even in developed countries in immunocompromised or other risk groups could be envisioned. implicit in the region's progress toward novel vaccine development is a maturing capacity to conduct clinical trials and improvements toward more robust regulatory processes and capacity, including pharmacovigilance systems. in addition, multinational companies increasingly have turned to countries in asia to conduct clinical trials because of lower costs and more streamlined regulatory approvals of clinical trial applications. international contract research organizations operate in many countries, and a growing local infrastructure to conduct clinical trials in compliance with the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use and good clinical practices standards will improve clinical research conducted in the region. unlike europe, asian countries are not unified in a central regulatory approval process. nor is there a regional public health presence as in latin america, where the pan american health organization (paho) leads regional vaccination programs and also provides central purchasing of certain qualified vaccines. however, the -nation association of southeast asian nations (asean; includes brunei-darussalam, cambodia, indonesia, lao pdr, malaysia, myanmar, philippines, singapore, thailand, and vietnam) in initiated efforts for a subregional regulatory harmonization scheme to reduce differences in technical requirements and regulatory procedures for pharmaceuticals. a harmonization initiative, under auspices of a pharmaceutical product working group, aimed to remove barriers to regional commerce and to eliminate technical barriers to trade without compromising product quality, efficacy, and safety. eventually, a subregional central or mutual-recognition procedure similar to that of the european union could be envisioned. importantly, local clinical trials are not required for registration under abbreviated pathways specified by the asean common technical dossier if the vaccine was approved and licensed by a benchmark regulatory agency, resulting in a certificate of pharmaceutical product. by contrast, the national regulatory authorities of china, india, japan, korea, and taiwan have required local clinical trials before or after registration, and in other countries, while data in local populations may not be required for registration, those data are important in deliberations on a vaccine's inclusion in the national schedule. for dengue, a disease of special public health urgency regionally, the global debut of candidate vaccines in the region is being considered, with individual country vaccine registrations ahead of approval by a benchmark agency and provision of a certificate of pharmaceutical product. the text continued on p. various countries in the region have had the effect of delaying the registration of proven vaccines that otherwise could have prevented significant morbidity and mortality with more timely introductions. descriptions of individual regulatory requirements for clinical trial applications and new product approvals are beyond the scope of this chapter; see the previous edition for a more detailed introduction. governments have had a greater role in vaccine manufacturing in the region than elsewhere, although devolution toward privatized or state-owned enterprises (i.e., government-owned corporations) has occurred (e.g., commonwealth serum laboratories in australia was privatized, and the six major government vaccine institutes in china now operate as a state-owned enterprise, china national biotech group; see table . ). although a growing number of private manufacturers have emerged, especially in china and india, in other countries, national and local government manufacturers continue to be important sources of certain vaccines and biologicals for domestic needs (e.g., the government pharmaceutical office in thailand, research institute for tropical medicine in the philippines, biofarma in indonesia, the national institute of hygiene and epidemiology in vietnam, and the central research institute and local government institutes in india). these and other facilities also fill and distribute bulk vaccines supplied by international manufacturers. several private and state-owned enterprise manufacturers in the region are members of the dcvmn, a consortium that seeks to identify and develop solutions to common challenges faced by manufacturers in developing countries. , , , a number of manufacturers (including in five asia-pacific countries) operate under practices and procedures that have prequalified them to produce certain vaccines for unicef, paho, and gavi purchases (e.g., pentavalent dtp combinations, oral polio vaccine, inactivated polio vaccine, hepb, rabies, influenza, oral cholera, and measles-containing vaccines) or that allow them to export vaccine to other countries in the region. a reliable supply of inexpensive diphtheria and tetanus toxoids combined with whole-cell pertussis (dtwp)-hib-hepb combination vaccines, made possible largely by indian and korean manufacturers, has facilitated the introduction of hib antigen into schedules of economically disadvantaged countries that otherwise would not have adopted the monovalent vaccine. similarly, provision of measles and measles containing vaccine by indian manufacturers was key to the elimination of that disease in latin america and the current state of polio elimination could not have been achieved without supplies from asian regional manufacturers. the provision of oral cholera vaccine for outbreak control in haiti, pakistan and other countries is an important example of the increasing ability of and global dependence on these manufacturers. who prequalification requires that the manufacturers and plants not only must satisfy who good manufacturing practices inspections, but, in addition, that national notifications of adverse events following immunization are captured and analyzed satisfactorily. this last requirement has been the principal impediment to prequalification of products from some countries and prequalification aided by who blueprint and other vaccine safety-related guidelines have facilitated the improvement of vaccine-related pharmacovigilance in the region. in china, the state-owned china national biotec group is the dominant supplier of vaccines in the country, providing % of doses used in the public program and % taken up initiative is a collaboration among the nongovernmental organizations, the dengue vaccine initiative, and the world health organization (who) developing countries vaccine regulatory network (dcvrn). the who through the dcvrn has been actively working toward harmonizing procedures in affiliated countries, including china, india, and indonesia, to bring those regulators under the who prequalification umbrella and to facilitate approval and supply of their products for gavi and united nations children's fund (unicef). the requirement of some national regulatory authorities for clinical data in local populations is based on a concern that racial, ethnic, or environmental differences could affect responses of the local population, both immunologically and in their risk for adverse events. genetically based differences in drug pharmacokinetics and pharmacodynamics, as well as disease risk, increasingly have been recognized, including immune responses to vaccines. studies of antibody responses to pneumococcal conjugate vaccines in asia, for example, have found higher prevaccination and postvaccination antibody titers among philippine and taiwanese infants compared with european or historical control subjects, and in korea, a considerably higher proportion of subjects were seropositive to meningococcal serogroup w polysaccharide at baseline than in the united states. [ ] [ ] [ ] while the basis for these differences may be an earlier exposure in life to cognate or crossreacting antigens (e.g., because of regional differences in host microbiomes), genetically restricted responses, as have been observed with hepb, measles, vaccinia, rubella, hib, and other antigens, or, in the case of oral rotavirus vaccine, in genetically determined viral attachment or receptor binding molecules, have been described. [ ] [ ] [ ] [ ] from the perspective of adverse events following immunization, the example of narcolepsy occurring in some recipients of an adjuvanted pandemic h n vaccine illustrates the role of genetic background as a cofactor in risk. in many examples, regulatory systems and processes in the region have had the effect of markedly slowing or effectively blocking the introduction of novel vaccines developed externally. in china, the introduction of an internationally registered and otherwise widely used product nevertheless necessitates recapitulating the entire clinical development program in china, including phase i studies, despite an abundance of previously scrutinized evidence. this requirement introduces a delay of a decade or more for registration of internationally developed, as opposed to domestically developed, vaccines. specifications in national pharmacopeias that deviate from established compendia, for example, exclusion of well-accepted excipients or methods, also have seriously impeded or prevented registration of foreign products or, when imposed with a revision of the pharmacopeia, have led to withdrawal of a previously registered product. clinical trial processes also have hindered local introduction of established or novel products (e.g., a indian supreme court ordered suspension of ongoing clinical trials and reexamination of previously approved trials was followed by a wholesale revision of clinical trial guidelines, leading to a temporary cessation of all industry-sponsored clinical trial activity). the potential inclusion of video recording of the informed consent process, newer insurance requirements and further proposed but unclear amendments to the drug and cosmetics act that could impose criminal penalties against trial investigators for poorly defined violations may further limit trial activity. china and indonesia place severe restrictions on the exportation of clinical samples from study subjects, thereby requiring that validated laboratories and procedures are established locally, adding a barrier that has led to delays of or avoidance of clinical studies in those countries. whether resulting from inexperience, a dearth of trained personnel, trade protection, or other reasons, administrative mechanisms in in most countries in the region, public health authorities now draw on external advisors to help formulate national vaccine recommendations in national immunization technical advisory groups (nitag), resulting, in part, from activities of the supporting independent immunization and vaccine advisory committees initiative (at the agence de médecine préventive). [ ] [ ] [ ] [ ] the advisory committee on immunization practices (acip) in taiwan and korea, expert committee on immunization in singapore, chinese expert committee on epi, hong kong scientific committee on vaccine preventable diseases, immunization committee of the indonesian pediatric society, national technical advisory group for immunization in india, and the australian technical advisory group on immunization are examples of such medical advisory groups. in china, vaccine recommendations are made through the national centers for disease control based on recommendations of the chinese expert committee on epi under the ministry of health and family welfare; however, provincial or local centers for disease control may issue independent recommendations for specific vaccines or modify the national recommendation for routine vaccines (see tables . the issues considered by asian nitags in formulating vaccine recommendations parallel those of other nitags, focusing on medical need, vaccine safety and efficacy, national resources, as well as implementation issues, including supply, cold-chain, fit within the national schedule, vaccine presentation, etc. health economic analyses are considered in the deliberation of some committees or are provided by an independent body (e.g., the health intervention and technology assessment program in thailand); although, in general, the use of health technology assessments in the region lag behind the united states and united kingdom. in some cases, industry sponsors, in providing such analyses to nitags in their justifications to include new vaccines into national programs, have played a role in introducing cost-to-benefit analyses to the recommendation process. in indonesia and malaysia, the halal status of vaccines is an important factor in public acceptance of a product and also is a consideration in the vaccine recommendation process, although there is movement to remove this consideration from debate. in certain asian countries, as well as in latin america, the approval process to include a new vaccine into the national program is used to leverage multinational companies to foster local manufacturing expertise. in brazil, technology transfer of the vaccine production process is required in turn for the vaccine's inclusion into the national schedule while, in indonesia, all epi vaccines are locally produced by biofarma, and no new vaccine has been introduced into the national schedule unless it was produced locally. technology transfer of some element of the manufacturing process also is a factor in introduction of new vaccines to thailand and malaysia. such requirements may be tested as costly vaccines manufactured by more complex technologies are introduced to the region. the recommendation process in japan illustrates how, even after registration, organizational and administrative processes can result in a lengthy interval before a new vaccine is introduced to the national schedule. although, since , japan has recovered from a "vaccine gap"-the self-acknowledged interval during which antigens such as hib and pneumococcal conjugate, rotavirus, hpv, inactivated polio and various combination vaccines were not introduced into japan despite their widespread use in other developed countries-adoption of new vaccines into the national immunization program after their registration still lags several years. a number of sequential approvals lengthens the process: the immunization policy and vaccination committee provides an initial recommendation whether the newly registered vaccine should privately. the group comprises manufacturing sites, which produce some products, including the first who prequalified vaccine produced in china (sa - - je vaccine). other private companies compete principally to provide vaccines for out-of-pocket sales at local centers for disease control and prevention and hospitals. within the asean community, comprised principally of low-and middle-income countries, regional vaccine security has been a focus of discussion, reflected in the establishment of the asean-network for drug, diagnostics and vaccines innovation that focuses on a broad agenda of health technology development and collaborations on vaccine manufacturing and plans for regional vaccine purchasing-similar to paho's revolving fund. similarly, the eight-nation south asian association for regional cooperation includes biotechnology in its agenda for cooperative research. a goal to achieve self-reliance in vaccine supply also has been articulated in korea, in its horizon-setting. to a growing extent, multinational companies are acquiring or partnering with local companies in the region, with the result that manufacturing standards and their regulation should improve toward meeting international specifications. table . lists the region's principal vaccine manufacturers and their licensed products. the list is not intended to be comprehensive, as the sometimes rapid emergence or disappearance of pharmaceutical and vaccine companies in china and elsewhere is difficult to track. vaccines that are manufactured elsewhere and refilled and distributed by local manufacturers are not listed. countries in the region can be divided broadly into countries with a single national schedule and countries in which a basic schedule of free epi vaccines is supplemented by recommendations of a professional organization (such as the national pediatric society) for additional antigens that are paid for outof-pocket. countries in the first group include, on the one hand, mainly developing countries offering a basic epi schedule and, on the other, countries like australia, new zealand, and taiwan that provide a universal vaccination program that includes an array of antigens or combination vaccines paralleling those of european and u.s. schedules. the continued introduction of new and frequently expensive vaccines is an ongoing tension for vaccine recommending and funding entities that must weigh the relative value of such innovations against other preventive and therapeutic health expenditures. even for low-middle-income countries in the region, the total per capita expenditure for all healthcare may be less than the cost of a full course of a novel vaccine! on the other hand, national schedules in the region can be as comprehensive as to include the hpv vaccine (australia) and influenza and varicella vaccines (e.g., korea, taiwan). at the same time, hib vaccine still is not recommended in some jurisdictions with high per capita income (hong kong, singapore). to some degree, the seemingly paradoxical recommendations of relatively high-income countries in the region reflect different social expectations of personal responsibility in healthcare purchases (see subsequent text). as shown in table . , some national schedules provide optional recommendations for some antigens; in many countries where government tenders choose specific manufacturer products, specific combinations are recommended in the national schedule. in addition, for some antigens, provincial-specific recommendations address regional differences in risk (e.g., for routine group ac meningococcal vaccine in china; for je vaccine in sarawak, malaysia, and for the torres straits, australia; and for rabies vaccine [preexposure] in areas of the philippines). be classified as either "routine" or "voluntary," based on available data; the technical recommendation is considered by the tuberculosis & infectious diseases control division which makes the administrative decision for the vaccine's inclusion in the national schedule; however, that decision requires additional legislative approval whether the disease (category a or b) qualifies for full or partial vaccine funding (up to circa %), respectively. the recommendation process is even lengthier than appearances suggest, as the immunization policy and vaccination committee does not convene a deliberative vaccine working group until after the product is registered, unlike the parallel activities of the u.s. acip and food and drug administration. only then does the committee assemble a dossier (fact sheet) that establishes the epidemiology of the disease and its local burden; if insufficient data are available, de novo studies might be required to establish need. the overall interval between vaccine approval and issuance of a recommendation typically is years. other asian countries have similar or even lengthier intervals between vaccine registration and full epi implementation. in thailand, for example, after a preliminary nitag recommendation, a new vaccine is implemented in a pilot program to establish effectiveness and to collect additional safety experience. such a program may be gradually extended to other localities over a period of as long as a decade before the antigen is provided nationally. for diseases with regional differences in disease burden, high-risk provinces may be covered first (e.g., je vaccine initially was introduced in thailand to eight high-incidence provinces and progressively, from to , to all provinces, while local production was established and expanded). for new, often costly vaccines, phased introduction provides a mechanism to accommodate their full epi coverage costs over time. in the interim, local governments of wealthier provinces or municipalities have issued their own recommendations for vaccines to be reimbursed (e.g., shanghai provides pneumococcal polysaccharide vaccine free of cost to older adults and bangkok established a school-based hpv vaccination program, while neither vaccine is included in respective national schedules). innovative financing mechanisms have played an important role in the introduction of vaccines to low-income countries, and their extension to graduating gavi will enable more rapid adoption of new vaccines in those countries. at the same time, tiered pricing, negotiated between sponsors, local government and other entities will aid middle income countries to accelerate vaccine adoption, as exemplified by introductions of pcv and rotavirus vaccines. during the interval between a vaccine's registration and its inclusion in the national schedule, after which it is available without cost, out-of-pocket sales still may result in considerable uptake. while rotavirus vaccine is still considered a voluntary vaccine in japan, coverage among infants is estimated to be approximately %. in korea, although almost all pediatric vaccines are self-paid by parents, vaccine coverage for antigens such as hib and pneumococcal conjugate vaccine rapidly reached coverage rates of approximately % that, with herd effects, led to disappearance of the respective diseases as quickly as in other countries. although in japan, the "voluntary" vaccine recommendation emanates from a government committee, in other countries, academic societies play the principal role in recommending vaccines that are not included in the epi schedule. the malaysian pediatric association, the pediatric society of thailand, and the philippines foundation for vaccination not only advise their respective ministries and nitags in formulating national recommendations, but also promulgate recommended schedules of administration for other approved but not epi-covered antigens, emulating in large part or entirely from u.s., australian, or european schedules. vaccines are delivered in varying proportions through public or private channels, depending mainly on local income levels and accessibility to private practitioners. in general, vaccines on national schedules are available at no cost in primary health centers or their equivalent (e.g., puskesmas in indonesia; polyclinics or government hospital clinics in singapore, malaysia, and thailand; village and county level centers for disease control in china; village communes in vietnam; public health centers and clinics in india and japan; and at general practitioner offices in japan, and australia). as vaccines generally are available free in public clinics, even in affluent countries, families may obtain them in government clinics or hospitals (e.g., in singapore, ≈ % of families obtain vaccines through the government system of polyclinics and hospitals). however, to avoid long waiting times and rotating staff at public clinics, many families opt to obtain these otherwise free vaccines privately and to pay out-of-pocket at pediatric, general practitioner, or other private clinics. in addition, as newer vaccines may be delayed in their introduction to the national reimbursement scheme, it is common for parents to pay voluntarily for these vaccines (see earlier). as might be expected from the distribution of income, the proportion of children vaccinated in government primary health centers is higher in rural areas. overall, approximately % of children in thailand and % in malaysia are vaccinated through public channels. in china, all vaccinations are under control of centers for disease control and prevention; therefore, nearly all chinese children receive free epi vaccines, as well as payable optional vaccines (e.g., hib, pneumococcal conjugate vaccines, varicella, rotavirus, and others) at public clinics. fig. . summarizes the coverage for epi vaccines for selected countries. supplementary immunization activities have played a critical role in the elimination of polio from the who southeast asian region that was achieved in , and in ongoing efforts to eliminate measles and congenital rubella syndrome. routine and supplementary immunization activities tetanus vaccinations have eliminated maternal and neonatal tetanus in all but four countries in the region: cambodia, indonesia, papua new guinea, and pakistan. economic growth and development in asia and secular trends in population structure and the evolution of healthcare systems are forces that inevitably will change various aspects of immunization in the region, if in as-yet unforeseeable ways. , the population of asia, as in other regions, is aging and shifting toward a structure with a larger proportion of adults and elderly persons. between and , the birth cohort of asia will decrease slightly from . to . million, and the population of children to years old will hold nearly constant while the number and proportion of adults from to years will increase dramatically, and the number of people older than years of age will nearly double, from . to . million. a demographic crossover point with more adults + years of age than children younger than years of age was reached in europe in the s, and will occur within another generation in asia (fig. . ) . with the exception of almost universal epi programs of tetanus toxoid vaccination of pregnant women, adult vaccination has been viewed mainly in the context of travel, as in group a meningococcal vaccine for the hajj, and in tropical asia, influenza vaccine alliance for vaccines and immunization-eligible countries (shaded) have similar coverage rates of basic vaccines as countries at higher levels of economic development, illustrating the success of expanded programme on immunization. hepatitis a, interestingly, is now principally a risk in the cohort of young adults who were raised in an era of economic development and improved sanitation and who therefore lack natural immunity but were born before routine childhood vaccination was implemented. a catch-up program to address this epidemiological shift has been recognized by adult vaccination recommendations in some countries (see table . ). in china, adult measles vaccination is under discussion, as more than , cases have occurred annually in recent years, in equal proportion in adults older than years and in infants who had not received their first vaccine dose. growing awareness of adult vaccination is reflected in an increasing number of countries with adult vaccination recommendations (see table . ). two other population trends that will influence the demand for vaccines and channels for their delivery are urbanization and income disparity. the urban-dwelling population in asia is projected to increase by almost a billion persons between and , from . billion to . billion, while the rural population will decline only slightly. urban crowding is likely to affect the transmission patterns of certain person-to-person transmitted diseases and even of infections acquired from environmental sources. dengue, for example, is transmitted by mosquito vectors that are more prevalent in urban environments; the already great need for a dengue vaccine will almost certainly increase with the growth of urban centers. while the growing size and number of large cities may increase transmission of certain infections, delivery of vaccine and of healthcare in general is better organized in cities than in rural areas. specific interventions are needed to ensure that the existing disparity in access to healthcare between urban and rural dwellers does not widen. associated with urbanization is the increasing income gap in many countries that, in the health arena, has translated into a two-tiered system of healthcare, including preventive medicine. while vaccines are regarded by many as a public good to be provided as a government service, as mentioned, access to the increasing number of new vaccines is likely to be stratified by income level and ability to pay, as governments must choose among increasingly costly vaccines and other health interventions. as shown in table . , pediatric societies in a number of countries promulgate recommendations emulating those of the u.s. acip, and these schedules, aimed at practitioners serving private-paying families, may diverge increasingly from the national epi schedules benefiting the majority of children in those countries. how the public and governments will respond to an increasing disparity of what has been perceived as a basic medical service remains to be seen. in coming years, more novel vaccines are likely to be developed in asia or licensed first in asia for a regional, developing world, or international market. governments and asean have expressed increased interest in providing for national and regional vaccine security. the collaboration of industry sponsors with nongovernmental organizations and government in public private partnerships for new product development has been highlighted by the successful introduction of vaccines and drugs for several neglected diseases, for which the dcvmn view a responsibility. for example, the japan international cooperation agency and kitasato daiichi sankyo provided technical assistance to establish domestic measlesrubella vaccine production in vietnam's public corporation, center for research and production of vaccines and biologicals, polyvac. at the same time, the entry of nongovernmental organizations as actual sponsors of novel vaccine development for certain target diseases introduces competition with dcvmn manufacturers and multinational companies that might also consider similar development programs. asian academic institutions and companies possess elements the crossover point when the population of adults older than years of age exceeded the population of children younger than years of age was crossed in europe around ; that crossover is projected to occur in asia around , within a generation from now. for travelers to temperate locations. however, the severe acute respiratory syndrome and pandemic h n outbreaks and the regional threat of h n influenza have focused attention on routine seasonal influenza vaccination for the first time in many countries, beginning with elderly populations, and the role of children in influenza transmission is being recognized while it is rediscovered in japan. as a result of high pediatric vaccination coverage in developed countries in the region, je has become almost exclusively a disease of adults older than years of age, reflecting the intrinsic biological susceptibility of older adults to neurotropic flaviviruses and suggesting a of the scientific and technical expertise needed to develop vaccines for current and emergent needs and, seemingly, the will to establish themselves on the global stage and contribute to their development. regional institutions responded rapidly to threats of middle east respiratory syndrome virus and ebola virus with candidate vaccine development even when transmission was geographically remote. further participation of regional institutions in global responses in the future is likely. trends toward increasing local development and manufacturing in the region and the accompanying need to strengthen respective regulatory agencies have been recognized by the who and local national regulatory authorities. revising and harmonizing guidelines and procedures to international standards and enforcing procedures in a consistent and predictable manner will improve the timely regional introduction of vaccines developed internationally. as important, compliance with international standards will be required of regional manufacturers hoping to license locally developed vaccines more broadly. indian and chinese manufacturers currently export a limited number of vaccines, mainly regionally and to african and latin american countries, but their horizons undoubtedly will expand. in the six-component framework of product development capability-manufacturing; national and international distribution systems; private and public r&d capabilities; intellectual property system; and drug and vaccine regulation-regional manufacturers are at different stages of maturation. in its ascendance to an advanced country producing complex biologicals as well as other high technology products, korea followed a path that might be emulated by others in the region, highlighted by its arrival at a stage with a national system of innovation in science and technology, linking government, universities and industry, a strong regulatory system and observation of intellectual property rights, including adherence to trade-related aspects of intellectual property rights (trips agreement). a specific area of regulatory control needing particular attention is the strengthening of national control laboratories. many countries lack the laboratory capacity to test samples for lot release, and because manufacturing and testing technologies change rapidly, keeping up with new procedures and purchasing needed equipment are ongoing challenges. continuous support also is needed to produce working quantities of reference standards, validation of new assays, staff training, and proficiency testing. as resources are unavailable in many countries to establish and maintain a fully functioning national control laboratory, a regional network has been proposed as an approach to share expertise and to divide workload, while at the same time standardizing methods and criteria. field surveillance of adverse events following immunization is another area requiring strengthening. investigations of adjuvanted h n and h n pandemic and prepandemic vaccines administered in korea and taiwan, respectively, illustrated the interest in and epidemiological capacity of local investigators but also the limitations of existing systems and databases. japan is establishing a database of clinical encounters that if linked to immunization records could be used as a future adverse events surveillance system. regulatory oversight of clinical trials and human subjects protection are other areas that are under growing pressure for improvement. multinational companies have increased the number of clinical trials in asian countries to reduce costs and to obtain local registration of products. their activities serve an important role in strengthening local compliance with good clinical practices, as many groups conducting trials in the region have limited experience with these precepts and procedures. countries in the region have an interest to establish and enforce clear guidelines, not only as hosts to an increasing number of trials but also because their manufacturers, as future sponsors of new products, will be accountable internationally to uphold recognized standards. references for this chapter are available at expertconsult.com. immunization in the asia-pacific region .e references . country hub. gavi, the vaccine alliance role of vaccine manufacturers in developing countries towards global healthcare by providing quality vaccines at affordable prices asia's ascent-global trends in biomedical r&d expenditures emergence of biopharmaceutical innovators in china, india, brazil and south africa as global competitors and collaborators developing countries can contribute to global health innovation the indian and chinese health biotechnology industries: potential champions of global health? chinese health biotech and the biollion-patient market indian vaccine innovation: the case of shantha 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long-term epidemiological effect of vaccine against hemorrhagic fever with renal syndrome in a large population harmonization of standards and technical requirements in asean dengue vaccines regulatory pathways: a report on two meetings with regulators of developing countries greater antibody responses to an eleven valent mixed carrier diphtheriaor tetanus-conjugated pneumococcal vaccine in filipino than in finnish or israeli infants safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in taiwanese infants immunogenicity and safety of a novel quadrivalent meningococcal conjugate vaccine (menacwy-crm) in healthy korean adolescents and adults identification of antigen-specific b cell receptor sequences using public repertoire analysis profiling of measles-specific humoral immunity in individuals following two doses of mmr vaccine using proteome microarrays both lewis and secretor status mediate susceptibility to rotavirus infections in a rotavirus genotype-dependent manner genetic polymorphisms of cxcr and cxcl are associated with non-responsiveness to the hepatitis b vaccine india's proposed amendments to the drug and cosmetics act: compensation for injuries to clinical trial participants and the criminalization of clinical research. life sciences law and industry report. lslr , / / bloomberg bna vaccines, our shared responsibility immunization policy development in thailand: the role of the advisory committee on immunization practice an overview of the national immunization policy making process: the role of the korea expert committee on immunization practices progress in the establishment and strengthening of national immunization technical advisory groups: analysis from the who/unicef joint reporting form, data for recent progress and concerns regarding the japanese immunization program: addressing the "vaccine gap who-unicef estimates of dtp coverage world health organization department of economic and social affairs population division vaccine preventable diseases surveillance program of japan. japanese encephalitis: surveillance and elimination effort in japan from to seroprevalence of hepatitis a and associated socioeconomic factors in young healthy korean adults urbanization and geographic expansion of zoonotic arboviral diseases: mechanisms and potential strategies for prevention development of health biotechnology in developing countries: can private-sector players be the prime movers? we are grateful for the help of john o'shea, jason humphries, takashi sugimoto, and hyun-ah chang. key: cord- -l za w authors: anand, sudhir title: human security and universal health insurance date: - - journal: lancet doi: . /s - ( ) - sha: doc_id: cord_uid: l za w nan human security is a multidimensional concept that has been a cornerstone of japanese development cooperation for more than a decade. at the heart of security is the idea of protection or insurance against downside risk. , three distinct questions arise from the concept of human security. first, protection of what? second, insurance against what? and, third, security for whom? the fi rst question relates to the specifi cation of what is to be protected. the defi nition of human security off ered by the commission on human security is: "to protect the vital core of all human lives in ways that enhance human freedoms and human fulfi lment". the core of a person's life is closely concerned with the person's wellbeing and agency, which is best viewed in terms of his or her "capability" to achieve alternative "beings and doings". in this context, health assumes central importance for two reasons: it is directly constitutive of a person's wellbeing; and it enables a person to function as an agent-that is, to pursue the various goals and projects in life that he or she has reason to value. this view deploys the notion of well-functioning, but it is not grounded in notions of economic welfare that are based on utility or income. it is, rather, an agency-centred view of a person, for whom ill health restricts the scope of human agency. since our ability to do things typically depends on our being alive, the capability to lead a long and healthy life must itself be regarded as a basic capability. the second question related to human security is insurance against what. here the concern is to insure against falling below an adequate threshold of human capabilities-in the case of a person's health, a minimum acceptable level. the probability of falling below a minimum threshold depends on both how vulnerable a person is-the degree of downside risk the person facesand how much above the threshold he or she is in the relevant dimension. the extreme case of insecurity is certainty of being below a specifi ed threshold, and the absence of any chance of avoiding that fate. threats to human security can arise, for example, from natural disasters and environmental catastrophessuch as the earthquake and tsunami in japan, and the consequent leakage of radioactive material from the fukushima daiichi nuclear plant. they can arise from disease outbreaks such as hiv/aids, severe acute respiratory syndrome and drug-resistant tuberculosis; from personal accidents and illness; from economic downturns as in the asian fi nancial crisis of - ; and from various other hazards that people face. , , the vulnerability of a person to such risks will depend on his or her individual circumstances-including location, epidemiological environment, health status, and economic position. a person's health is aff ected by health care and various other determinants-eg, socioeconomic, behavioural, continuing commitment. otherwise, we risk alienating patients and damaging the therapeutic alliance. as part of the eradication eff ort, who requested that countries and laboratories either destroy the remainder of their stocks of variola, the causative agent of occupational, and dietary. but access to appropriate health care is also a vital factor in protecting a person from the risk of ill health, and especially of catastrophic ill health. comprehensive health care is thus important both in promotion of health and in response to health crises. without health insurance, a severe medical crisis that threatens survival, for example, can have disastrous fi nancial implications-that can aff ect human security in many other dimensions. the third question concerns security for whom-the entire population or a subset of it? universalism can be defended through a variety of diff erent approaches, which all invoke equity, fairness, or impartiality in some form or other. for instance, we can appeal to impartiality through the device of rawls's "veil of ignorance" in the "original position". behind the veil of ignorance, i do not know who i will turn out to be and what serious illness or health threat i might encounter, which could require extensive medical attention. given this uncertainty, the institutional arrangement for health care i am likely to favour is one that ensures comprehensive coverage for all. the concept of human security has wide reach and includes multiple concerns. a major concern is protection of people's health, for which comprehensive health coverage for all is an essential requirement. universal health insurance thus contributes directly to furthering human security. this implication is as valid for japan as for other countries in the world. the tragic events of, and responses to, the earthquake of are a powerful reminder of japan's concern for human security. in the past, several distinguished politicians, civil servants, and academics in japan have drawn on and developed the concept of human security-including former prime minister keizo obuchi, japan international cooperation agency president sadako ogata, global health expert keizo takemi, and japan center for international exchange president tadashi yamamoto. , [ ] [ ] [ ] [ ] indeed, universal health coverage in japan, now in existence for years, is indicative of the priority that japan accords to human security. over the decades, japan has also undertaken policies to advance human security in other dimensions, such as basic education, social protection, and economic safety nets. internationally, japan has used the concept of human security to guide assistance to developing countries through bilateral aid and multilateral policies. the range and reach of the idea of human security are extensive, as japanese actions have shown. a central manifestation of these actions is the country's commitment to universal health insurance. department of economics, university of oxford, oxford ox uq, uk sudhir.anand@economics.ox.ac.uk i declare that i have no confl icts of interest. the "impossible patient": organizational response to a clinical problem long-term opioid contract use for chronic pain management in primary care practice: a fi ve year experience contracting for safety with patients: clinical practice and forensic implications contracts between patients and healthcare practitioners for improving patients' adherence to treatment, prevention and health promotion activities limiting exposure to medical malpractice claims and defamatory cyber postings via patient contracts systematic review: treatment agreements and urine drug testing to reduce opioid misuse in patients with chronic pain are no-suicide contracts eff ective in preventing suicide in suicidal patients seen by primary care physicians? partnerships in patient care: a contractual approach a rose by any other name: pain contracts/agreements nonabandonment: a central obligation for physicians the use of contracts in the inpatient treatment of the borderline personality disorder motivational interviewing in health care: helping patients change behavior economic security. paper presented at common security forum conference concepts of human development and poverty: a multidimensional perspective human security now: protecting and empowering people commodities and capabilities why human security? health as a human security priority for the st century. human security track iii. the helsinki process a theory of justice opening remarks common security in asia: new concepts of human security common security in asia: new concepts of human security the asian crisis and human security: an intellectual dialogue on building asia's tomorrow key: cord- - ah w o authors: sakurai, mihoko; adu-gyamfi, bismark title: disaster-resilient communication ecosystem in an inclusive society – a case of foreigners in japan date: - - journal: int j disaster risk reduct doi: . /j.ijdrr. . sha: doc_id: cord_uid: ah w o the number of foreign residents and tourists in japan has been dramatically increasing in recent years. despite the fact that japan is prone to natural disasters, with each climate-related event turning into an emergency such as with record rainfalls, floods and mudslides almost every year, non-japanese communication infrastructure and everyday disaster drills for foreigners have received little attention. this study aims to understand how a resilient communication ecosystem forms in various disaster contexts involving foreigners. within a framework of information ecology we try to get an overview of the communication ecosystem in literature and outline its structure and trends in social media use. our empirical case study uses twitter api and r programming software to extract and analyze tweets in english during typhoon (hagibis) in october . it reveals that many information sources transmit warnings and evacuation orders through social media but do not convey a sense of locality and precise instructions on how to act. for future disaster preparedness, we argue that the municipal government, as a responsible agent, should ( ) make available instructional information in foreign languages on social media, ( ) transfer such information through collaboration with transmitters, and ( ) examine the use of local hashtags in social media to strengthen non-japanese speaker’s capacity to adapt. the geographic characteristics of japan makes the country highly vulnerable to disasters such as earthquakes, typhoons, volcanic eruptions, flash floods and landslides [ ] . therefore, disaster risk reduction and resilience measures are enshrined in the every-day activities of the japanese people including school curriculum, building regulations and design, as well as in corporate organization setup [ ] . disaster risk reduction drills often take place in schools, work places, homes, and include the publication of detailed evacuation plans and procedures in all local communities [ ] . however, these efforts of building the capacity of residents facing emergencies could be falling short in terms of coverage and results due to the influx of foreigners who may neither be enrolled in schools or engaged with japanese establishments or are staying only for a short period of time [ ] . the population of foreign residents in japan has been increasing exponentially in recent years according to a report by mizuho research institute [ ] , with their number reaching a record high of . million people by january , . the report reveals an increase by , residents from january to december in . additionally, the japan national tourism organization reports that the number of foreign tourists visiting japan is rapidly increasing. it recorded million in , which was four times as many as visiting in , and six times over the number visiting in . this trend of increased foreign tourists may partly stem from the influence of numerous sporting and other events across the country which include the formula grand prix, the fivb volleyball women's championship and the japan tennis open [ ] . the tokyo olympic and paralympic games, which has been postponed to , as well as the push by government stakeholders to amend bills to relax immigration laws can also be expected to boost the number of foreign residents and tourists in the country [ , ] . even though some foreigners become permanent residents, the qualification for permanent residence status requires a ten-year continuous stay in japan including five years of work, but does not necessarily require the acquisition of japanese language skills [ ] although it may matter at some points. living in japan briefly or as a permanent resident does not necessarily mean the person speaks japanese or is accustomed to disaster risk reduction culture and procedures in japan. although the composition and dynamics of the japanese population is gradually changing in terms of the non-japanese population, existing infrastructure and systems that support non-japanese speakers and foreigners as a whole during disasters seem to last accessed on march , . https://www.jnto.go.jp/jpn/statistics/visitor_trends/ j o u r n a l p r e -p r o o f be inadequate or lacking . the current japanese disaster management system is composed of both vertical and horizontal coordination mechanisms and, depending on the scale of the disaster, activities flow vertically from the national level, through the prefectural to the municipal level before getting down to the communities themselves [ ] . the municipal disaster management council together with other parties are close to disaster victims and responsible for municipal disaster management plans and information [ ] . when necessary, the municipality is also responsible for the issue of evacuation orders to residents during disasters [ ] . again, when it comes to emergency alerts or risk information, there is an existing mechanism where warning messages proceed from the national government (the cabinet, japan meteorological agency (jma)) to local administrations (prefecture, municipalities) and from there to the people. messages may also be transmitted directly top-down through base stations and are received on cell phones. however, reports by news outlets suggest that language barriers, coupled with the inexperience of many foreigners with regard to japanese disaster procedure protocols, create a huge sense of panic and confusion during disasters [ , ] . lack of appropriate risk information and procedural evacuation or alert actions create this confusion amongst the foreigners. therefore, most foreigners access news outlets in their respective countries or rely on social media for disaster risk particulars, alert instructions, or evacuation information [ ] . the use of social media is seen as a significant trend in accessing swift, precise and easy feedback information in critical situations [ ] [ ] [ ] . social media has become the most sourced avenue for information during disasters [ ] . this applies to both japanese and non-japanese users. irrespective of the chaotic nature or confusion that erupts due to limited risk information accessibility and delivery, a certain level of resilience is achieved through instinctive user responses. thus, in extreme events, a spontaneous system of resilience is often formed within the context of all the actors involved in the situation [ ] . this process usually develops because the participating actors share a common interest or find themselves in a situation that requires urgent solutions. the characteristics of this phenomenon create a state of ecosystem which becomes unique to the area affected and to the nature of the event; it generates interactions and communication procedures or methods to reduce vulnerabilities to the disaster [ , ] . the interactions within this ecosystem could be formal or informal, depending on the situation and different communication structures, modes and tools employed. the high frequency of disaster occurrences in japan in conjunction with a booming foreign population up to covid- , and an anticipated, fast rising population again thereafter, provides an ideal case for trying to understand how this ecosystem is established within the context of resilience and communication. resilience in this study is defined as a system's ability to absorb disturbances [ ] . to analyze this ability, we define the system in terms of the information ecology framework. we regard disaster resilience in the information ecology framework to encompass the efforts of collaboration and communication dependencies that exist amongst stakeholders engaged in the situation within a local context. we want to investigate how foreigners in japan obtain disaster related information while facing a language barrier and inexperience with disaster management procedures. this will guide us to understand the characteristics of the communication ecosystem for a foreign population. this paper is divided into the following parts: first, we introduce the information ecology framework as the theoretical premise of a resilient communication ecosystem. a literature review should give us insights into current studies on the topic and their deployment. a key question is how a resilient communication ecosystem is formed in different disaster contexts. to better understand this question, we seek cases amongst the literature which highlight disaster resilience through collaboration, communication and stakeholder participation. cases with such attributes are selected, reviewed and discussed from the view point of information ecology. following the case review, we use the twitter api (application programming interface) application and the r programing language to collect and analyze english language tweets shared on the twitter platform during typhoon hagibis which hit japan in . this gives us an empirical understanding of a resilient communication ecosystem. it is assumed that information shared on twitter in english is meant for consumption by non-japanese. again, media coverage of the typhoon is also monitored to serve as supplementary information to our analysis. based on insights from literature review and findings from tweet analysis, we try to describe a structure which guides our understanding of a communication system applicable to foreigners during a disaster. we conclude the paper with observed limitations and future research directions. information ecology is composed of a framework which is defined as "a system of people, practices, values, and technologies in a particular local environment" [ ]. previous research uses the notion of ecosystem to describe the nature of resilience in a societal context [ ] . information ecology framework, which is an extended notion of the ecosystem, helps us to examine the capabilities of each element within the system. the framework contains five components, including: system, diversity, coevolution, keystone species and locality. table summarizes the elements of information ecology. not shown but also included is the perspective of a technology role which focuses on human activities that are enabled by the given technology implementation. table table table table the presence of keystone species is crucial to the survival of the ecology itself, e.g., skilled people/groups whose presence is necessary to support the effective use of technology. locality locality locality local settings or attributes that give people the meaning of the ecology. given that resilience is defined as a system's ability to absorb disturbances [ ] , information ecology believes that this ability emerges through interrelationships or dependencies between system entities [ ] . these interrelationships exist between a diversity of players equipped with relevant technologies. keystone species play a central role in creating interrelationships, which, in turn, may lead to new forms of coevolution. the notion of locality reminds us of interrelationships and coevolution emerging in a local setting [ ] . the information ecology framework comes down to a set of key elements providing a systematic view of resilience. it enables us to understand who within a network of relationships is a key for realizing resilience, and what relations can be observed within a system and under which local contexts. coevolution is a driving force for forming the resilient ecosystem in times of disaster and is produced by collaboration of systems with j o u r n a l p r e -p r o o f diverse species that provide local context or knowledge [ ] . to this we need to add the role of technology in helping human activities and support the formation of coevolution. we notice a similar framework exists as communication ecology [ ] . it refers to individual or socio-demographic group connections that strengthen neighborhood communication infrastructure. it helps identify communication patterns of local communities or groups of people. in a disaster context, communication ecology includes community organizations, local media and disaster-specific communication sources [ ] . such communication fosters community resilience [ ] , which can be described as the ability of a neighborhood or geographically defined area to collectively optimize their connected interactions for their benefit [ ] . collective ability is required to deal with stressors and resume daily life through cooperation, following unforeseen shocks. hence, community resilience and its collective ability aim to empower individuals in a given community [ ] . resilience is enhanced through economic development, social capital, information and communication, and community competence [ ] . provision of or ensuring access to vital information through proper communication systems is essential to strengthen community capacity towards the unexpected. in this regard, a resilient communication ecosystem that this paper tries to get an understanding of, encompasses the dynamically evolving processes that empower the collective ability of information gathering and provision, as well as interactions and collective communication structures among individuals, communities, and local organizations. a literature review in the following section will help us extract the essential elements of a resilient communication ecosystem based on information ecology. this study follows a systematic mapping approach [ ] as it has the flexibility, robustness and ability to chronologically categorize and classify related materials in research contexts. the process was based on papers which applied the same approach, i.e., [ ] [ ] [ ] . we set the following research question (rq); rq: how is a resilient communication ecosystem formed in time of a disaster? we want to understand how resilience mechanisms are able to evolve spontaneously during disasters through communication, collaboration and the roles of different j o u r n a l p r e -p r o o f stakeholders affected by them. to accomplish this, this study categorized the process into three main activities [ ] . they are ( ) search for relevant publications, ( ) definition of a classification scheme, and ( ) review of relevant journal papers. the search for relevant publications was undertaken in two steps. first, an online database search was conducted with the keywords "collaboration in disaster", "communication in disaster" and "institutional role in disaster" across a number of journal databases such as sciencedirect (elsevier), springerlink, and emerald this procedure resulted in the selection of papers from all the sampled papers. the classification used in this research was to read, clearly identify and arrange the contents of the potential papers to extract the following content; . research titles, . source (type of journal database), . research question of the article . purpose (which keyword is dominant), . type of disaster, . country or region of disaster, . identified stakeholders, . communication tools used, and . communication structure of the case. this stage identified the contents relevant to this study. the communication ecosystem found to be prevalent within the reviewed cases highlights the presence of institutions, individuals and other actors who become involved in disaster events and perform various tasks or activities aimed at reducing either their own risk or that of the stakeholders. in most cases, a system evolved consisting of actors receiving and sharing information when a disaster or crisis took place [ , ] . information ecology promotes sharing and learning; particularly about the use of new technologies, and to reduce given levels of confusion, frustration and ambiguities [ ] . in this review, social media emerges as a new trend in technology and rather becomes the medium for sharing information with the aim to reduce anxiety about a disaster situation that could negatively affect the people involved [ ] . actors include government agencies, non-government agencies, and other actors who evolve to become part of the resilient structure of the framework, matching the nature of each situation. actors can be clearly distinguished based on the roles they play within the system. they include ( ) remote actors, ( ) responsible agents and ( ) transmitters, and ( ) austria [ ] . the world health organization also supports local communities by giving constant guidance and recommendations as a remote actor during ebola outbreak in west africa [ ] . responsible agents have the highest authority and are responsible for issuing needed guidance for people to stay out of risk zones, advising rescue efforts, and providing information on how to reduce risk [ ] . they are the national agencies such as at the level of ministry or governmental organization [ ] , including security agencies and fire brigades [ ] . and information flow between them and other parties [ ] . keystone species identified in the described disasters represent the group or actors whose presence is crucial for all resilience activities. although they are usually described as highly skilled actors, evidence from the reviewed cases also proves that their activities would not be successful without the cooperation and support from other entities [ ] . for instance, staff of the oslo university hospital can be described as "keystone species" in the case of terrorist attacks in norway [ ] . however, actors such as blood donors to the hospital and patients, news agencies who covered all events, individual volunteers who help spread "blood request" alerts by the hospital on social j o u r n a l p r e -p r o o f media, as well as others who sent time situational information on social media to update others can be said to be the auxiliary actors who complemented the "keystone species" effort. they give rise to the notion that resilience efforts are often created by a system of actors or groups who are connected through information sharing and play specific roles in a cohesive and coordinated manner during events or disasters [ ] . furthermore, the key to all these systems are the space or context in which their actions take place [ , ] . in information ecology, it is the "locality" or the setting that initiates all processes. this gives the specification of the context and guides the actions that follow. in some cases, factors such as the geographical location, messaging or information content, characteristics of actors, the medium of information delivery, duration of the event, and the actions taken give the "local identity" of the system [ , , ] . during the ebola crises, this meant the names of the affected countries [ ], while for earthquakes and disasters such as fires it refers to their exact locations. for the purpose of this study, locality refers to a system of individuals, agencies and local communities that refer to a local information and actions to be taken in order to reduce risk within a given area. table summarizes insights from our case review which illustrate the structure of a resilient communication ecosystem. table table table table as stated previously, the japanese governance system is composed of three tiers, national, prefectural, and municipal. among these, municipal government is closest to people at large and therefore is in charge of issuing evacuation orders, opening and operating evacuation centers, and managing relief efforts [ ] . when an evacuation order is issued by a local municipality, residents who live in specific zones are supposed data collection was done using twitter api in r package to scout tweets in the english language. to extract the tweets, the following steps were used: a. registration with twitter api to secure api credentials b. integrating twitter cran (programming language for r package) into r statistical package c. searching by tweets. this was conducted by searching the hashtags "#hagibis" and "#typhoon_ ". to restrict tweets to only english language, the code "en" was added. typhoon hagibis made a landfall on october , . therefore, the tweet search was restricted to search for information of tweets made on that day. the entire code use for the search was (hagibis <-searchtwitter ("#hagibis","#typhoon_ ", n=" ", lang = "en", since = " - - : ", until = " - - : "). five thousand tweets were collected from this search. d. export and further analysis: the results were exported to microsoft excel for further analysis. data analysis was based on the framework of a resilient communication ecosystem. the first aim was to find keystone species supporting the communication ecosystem. therefore, we focused on the number of tweets and twitter id accounts with a high number of retweets. as a result of retweet number analysis, we established the formation of coevolution within the ecosystem. we also aimed to explore the kind of information content exchanged in the ecosystem. hence we conducted thematic analysis [ ] which allowed us to apply key elements of a communication ecosystem. it shows how the communication ecosystem for english language was formed when the typhoon hit japan. in order to extract a sense of locality, we picked up the name of a region or city, and searched for local information such as evacuation orders or emergency alerts within a certain tweet. diversity in this case reflects the number of tweets and different account id information that posted or retweeted with #hagibis or #typhoon_ during the sampled time frame. frame, but could be the n th retweet for that particular tweet. from our findings, the highest number of original tweets from a single account was out of the original tweets. they were sent by @nhkworld_news, a public broadcaster in japan (figure ). this was followed by tweets created through a twitter handle name @earthuncuttv; which is leading online portal, documenting extreme natural events or disasters in the asia pacific region and around the world. around half the tweets within the original tweets were made by individuals. this was determined based on their handle ids. the total aggregated number of retweets we found from our time frame was , . amongst them, % were retweets which originated from @nhkworld_news ( figure ). @earthuncuttv covered % of total retweets while % came from other origins. the results show that within our sample framework, @nhkworld_news is the most dominant tweeter account. the ranking of retweets exceeding one hundred per source is shown in figure . the most retweeted tweet recorded , retweets. the original tweet was posted by @nhkworld_news saying "hagibis is expected to bring violent winds to some areas. a maximum wind speed of kilometers for the kanto area, and kilometers for the tohoku region." the second most retweeted one was posted by an individual account saying "ghost town //shibuya at pm before a typhoon" with a photo taken in shibuya city. the top ten retweeted tweets are shown in table . names in italics stand for region or city name. table table table table the th and th tweets mentioned evacuation orders issued by local municipalities while other tweets shared the event as it happened in different places around japan, or they reported on its effects. we found only three among original tweets that captured the emergency alert issued by jma (figure ). those three were tweeted by individuals and generated only a few retweets. the emergency alert was described in japanese which is translated as "special alert at level for heavy rain has been issued to tokyo area. level alert means the highest level of warning. take action for saving your life. some areas have been newly added under the special alert. check tv, radio, or local municipalities' information," as of october , : pm. the alert requested people to refer to information from local municipalities but all of it was in japanese. our constant monitoring of the event on the internet also reveals that jma was giving constant updates of the real-time weather status and alerts on its website. this information was provided in english. however, most of it did not appear in our sample frame as it did not utilize twitter in english. as the japanese disaster management system also grants direct access of emergency information from jma to the population, an emergency alert was sent to people's cell phones from jma, but this was written in japanese ( figure ) . hence, people who do not speak japanese may not have understood this, and the real time information could only be solicited by a few who originally knew about such a website or the information provided. on the other hand, much evacuation information is issued by municipal government. we found five tweets mentioning evacuation orders. two of those were the j o u r n a l p r e -p r o o f @nhkworld_news (italics refers to location), as follows. as to take [ ] . for example, a dutch man, who had been living in japan for a half year, received an emergency message but he could not find information where to evacuate. another american woman was supported by her friend in translating disaster information. emergency situations generate exceptional communication features which contravene the norm [ ] . therefore, a communication ecosystem forms and breaks up every time a disaster occurs. we observed dynamic characteristics of crisis communication structure with the involvement of various organizations [ ] and tools [ ] . the tools greatly hinge on social media which have become essential for digital crisis communication [ ] . however, more research is required to better capture the nature of social media use in crisis communication [ ] . several studies reported that j o u r n a l p r e -p r o o f local governments especially in thailand and the u.s. are reluctant to utilize social media in disaster situations [ , ] . they prefer to use traditional media such as tv and radio though those mass media tend to allow only one-way communication during a crisis [ ] . as our study shows, information through mass media becomes limited or challenged to meet specific needs due to its broader audience base [ ] compared to the multiple-interaction communication style that characterizes social media [ ] . government, as a responsible agency, needs to understand the characteristics of mass media and social media communication and create an appropriate communication strategy or collaboration scheme when preparing for future catastrophes. in the recent covid- crisis, the need for more targeted health information within a community and the importance of strong partnerships across authorities and trusted organizations are being discussed [ ] . we hold that more consideration should be paid to the provision of targeted information to those who do not share a common context or background of disaster preparedness. on daily-basis practices in japan, almost all local municipal governments prepare a so-called "hazard map" and distribute it to residents. ordinarily, municipalities only issue evacuation orders and do not provide residents with specific information where to evacuate to. from a regular everyday disaster drill, trained residents are expected to know where the evacuation centers are in their area. as people interpret new information based on previously acquired knowledge [ ] , a responsible agent should also consider how to provide information to people who haven't had any disaster training. beside the feature of interactive communication characterizing social media, the pull of multiple information sources and agents creates a sense of assurance and security in coping with or adjusting to events. this was experienced in the haiti earthquake, when social media contributed swiftly to creating a common operating picture of the situation through the collection of information from individuals rather than from a hierarchical communication structure [ ] . in addition, what is important in crisis communication is who distributes a message and how it is mediated in a population [ ] . our study shows that news sources who already seem to have gained social trust played a role as a keystone species in foreign language communication minorities prioritize personal information sources over the media [ ] . these cases suggest that social media promote effective resilience in communication, and that the delivery of information to foreigners in japan from different language backgrounds and cultures further creates traits where personal connection contributes to information accessibility choices. it is also true that social media-based communication raises situation awareness [ ] by receiving warning alerts as well as emotionally-oriented messages such as from friends and relatives [ ] . language barrier is likely to have been an obstacle that actually helped to shape the ecosystem identified in this study. during typhoon hagibis, responsible agents sent warning messages only in japanese language as shown in figure , so this could have been a major factor in harnessing the full potential of social media for disaster communication. results from this study reveal a lack of local knowledge or reference points across twitter communication, which shapes discussion within the ecosystem. for instance, in the case of the chennai flooding in india, tweets covered subjects such as road updates, shelters, family searches and emergency contact information [ ] , none of which were much referred to in our sampled tweets. while in the and philippine flooding, the most tweeted topic was prayer and rescue, followed by traffic updates and weather reports with more than half the tweets written in english [ ] . moreover, around % of those tweets were secondary information, i.e., retweets and information from other sources [ ] . our study recognizes the importance of secondary information. information exchange between agencies and affected residents and tourists assists in reducing risks such as, for example, during hurricane sandy in , where the top five twitter accounts with a high number of followers were storm related organizations posting relevant news and the status of the hurricane [ ] . as a remote actor becomes a source of secondary information, however, such information should guide people in what they should do next, rather than just point to the disaster situation. in this context, we note the importance of instructional messages whose content guides risk reduction behaviors [ ] . an example of such a message is "do not drive a car" which was tweeted by police during a snowstorm in lexington [ ] . it tells what specific life-saving action should be taken [ ] . generally, however, there appears to be little investigation of instructional information in crisis communication [ ] . the findings of this study imply that there could be two types of disaster information: a) risk information that refers to the potential effect of the disaster, i.e., emergency alert or warning, and b) action-oriented information that carries instructions for reducing the risk, i.e., an evacuation order and itinerary to be followed. risk information is published by remote actors while action-oriented information is provided by responsible agents. both types of information must contain local knowledge and allow easy access when people search for that information [ ] . in order to make sure people can reach instructional messages, it is not enough to just point to the information on organizational websites [ ] , as jma did during typhoon hagibis. instead, "localized hashtags" [ , , ] can support people to find life-saving information as quickly as possible. a previously cited study argues that social media can be useful when sending a request for help [ ] . "typhoon damage of nagano" is an example of a localized hashtag, assisting a fire brigade in nagano prefecture utilizing twitter to respond to calls for help from residents during typhoon hagibis . a localized hashtag can also be used as a point of reference where people can find relevant information [ ] . last accessed on july , , http://www.nhk.or.jp/politics/articles/lastweek/ .html. in future disaster communication, local information should be distributed using geographical information with instructions as to what to do next [ ] . responsible agencies should take this into account, while also analyzing which information was shared across social media in previous disaster cases [ ] . furthermore, cultural differences can also be considered, as western culture emphasizes individual action [ ] while asian cultures prioritize collective actions or community commitment. as this may generate behavioral differences among japanese and foreign residents or tourists, further investigation will be beneficial. based on information ecology framework and literature review, the structure of resilient communication ecosystem is proposed and verified through empirical data analysis. the resilient communication ecosystem is structured with heterogeneous actors who could be a driving force for collaboration or coevolution. the empirical case revealed that a media source might transmit warnings and evacuation orders through social media but that such information does not contain points of reference. limited delivery of such information particularly in the english language results in confusion to non-japanese communities who need it most. based on study results and discussions, we suggest that in any disaster a form of ecosystem is spontaneously generated. municipalities, which are often responsible agents should ( ) produce instructional information in foreign languages on social media, ( ) transfer such information through collaboration with transmitters who may have a strong base on social media to assist translating it to reach the wider audience, and ( ) examine, in association with the mass media and weather forecast agencies, the use of local hashtags in social media communication for future disaster preparation. it brings various actors together, creates a sense of locality, and supplements individual efforts in risk reduction. our empirical data is limited to some extent as we only observed twitter for one day, october , . again, we intended to extract tweets in english language just before and after the typhoon hagibis hit japan. hence, we chose hashtag #hagibis and "#tyhoon_ " when we were crawling tweets. we are also aware that our data is based on only a single disaster case and requires further data sets from other disaster events for corroboration. we may consider different characteristics of information and actors in the resilient communication ecosystem in future research. nevertheless, our findings provide interesting insights into disaster communication which may guide the direction of future research. as instructional information sometimes contains ethics and privacy issues [ ] , rules for dealing with specific local or personal information should be taken j o u r n a l p r e -p r o o f into account. behavioral differences are also worth investigating because of increasing human diversity in japanese society. the case of japan reveals the importance and potential of communication as a main mechanism for offering information and response activities to promote resilience and reduce the risk of disasters. social media space serves as a major platform that brings numerous stakeholders together. this platform creates the main avenue for information sharing on events and offers feedback mechanisms for assessment and improvement. for future disaster preparedness, then, we may benefit from a closer understanding of the nature of a resilient communication ecosystem, its structure and the knowledge that it allows us to share. 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strategies in japan date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: vvgov covid- has globally spread to over million people and the epidemic situation in japan is very serious. the purpose of this research was to assess the risk of covid- epidemic dissemination in japan by estimating the current state of epidemic dissemination and providing some epidemic prevention and control recommendations. firstly, the period from january to march was divided into four stages and the relevant parameters were estimated according to the imported cases in japan. the basic reproduction number of the current stage is . ( % confidence interval (ci) . – . ), which means covid- will spread quickly, and the self-healing rate of japanese is about . ( % ci . – . ), with small variations in the four stages. secondly, the results were applied to the actual reported cases from to april , verifying the reliability of the estimated data using the accumulated reported cases located within the % confidence interval and the relative error of forecast data of five days being less than [formula: see text]. thirdly, considering the medical resources in japan, the times the epidemic beds and ventilators become fully occupied are predicted as and may , respectively. keeping with the current situation, the final death toll in japan may reach into the millions. finally, based on experience with covid- prevention and control in china, robust measures such as nationwide shutdown, store closures, citizens isolating themselves at home, and increasing pcr testing would quickly and effectively prevent covid- spread. since the first covid- case was diagnosed in december , covid- had quickly spread to all chinese provinces by january [ ] . on april , the world health organization (who) reported , confirmed cases and , deaths globally [ ] . among these countries, the most serious epidemic situations were occurring in the united states, italy, china, spain, germany, france, and iran, which each exceeded , infected people. in particular, the number of cases in the united states has grown quickly, with the number of reported cases increasing from to , over days. contemporaneously, the first covid- case was reported on february in japan and the number of accumulated reported cases was on april . the undocumented infected individuals will facilitate the rapid dissemination of covid- [ ] . therefore, estimation of current infected cases plays an essential role in controlling epidemic development and will help us to evaluate the strategies that should be implemented to adjust the prevention and control measures for mitigating the spread of covid- in japan. from to january , the reported cases in japan were due to imported cases. for instance, the first reported case was a person from wuhan on january, who had been experiencing symptoms on january. this means that the covid- starting time in japan was january due to the first imported case not being diagnosed and isolated. some imported infected or exposed people were in a state of natural transmission until confirmed. according to news reports, the first imported cases, with their serial number, imported days, the symptom days, and confirmed days, are listed in table . january january january january january january january january january january january january january january january january january january january january january january january january the predominant route of transmission of covid- is person-to-person and dynamic methods can better reflect epidemic law from the aspect of the disease transmission mechanism. to provide theoretical and quantitative bases for making prevention and control decisions, we aimed to establish a mathematical model that reflects the dynamics of covid- propagation, and to qualitatively and quantitatively analyze and numerically simulate the dynamics of the model to show the development process of the disease, reveal the epidemic law, predict the development trend, and analyze the causes and key factors of the epidemic. recently, toshkazu [ ] predicted the epidemic peak of covid- in japan based on a susceptible-exposed-infected-recovered (seir) model. this work forecasted that the epidemic peak would be reached in the early to mid-summer and the peak number of confirmed cases in a single day would be . × . the works [ , ] on the diamond princess cruise ship helped officials to make strategic decisions to prevent covid- from spreading from the ship. these works have a limited impact on future disease prevention and control in japan and cannot directly explain the key tasks of disease prevention and control. the current state estimation generated by the right model can be useful to govern the epidemic battle in japan. in this paper, we establish a seven-compartment dynamic model, and adopt the piecewise method and nonlinear least squares to obtain the parameters in the epidemic model. according to the calculation results, we analyze the key factors that affect the covid- outbreak in japan. through numerical examples, we forecast the time when medical resources are scarce and the number of deaths without improving the current situation. we divided the total human population into seven compartments, as shown in figure , named the seihrd model: the susceptible (s), the exposed (e), the infected (i), the hospitalized (h), the recovered by self-healing (r i ), the recovered by hospital curing (r h ), and the deaths (d). the mathematical model is given by: where s(t), e(t), i(t), h(t), r i (t), r h (t), and d(t) denote the proportion of susceptible, exposed, infected, hospitalized, removed by hospital cure, removed by self-healing, and deaths in the total populations at time t, respectively. thus, the following equation holds: where β se and β si denote the transmission rates from the exposed and infected, respectively. because covid- is an infectious person-to-person virus, the growth of exposed and infected cases increases the transmission rates β se and β si . therefore, effective isolation measures and medical screening with the polymerase chain reaction (pcr) test can reduce β se and β si . the exposed individuals become infected after incubation period /µ. in reality, a proportion ζ r recovers from infection through self-cure; ζ h is the confirmed rate from an infected individual. the hospital cure recovery rate is and d is the proportion of deaths. based on previous research [ , ] , /µ = , and hence µ = . . in [ ] , β se = κβ si where κ = . . we assume . ≤ κ ≤ . because the transmission rate of infected individual is more powerful than that of exposed individuals. due to the different national fitness and medical conditions of different countries, the hospital cure rate and disease-related mortality rate are also different. according to reported data in figure , we estimated the cure and death rates as = . ( % confidence interval (ci), . - . ) and d = . ( % ci . - . ), as exhibited in figure . we assumed that the total population is n = . × in japan [ ] . the basic reproduction number r , which is the expected number of secondary cases produced by one infected individual [ ] , is described as: the basic reproduction number r is independent of the hospital cured rate and the covid- -related death rate d. from equation ( ), the number n × (h(t) + r i (t) + d(t)) is the accumulated reported cases, which can be called the documented cases at time t. n × i(t) is the undocumented infection cases at time t. we used data from the ministry of health, labor, and welfare, which reports the data related to covid- in japan [ ] . we also collected reported cases from news reports containing more details to guarantee the validity of the data. in table , covid- spread started in japan on january , which was set as t = . we assumed that no. , no. , and no. are the unconfirmed imported infected individuals, and that nos. - and - are imported exposed individuals. we assumed the uniform distribution of the imported exposed individuals that landed from the diamond princess cruise ship from to february [ ] . thus, the states of e(t) and i(t) should be updated by the following equations: and whereĒ(t) ands(t) indicate the imported proportion at time t. because the spread of covid- is affected by many factors, different interventions produce different results. therefore, we estimated the parameters of the model for different stages of the epidemic. we divided the period from january to march into four stages to estimate the model parameters. the first dividing point is february [ ] , the second dividing point is the implementation of the school suspension order on march [ ] , and the third is the landing of the diamond princess. . . estimation of the parameters β se , β si , ζ h , and ζ r covid- is a new type of coronavirus, and its detection methods and diagnostic techniques are limited, thus we could not guarantee the integrity of the documented data. thus, the nonlinear least square method was used to estimate the parameters, and random simulations under isometric observations were used to obtain the confidence intervals of the parameters. we appointed y(t), t = , , · · · , as the accumulated reported cases of covid- in japan from january to march . we solved the following least-square-based optimization problem: for given parameters β se , β si , ζ h and ζ r , the numerical value of y(t) is: by employing the model in equation ( ) and the assumption in section . . we constructed the objective function as: then, based on the previous statement, the optimization problem is given by: subject to: and: by solving the above nonlinear optimization problem employing the lsqnonlin toolbox in matlab (mathworks, natick, ma, usa) [ ] , the parameters β se , β si , ζ h , and ζ r can be determined. however, the approximations cannot reflect the error ranges, and the estimation intervals need to be given to confirm that the true values of the unknown parameters are included in these intervals. based on the bootstrap method [ ] , we constructed isometric random observations as: where χ(t) denotes random variables from a normal distribution with a mean of zero and variance σ i in the ith stage. y(t) and χ(t) are calculated by applying β se , β si , ζ h , and ζ r . after simulations, we obtained the calculation results shown in figure and table . r was calculated according to equation ( ). the root-mean-square error (rmse) is . and the goodness of fit (gof) r is . from january to march. as assumed in section . , the first dividing point was february and the people disembarked from the diamond princess cruise ship were required to isolate for days. this aroused public attention and strengthened self-protection awareness. the transmission rates β se and β si slightly decreased from the first to the second stage. the second dividing point was the implementation of the decree suspending school and provision of financial assistance to parents. the transmission rates β se and β si dramatically decreased and the basic reproduction number was close to . this measure strengthened the isolation measures and effectively prevented the spread of covid- . after the third dividing point, the transmission rates and confirmed rate substantially changed. during the nine days of the third stage, the number of pcr tests and documented cases were and , respectively. during the days of the last stage, the number of pcr tests and documented cases were , and , receptively. we defined an average index Υ for the daily pcr tests as: where ∆n pcr , ∆h, and t denote the number of pcr tests, the number of documented cases, and the days of the different stages, respectively. we calculated the values of Υ in the last two stages as . and . , respectively, which showed the confirmed rate decreased sharply and resulted in the growth of transmission rates. the self-healing rate is within [ . , . ] with small variations, showing that this parameter relies on the national constitution. from the trend of the basic reproduction number r in table , direct and indirect isolation and pcr medical screening measures are effective prevention and control strategies. with the development of the epidemic, the implemented prevention and control strategies are changing daily. we applied the accumulated reported cases of five days to examine the reliability of our approach. employing the model in equation ( ) and the parameters in table , the numerical accumulated reported cases and their confidence intervals are ( % ci - ), ( % ci - ), ( % ci - ), ( % ci - ), and ( % ci - ). all real reported cases exist within the % confidence intervals, as proven in figure a . as demonstrated in figure b , the largest relative error of the five-day forecast data is less than . %, which means the forecast data have high reliability under the current measures. in comparison with the results in [ ] , the relative errors of forecast data are around %, indicating the model and method in this paper are closer to the actual situation. to show that the current control strategies may lead japan to a massive crisis, we combined the existing epidemic bed statistics and the number of ventilators to predict the future medical state. according to a previous investigation [ ] , the number of existing epidemic beds is about , and we assumed % of the beds are used for other epidemic cases. the number of all kinds of ventilators on standby is about , including ecmo devices. according to the real data, we estimated that the proportion of severe cases of covid- in japan is % of the reported cases [ ] . when the use of ventilators and/or epidemic beds is saturated, the mortality rate will increase dramatically. consequently, we estimated the ventilator and epidemic bed saturation times. as estimated in figure a ,b, the hospital cases would reach , , with epidemic beds being insufficient on may and the respirator shortage occurring on may, with about , critical patients. the results show that makeshift hospitals and shelter hospitals should be arranged in late april at the latest. if we continue with the current situation and adjust the death rate to %, %, and % after the saturation, the number of deaths, as exhibited in figure c , would reach . × , . × , and . × , respectively. to avoid this serious situation, some prevention and control measures that could change the current situation were simulated to predict the epidemic trend. in this section, we discuss some effective strategies for mitigating covid- dissemination. from equation ( ), some parameters affect the value of the basic reproduction number, such as β se , β si , and ζ h . the parameters µ and ζ r are subject to the characteristics of covid- and the national physique. hence, we can adopt some appropriate control strategies to impact the transmission rate and the confirmed rate via β se , β si , and ζ h . given china's experience, the implementation of nationwide shutdowns can effectively reduce the values of β se and β si . increasing the number of pcr tests and enhancing the investigation of those with direct or indirect contact history can quickly increase the percentage of diagnoses ζ h . we assumed that nationwide shutdown, store closures, and citizens isolating themselves for at least days are implemented, and that the β si decreased to . starting from march . the numerical result exhibited in figure a shows that the peak value decreases and the peak is delayed to august. with the parameters set to β se = . and β si = . , covid- would be extinct on august in japan. the above measures appear to be valid strategies for restricting covid- dissemination in japan. essentially, the transmission rate β si is decreased when the confirmed rate ζ h increases. therefore, in the second numerical example, we changed the parameters β se , β si , and ζ h simultaneously. figure b shows that this effectively shortens the peak time and reduces the peak size, resulting in the earliest covid- virus extinction. compared with the first simulation, the end of the epidemic would occur on july with β se , β si = . , and ζ h = . , whereas the limited value ζ h in china is % [ ] . we applied the seihrd model to analyze the development of the covid- epidemic situation in japan. to eliminate the impact of the uncertainty in the early data, the first imported cases were extracted based on news reports. table details the days the cases appeared in japan as infected or exposed individuals. the parameters of the seihrd model were estimated by dividing the period from january to march into four segments, and the basic regeneration number r in the first segment was estimated as . ( % ci . - . ), as illustrated in table . on january, due to the landing of the diamond princess cruise ship, the officials began to pay attention to the epidemic and introduced relevant isolation measures, which reduced the infection rate and the basic reproduction number r to . ( % ci . - . ). with japan's large-scale suspension of schooling on march, the transmission rates weakened to β se = . ( % ci . - . ) and β si = . ( % ci . - . ). the basic number of r became . ( % ci . - . ). we constructed an index of pcr testing and the proportion of diagnoses ζ h degraded to . ( % ci . - . ) when the index reduced from . to . . the transmission rates β se and β si increased as the index decreased. these results show that strengthening the isolation measures and increasing pcr medical screening would help to effectively mitigate covid- spread. to examine the validity of our approach, applying the seihrd model, we generated five-day forecast data and compared the results with the actual accumulated reported cases. the five days of accumulated reported cases exist within the % confidence intervals, as shown in figure a . the five-day relative error of the forecasted accumulated reported cases is less than . %, which means this seihrd model and the piecewise estimation method can be effective applied to covid- . according to the existing medical resources [ ] , we predicted the time of medical saturation under the current situation and forecast the number of deaths, as shown in figure a . numerical examples proved that the suspension of production and business, school shut downs, and large-scale implementation of pcr testing for individuals who may have directly or indirectly come into contact with confirmed cases are the success strategies for preventing the further spread of covid- . it may be possible to end the epidemic in early or mid-july by combining isolation measures and increasing pcr testing, as shown in figure b . in conclusion, there is the possibility of a large-scale outbreak of covid- in japan given the current situation. some control strategies such as strengthening the isolation measures and increasing pcr medical screening should be taken to prevent the covid- further spreading, as shown in section . the authors declare no conflict of interest. the latest situation of the pneumonia epidemic infected with new coronavirus as of : on coronavirus covid- global cases by the center for systems science and engineering (csse) at johns hopkins university (jhu). available online substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (covid- ) prediction of the epidemic peak of coronavirus disease in japan transmission potential of the novel coronavirus (covid- ) onboard the diamond princess cruises ship estimating the asymptomatic proportion of coronavirus disease (covid- ) cases on board the diamond princess cruise ship incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data tracking and predicting covid- epidemic in china mainland modified seir and ai prediction of the epidemics trend of covid- in china under public health interventions reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission the japan times. nearly all prefectures in japan shut schools amid coronavirus outbreak passengers disembarking from diamond princess need to be quarantined for days nonlinear observer using mean value theorem and lsqnonlin matlab algorithm exact bootstrap confidence intervals for regression coefficients in small samples characteristics of and imoprtant lessons from the coronavirus disease urgent investigation on the number of respirators and ecmo devices handled key: cord- - z ob authors: morimoto, tomoko; ishikawa, hirofumi title: assessment of intervention strategies against a novel influenza epidemic using an individual-based model date: - - journal: environmental health and preventive medicine doi: . /s - - - sha: doc_id: cord_uid: z ob objectives: the objective of this study was to assess intervention strategies against a novel influenza epidemic through simulations of various scenarios in sapporo city, hokkaido, japan. a series of interventions were examined: administration of antiviral drugs by two approaches [targeted antiviral prophylaxis (tap) and school-age targeted antiviral prophylaxis (stap)], school closure, restraint, and combinations of these four interventions. methods: in order to generate a more realistic situation, we constructed an individual-based model (ibm) for the transmission of influenza in which each individual was assigned personal information on the basis of the national census and employment status survey of sapporo city. in addition, data on high-risk casual contact groups commuting in crowded trains and buses were obtained from a census on transportation modes and introduced into the model. observational data from previous pandemics were used for the epidemiological parameters. results: both tap and stap interventions were highly effective in suppressing the spread of infection during the early period of an outbreak, but stap was inferior to tap in terms of the ripple effect of the administration of antiviral drugs. school closure and restraint were able to bring about a delay in the peak of infection. the combination of tap, school closure, and restraint interventions were highly effective in decreasing the total number of patients and shortening the epidemic period. conclusions: based on the simulation results, we recommend implementing tap together with both school closure and restraint as strategies against a future novel influenza outbreak. the recent spread of avian influenza a (h n ) in wild and domestic poultry has raised global concern over a pandemic outbreak in humans [ ] . since , about cases of avian influenza a (h n ) infection in humans have been reported in countries, mainly in southeast asia [ ] . in the study reported here, we have attempted to predict the effectiveness of intervention strategies against an epidemic of a novel influenza that will be caused by new strains of influenza achieving the same transmission ability in humans as avian influenza a (h n ) by simulating various scenarios targeting sapporo city, the capital of hokkaido in japan, with a population of about . million. influenza a (h n ), which is a new flu virus of swine origin, was first detected in mexico in march [ ] and has spread rapidly across the globe. a total of , cases of influenza a (h n ) infection in over countries, including , deaths, had been officially reported by late august [ ] . the ministry of health, labor, and welfare of japan [ ] designed a ''pandemic influenza preparedness action plan'' which, among other measures, includes the administration of antiviral drugs and school closure in the case of an influenza pandemic. mathematical models have been developed for influenza transmission. a series of studies using stochastic models were carried out between and [ ] [ ] [ ] [ ] and, recently, there have been several analytical studies on various containment strategies against an influenza pandemic for southeast asia [ , ] and for the usa [ ] . using an individual-based model (ibm), ohkusa and sugawara [ ] recently investigated the spread of influenza in a metropolitan area in japan as a result of infection occurring on crowded trains. the objective of the study reported here was to evaluate the possibilities for suppressing a pandemic through interventions. to this end, we constructed an ibm for the transmission of a novel influenza virus in sapporo city that takes personal information, such as age, household, habitation, social activity group, casual contact group, and behavioral patterns, into account, thereby resulting in a more realistic model. a series of interventions were explored: ( ) targeted antiviral prophylaxis (tap) through prescribing antiviral drugs for symptomatic patients and persons in close contact with them; ( ) school-age targeted antiviral prophylaxis (stap) by prescribing antiviral drugs for school-aged children; ( ) school closure; ( ) restraint. the simulation results showed ( ) that both tap and stap interventions could be effective in suppressing an outbreak within an early period of days but that stap would be inferior to tap in terms of the ripple effect of the administration of antiviral drugs and ( ) school closure and restraint could bring about a delay in the peak of infection. the combination of three interventions-tap, school closure, and restraint-would decrease the number of total patients to . % of that with a no-intervention situation and would be highly effective in suppressing infection. in comparison, although the combination of tap with only school closure or restraint would have some degree of effectiveness in reducing the number of patients, this strategy would be unable to shorten the epidemic period. the results of this study will be helpful in planning intervention strategies against a future influenza pandemic. worldwide influenza pandemics have occurred at least three times in recorded history. the most serious pandemic is considered to have been the ''spanish flu'' a (h n ) in - , which affected large parts of the world's population and was assessed to have killed at least million people [ ] . the much later ''asian flu'' a (h n ) in - and ''hong kong flu'' a (h n ) in - also caused significant morbidity and mortality worldwide. to construct the model reported here, we used observational data from these previous pandemics for the latent and infectious periods, and illness attack and mortality rates as well as surveillance data for avian influenza a (h n ) infection for the rate of crisis due to the unknown natural history of a future influenza infection. following the latent period, a person infected with influenza has infectivity during the infectious period; thereafter, he/she recovers with immunity or dies. the latent and infectious periods lasted - and - days with means of . and . days, respectively, in the ''asian flu'' a (h n ) [ ] , and the latent period ranged mainly from to days, with a maximum of days for a few cases, in swine flu a (h n ) in [ ] . based data on ''asian flu'' a (h n ), the latent and infectious periods were assumed to range over - and - days, respectively; the associated probabilities were tested at , , and %, and , , , and %, respectively. infected states were distinguished as symptomatic and asymptomatic infection. in this model, it was assumed that the rate of crisis was % on average, while the other cases were asymptomatic on the basis of the surveillance of avian influenza a (h n ) infection [ ] , and that asymptomatic cases had half the infectivity of symptomatic cases [ ] . a confirmative rate in all infections was defined by the ratio of patients who sought medical care and were diagnosed with a novel influenza. because many cases of avian influenza a (h n ) appeared to have severe symptoms with vomiting and diarrhea [ ] , the confirmative rate was assumed to be higher, at %. the illness attack rate is different among different types of viruses [ ] , with that of the ''asian flu'' a (h n ) being much higher in children than in adults [ ] ; in contrast, the illness attack rate of the ''hong kong flu'' a (h n ) was approximately the same among all age groups [ ] . the illness attack rate of swine flu a (h n ) virus has been reported to be twice as high in children as in adults [ ] . a study of the sporadic transmissions of avian influenza a (h n ) virus from birds to humans revealed that children are more susceptible to this virus than adults [ ] . because the age-specific illness attack rate of avian influenza a (h n ) was similar to that of ''asian flu'' a (h n ) [ ] , this model adopted the estimated values in the ''asian flu'' [ ] as the age-specific illness attack rate. the mortality in ''spanish flu'' a (h n ) in - showed a w-shaped curve, i.e., it was high not among infants and elderly people but also among young adults ( - years), an age group which usually has a very low mortality with seasonal influenza [ , ] . although the profile for a future influenza pandemic is unclear, we assumed that a novel influenza infection would cause a high mortality similar to the situation of the ''spanish flu'' a (h n ) [ ] . the age-specific illness attack and mortality rates in this model are presented in table . there are currently two kinds of antiviral drugs, oseltamivir and zanamivir, and both are regarded as being highly effective for both the treatment and prophylaxis of influenza infection [ , ] . as treatment, a person must be treated with two tablets of oseltamivir a day for a course of days for treatment; as prophylaxis, treatment consists of one tablet a day over a course of - days. antiviral efficacy lasts only during the course, and no residual effect remains thereafter. the administration of antiviral drugs alleviates symptoms, reduces infectiveness, shortens the infectious period as a treatment effect, and also prevents infection as a prophylactical effect [ , ] . longini et al. [ ] estimated the effects of administering the antiviral drugs that were also adopted in this model and found: ( ) the illness attack rate in susceptible persons dosed with antiviral drugs for prophylaxis decreased to . on average compared with those without dosing (relative susceptibility = . ); ( ) the probability of developing symptoms of influenza in infected persons dosed with antiviral drugs for prophylaxis decreased to . on average compared with those without dosing; ( ) the infectiousness of infected persons dosed with antiviral drugs for treatment or prophylaxis decreased to . on average compared with those without dosing (relative infectiousness = . ); ( ) the infectious period in infected persons dosed with antiviral drugs for treatment or prophylaxis was reduce to day compared with those without dosing. study area and population structure of the ibm sapporo city, the capital of hokkaido in japan, was chosen as the targeted area because sapporo city has a number of characteristics that make it suitable for simulating the spread of infection, including relatively small daily influxes and outflows of people compared with other major cities in japan [ ] . the national census of japan [ , ] was used as the source of data on the age structure, resident population for each ward, household composition, places of schooling and work, and mode of transportation. the school basic survey [ ] was the source of data on the number of schools and the enrollment rate at college, while the employment status survey [ ] provided the employment rate. sapporo city, which had a population of , , in [ ] , is one of the ordinance-designated metropolis cities in japan and is divided into ten wards as administrative units [ ] . there is a substantial difference in the density of population among wards. there are also many commuters to school and work between wards during the daytime, which leads to the mass movement of people on a daily basis [ ] . to simulate the situations of an influenza epidemic on a realistic basis, we adopted an ibm that can reflect the heterogeneous population structure for the targeted area, in which each individual was assigned his/her personal information, such as age, household, residence district, social activity group, and casual contact group. the age structure in sapporo city [ ] is shown in fig. . the distribution of household size was determined on the basis of the national census of japan data [ ] , where each household was assumed to consist of at least one adult aged c years. we specified five categories of social activity groups that an individual may belong to according to age: playgroup ( - years), elementary school ( - years), high school ( - years), college group ( - years) , and work group ( - years). the age-specific rates of the enrollment of college students and employment were determined based on data in the school basic survey [ ] and the employment status survey [ ] . the size of social activity groups was assumed to be smaller than their actual size so that individuals in a group could come into close contact with each other and transmit the influenza virus among them. two kind of untraceable casual contact groups were considered: a low-risk group who came into contact with other people at markets, in the neighborhoods, etc. and a high-risk group who used public transportation facilities, such as crowded trains and buses. the ratio of high-risk casual contact groups was set at % on average in all casual contact groups based on a census of the mode of transportation [ ] . all casual contact groups were composed afresh twice a day with a size of individuals on average. we assumed that individuals would mix with each other randomly (in the household, during social activities, and in casual contact groups) and that individuals had a possibility of becoming infected when an infected person belonged to any of their groups. the average contact rate in a household was set to be higher than that for other groups, followed by that in social activity groups. the average contact rate in a playgroup was set to be higher than that in other social activity groups because children spent more time in close proximity to each other at a day nursery. the usual average contact rate in the low-risk casual contact group was set to be quite low because such contacts occur accidentally during outings [ ] . on the other hand, the average contact rate in high-risk casual contact groups, such as while commuting on crowded trains or buses, was assumed to be -fold higher than that in the low-risk casual contact group. this was assessed by the average commuting time to school and work (an hour per day) [ ] . the average contact rates for every group are tabulated in table . because it is difficult for symptomatic patients to follow their usual behavioral pattern [ ] , it was assumed that a symptomatic patient had a different behavioral pattern from the day after onset; a symptomatic patient retreats to the home, thereby exposing only his/her family [ ] . this article explored four kinds of interventions with the aim of analyzing their effectiveness in terms of suppressing an influenza epidemic: administration of antiviral drugs for prophylaxis through two methods of medication, tap and stap; school closure; restraint; combinations of these interventions were also analyzed. it was assumed that all interventions would be started days after the introduction of the initial patient. in the tap intervention strategy, patients with symptoms as well as persons in close contact with them are treated with antiviral drugs in accordance with the ''pandemic influenza preparedness action plan'' [ ] . tap is regarded as an important intervention in the early phase of an influenza epidemic in the ''guidelines for the prevention and control of pandemic influenza'' [ ] . in this model, it was assumed that a patient with symptoms would be treated with an antiviral drug just being diagnosed with a novel influenza infection, that all members of his/her household would be dosed with an antiviral drug for prophylaxis on that same day of diagnosis or thereafter, and that a person belonging to the same social activity group as the diagnosed patient would be traced, if possible, and treated with an antiviral drug within a few days of contact [ ] . in this strategy, no members of the casual contact group could be traced. one of the advantages of tap intervention is the small amount of antiviral drugs that are distributed for prophylaxis. the model limits the possible number of symptomatic cases for whom members of the social activity group can be traced for close contact to ten persons for each ward per day because of the limited capacity of the healthcare center (only one center for each ward in sapporo city) [ ] . during the swine flu a (h n ) outbreak in japan, not all of the persons in close contact with an infected person could be traced and, moreover, some traced persons refused to take antiviral drugs [ ] . various situations with respect to the proportion of identifiable close contact persons ( , , and %) and necessary tracing periods ( , , and days) were examined. in the stap intervention strategy, antiviral drugs are distributed to the children of a school once a child is diagnosed with a novel influenza at a medical institution to prevent infection among school-aged children, who have a high attack rate. naturally, all household members of the infected person would be dosed with an antiviral drug for prophylaxis from the day of diagnosis. proportions for the coverage of distribution to children in the affected schools of , , and % were investigated, as was the necessary tracing period of , , and days. school closure is regarded as an important intervention measure because an influenza epidemic can be spread widely among children at school [ ] . in this model, when a person is diagnosed with a novel influenza at a medical institution in a ward, school closure will be implemented from the day following the diagnosis for all schools in this ward. the ''guidelines for the prevention and control of pandemic influenza'' [ ] states that a judgment based on the epidemiological investigation will end the period of school closure; however, in our model, school closure was assumed to be continued until no patients were detected in the ward. requests to residents to refrain from going out unnecessarily have been noted as a containment policy [ ] . restraint was generally recognized as a preventive measure by residents in hong kong when they experienced an outbreak of severe acute respiratory syndrome (sars) [ ] . however, many people in japan are unwilling to practice a voluntary policy of restraint, and in one study, only . % of people expressed willingness to practice restraint in a ''survey of pandemic behavior: to stay at home or not.'' [ ] . when a person is diagnosed with a novel influenza at a medical institution in a ward, restraint will be performed in this ward from the day following diagnosis. various rates of restraint ( , , and %) were investigated. the period of restraint was assumed to continue for weeks, and a person who agreed to restraint once was not required to be restrained again for at least month. we investigated five scenarios with different combinations of interventions for their effects on suppression; these included the administration of antiviral drugs for treatment (tap/stap) with/without school closure and with/without restraint (table ). in tap and stap, the necessary tracing period and the coverage were set to days and %, respectively; for restraint, the rate was set to %. tap should be terminated when the number of patients reaches ten persons for each ward per day because the ''pandemic influenza preparedness action plan'' [ ] showed that the administration of medicine to persons in close contact with a patient should be stopped after an influenza infection has spread widely. the stochastic model was programmed using intel visual fortran to work on any computer using the microsoft windows platform, where the random number generators in the imsl libraries were used. the time step of the stochastic process was adopted to be day, and -trial simulations were carried out over days, or until the epidemic was eradicated. we carried out simulations of a novel influenza epidemic in sapporo city to estimate the suppressive effectiveness of the following interventions against an influenza attack: tap, stap, school closure, restraint, and various combinations thereof. in each scenario, the initial patient, an -year-old high school student with symptoms, was introduced into chuo ward of sapporo city, which has the highest day-time population of any of the other city's wards [ ] . the baseline scenario was the situation with no intervention. the number of patients reached a peak at about days after introduction of the initial patient; thereafter, the number decreased gradually, and the epidemic was eradicated after about months (fig. a) . the effective reproductive number (r t ) for days after introduction of the initial patient, which was calculated following the formula of germann et al. [ ] , was estimated to be . in the beginning and . about days after the introduction (fig. b) . infection spread throughout sapporo city from chuo ward for month, decreasing just after the peak of the epidemic in the kita and higashi wards, which have a greater residential population than the other wards in sapporo city, months following its introduction [ ] (fig. ) . the infected cases and deaths in the entire population were assessed to be, on average, and . %, respectively, in -trial simulations. four intervention measures were examined: medication of antiviral drugs by tap and stap, school closure, and restraint. the impact of changing the necessary tracing period and the actual coverage of tracing persons in contact with a patient for the tap intervention were investigated (fig. ) . the mean numbers of total patients and persons treated with antiviral drugs for prophylaxis with % confidence intervals (cis) for days after the introduction of the initial patient and also the numbers of cases prevented per person treated with antiviral drugs for prophylaxis are shown in table . for situations with a necessary tracing period of days with % coverage and days with %, the mean number of total patients for days would be decreased to . and . %, respectively, compared with the baseline. the ripple effect of the administration of antiviral drugs in tap on the reduction in patient numbers was estimated to be . -and . -fold, respectively, for these situations, indicating that the tap intervention would have a substantial effect in suppressing the spread of the infection. the impact of changes in the necessary tracing period and the actual coverage for the stap intervention were investigated (fig. ) . the mean numbers of total patients and persons treated with antiviral drugs for prophylaxis with % cis for days after the introduction of the initial patient and also the numbers of cases prevented per person treated with antiviral drugs for prophylaxis are shown in table . for situations with a necessary tracing period of days with % coverage, the mean number of total patients for days would be decreased to . % compared with the baseline. however, there were no significant differences in the number of patients for a necessary tracing period of , , and days, respectively or for a coverage of , , and %, respectively. the ripple effect of the administration of antiviral drugs in stap on the reduction of patient numbers was estimated to be . and . -fold for a necessary tracing period of days with % coverage and days with %, respectively, which indicates that stap intervention would have a weaker ripple effect than tap. the impact of school closure is shown in fig. . school closure would, on average, delay the peak of infection for an average of days compared to the baseline and decrease total and peak patients to and % of the baseline scenario (table ) . the impact of changes in the execution rate for restraint is shown in fig. . with mean proportions of , , and % of total patients restraining their behavior, on average, the number of cases would decrease to , , and % of the baseline, and the peak would be delayed for , , and days, respectively, compared with the baseline scenario (table ). at the same time, the epidemic period would be prolonged with respect to the baseline, which was similar to school closure. the mean number of total patients, deaths, and persons treated with antiviral drugs with % cis and the epidemic periods in scenarios - (table ) are shown in table . in scenarios - , which encompass the combination of the administration of antiviral drugs (tap/stap) with only school closure or restraint, the mean number of total patients would be decreased to . , . , and . %, respectively, of the baseline scenario. epidemic periods for (table ). on the other hand, scenarios and , which are two different combinations of three interventions [administration of antiviral drugs (tap/stap), school closure, and restraint] would have a greater effect on the suppression of infection in terms of the number of total patients, deaths, and persons treated with antiviral drugs and on shortening the epidemic period. in particular, the combination of tap, school closure, and restraint in scenario ( fig. ) would decrease the total number of patients to . % of that of the baseline scenario and shorten the epidemic period to days ( table ). in this article, sapporo city was chosen as a region suitable for investigating the transmission of a novel influenza through simulations because of the small daily influxes and outflows of people to and from the region in comparison with other major cities in japan [ ] . in many cities, including sapporo city, many students and workers commute in crowded trains and buses every day, which will accelerate the spread of infection [ ] . therefore, high-risk casual contact groups based on commuters among wards were introduced into the model. on the condition that the effective reproduction number r t = . , the baseline scenario showed that, on average, % of the entire population in the -trial simulations would be infected, including the % who would be asymptomatic. in past pandemics, the basic reproductive number (r ) was estimated to be . - . for ''spanish flu'' a (h n ) [ ] and . for ''asian flu'' a (h n ) [ ] . for the swine flu a (h n ) in , r has been estimated to be . - . [ ] and . - . [ ] based on data originating from the epidemic in mexico and to be . based on the epidemic in japan [ ] . therefore, further studies will be needed for a situation with a highly effective reproductive number that may cause more damage. the implementation of tap as a single intervention was highly effective in suppressing the outbreak during the early period of the first days. naturally, this effectiveness was enhanced by shortening the necessary tracing period and raising the coverage of tracing. for a necessary tracing period of or days, there were no differences in the amount of antiviral drugs needed for prophylaxis with a - % coverage; when the tracing period was increased to days, however, these amounts rose, on average, to . and . % for and % coverage, respectively, in comparison with % coverage. these large differences may result from having a wider spread of the influenza infections before the onset of treatment with antiviral drugs for the situation of a -day tracing period, while shorter tracing periods ( - days) can contain most persons with an infection risk at an early stage of the outbreak. the implementation of stap as a single intervention would also be highly effective in suppressing the outbreak during the early period of the first days, but stap was inferior to tap in terms of the ripple effect of the administration of antiviral drugs, most likely because the stap intervention, unlike tap, only distributes antiviral drugs to students, including those at a low risk of infection, notwithstanding influenza infection spreading among adults. school closure and restraint brought about a delay in the peak and a decrease in incidence at the peak. however, it is actually difficult to implement school closure for such a long period, as was assumed in this model, where closure would be continued until no further patients were detected in the ward. therefore, the effectiveness of school closure may be overestimated. in the outbreak of swine flu a (h n ) in japan in , it was reported that the rapid spread of infection disrupted medical institutions [ ] . implementing a policy of school closure and restraint may be one approach to preventing an acute disruption of services at medical institutions. however, school closure also brings about the long-term absence of parents from work, which leads to economic loss [ ] . the importance of restraint was recognized by only . % citizens in japan and the loss of precautions against infection may spread rapidly providing that a novel influenza infection has a low mortality [ ] ; therefore improved recognition of the necessity of restraint is needed. the specific combination of the three interventions tap, school closure, and restraint was shown to be the most effective of all the combined interventions (scenarios - ) in terms of suppressing infection. it resulted in a average decrease in total patients to . % of that of the baseline scenario because of concomitantly preventing the spread of infection at an early stage by prophylaxis and decreasing infection risk by school closure and restraint. on the other hand, the combination of prophylaxis with antiviral drugs (tap/stap) with either school closure or restraint would reduce the amount of patients to some extent but would not be able to shorten the epidemic period because the influenza infection would be spread among people, unlike the combination of prophylaxis with both school closure and restraint. the tap intervention should be stopped after the influenza infection has spread widely due to the priority shifting from prophylaxis to the treatment of patients [ ] . moreover, it may be difficult to identify persons in close contact. it has been reported that the spread of the infections made it difficult for doctors to diagnose swine flu a (h n ) cases and that it was also hard to trace persons in close contact during this outbreak [ ] . in contrast, stap is easy to implement because antiviral drugs are distributed only at schools, although stap does have a weak ripple effect due to drug wastage. therefore, it is necessary to improve the methods of distribution in stap, such as its range and frequency. the results of this study will be helpful in planning strategies for the suppression of an outbreak of a novel influenza not only sapporo city but also in other cities. looming flu pandemic has experts crying fowl a preliminary estimation of the reproduction ratio for new influenza a (h n ) from the outbreak in mexico pandemic (h n ) pandemic influenza preparedness action plan of the japanese government an extension of the reed-frost epidemic model for the study of competition between viral agents in the presence of interference simulation of mathematical models for public health problems a stochastic model for competition between viral agents in the presence of interference. . live virus vaccine in a randomly mixing population, model an influenza simulation model for immunization studies containing 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of school closure for mitigating an influenza pandemic acknowledgments the authors are grateful for the statistical assistance of dr. k. fueda of okayama university. this work was supported in part a grant-in-aid from the japan society for the promotion of science ( ) and a grant-in-aid from the ministry of health, labour and welfare of japan (h -sinkou-ippan- ). , ( , , , ) , ( , key: cord- -udtoutew authors: furuse, yuki; sando, eiichiro; tsuchiya, naho; miyahara, reiko; yasuda, ikkoh; ko, yura k.; saito, mayuko; morimoto, konosuke; imamura, takeaki; shobugawa, yugo; nagata, shohei; jindai, kazuaki; imamura, tadatsugu; sunagawa, tomimasa; suzuki, motoi; nishiura, hiroshi; oshitani, hitoshi title: clusters of coronavirus disease in communities, japan, january–april date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: udtoutew we analyzed , cases of coronavirus disease in japan and identified case-clusters in healthcare and other care facilities, restaurants and bars, workplaces, and music events. we also identified probable primary case-patients for the clusters; most were – years of age and presymptomatic or asymptomatic at virus transmission. we analyzed , cases of coronavirus disease in japan and identified case-clusters in healthcare and other care facilities, restaurants and bars, workplaces, and music events. we also identified probable primary case-patients for the clusters; most were - years of age and presymptomatic or asymptomatic at virus transmission. functions; and ( %) transportation-related incident in an airplane. most ( / ; %) clusters involved - cases ( figure , panel b). the largest cluster involved > cases in a hospital, including nosocomial infections and staff infections. the largest non-healthcare-related cluster we observed was among > persons who attended a live music concert, including performers, audience members, and event staff. healthcare and care facilities accounted for > % of clusters at epidemiologic weeks and ( figure , panel c). we identified probable primary case-patients who had symptoms develop before they had contact with other case-patients in a cluster or who had prior epidemiologic links before contact with a cluster. we did not identify probable primary cases for nosocomial clusters. we believe these case-patients contributed to the incidence of clusters. demographic data show that ( %) probable primary case-patients were female and ( %) were male. the most frequently observed age groups among probable primary cases were - years (n = ; %) and - years (n = , %) ( figure , panel a). for clusters, we determined the date of transmission from probable primary case-patients to other case-patients in a cluster and found % ( / ) of probable primary casepatients were presymptomatic or asymptomatic at the time of transmission; only had a cough at the time of transmission ( figure , panel b). of the probable primary case-patients, % ( / ) had cough at the time of diagnosis. of the probable primary case-patients with the determined date of transmission, transmission occurred one day before illness onset for ( %) case-patients and on the same day of illness onset for ( %) case-patients ( figure , panel c). all age groups demonstrated presymptomatic or asymptomatic transmission. we investigated clusters of covid- cases and probable primary cases in japan during january -april , . we found that healthcare facilities, such as hospitals, and care facilities, such as nursing homes, were the primary sources of clusters, some of which had > cases. japan experienced waves of imported cov-id- cases, after which local transmission occurred and the epidemic grew ( ) . of note, clusters of covid- cases at healthcare and care facilities predominated at epidemiologic weeks (march - ) and (march -april ), which corresponds to ≈ weeks after the waves of imported cases (figure , panel c) . healthcare and care facilities might be located at the end of the local transmission chain because clusters in those facilities only became evident several weeks after community transmission persisted. we noted many covid- clusters were associated with heavy breathing in close proximity, such as singing at karaoke parties, cheering at clubs, having conversations in bars, and exercising in gymnasiums. other studies have noted such activities can facilitate clusters of infection ( , ) . japan's prime minister's office and the ministry of health, labour and welfare announced situations that could increase the risk for covid- cases and advised the population to avoid the "three cs": closed spaces with poor ventilation, crowded places, and close-contact settings ( ) . among the probable primary covid- cases we identified from non-nosocomial clusters, half ( / ) were - years of age, which is younger than the age distribution of all covid- cases in japan (figure , panel a) . we do not know whether social, biological, or both factors play a role in the difference in transmission patterns between the younger and older persons. we also noted probable primary covid- case-patients appear to transmit the virus and generate clusters even in the absence of apparent respiratory symptoms, such as cough. our study has some limitations. the epidemiologic investigation mostly relied on voluntary cooperation. because some case-patients could not disclose contact history, epidemiologic links and clusters of cases might have missed. recall bias is likely because japan did not use digital devices for contact tracing and information was obtained only through interviews. in addition, we could not calculate a secondary attack rate from probable primary cases because data were unavailable for denominator, such as the number of persons present in the places where clusters of cases were detected. active case finding and investigation are key to establishing links to other cases or transmission events. detecting clusters of cases can lead to effective quarantine of close contacts and to the identification of risk factors for the formation of such clusters ( ) . our findings provide further information and insight on clusters of covid- cases in communities that can aid in the ongoing efforts to curb the global pandemic. age ranges of probable primary covid- cases in clusters. age distribution among all covid- cases in japan is provided as reference. b) proportions of symptoms among probable primary cases of covid- clusters at transmission (n = ) and among at laboratory confirmed diagnosis (n = ). , asymptomatic; , fever; , fatigue; , cough; , sore throat; , headache; , arthralgia or myalgia; , runny nose; , diarrhea; , difficulty breathing. c) distribution of probable primary cases of covid- clusters by time of transmission compared with illness onset by age groups (n = ). six cases were excluded because the time of transmission was undetermined. nin, kota ninomiya, yukiyo nitta, akiko sakai, kazuaki sano, asako sato, akiko sayama, ayaka takeuchi, hiroto tanaka, fumie tokuda, shogo yaegashi, yoko yamagiwa, lisa yamasaki, and fumi yoshimatsu. this study was supported in part by grants from the ministry of education, culture, sport, science and technology in japan (no. ) and the japan agency for medical research and development (no. fk h ). china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china clinical features of patients infected with novel coronavirus in wuhan world health organization. who director-general's opening remarks at the mission briefing on covid- - world health organization. who director-general's opening remarks at the mission briefing on covid- - investigation of three clusters of co-vid- in singapore: implications for surveillance and response measures transmission potential and severity of covid- in south korea about coronavirus disease (covid- ) press conference epidemiology of covid- outbreak in japan coronavirus disease cluster associated with fitness dance classes, south korea. emerg infect dis high sars-cov- attack rate following exposure at a choir practice prime minister's office of japan; ministry of health, labour and welfare. avoid the "three cs identifying and interrupting superspreading events-implications for control of severe acute respiratory syndrome coronavirus dr. furuse is an assistant professor at institute for frontier life and medical sciences and hakubi center for advanced research, kyoto university, japan. his primary research interests include the public health, epidemiology, bioinformatics, and molecular biology of viral diseases. key: cord- - av gx r authors: yamagishi, takuya; kamiya, hajime; kakimoto, kensaku; suzuki, motoi; wakita, takaji title: descriptive study of covid- outbreak among passengers and crew on diamond princess cruise ship, yokohama port, japan, january to february date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: av gx r an outbreak of coronavirus disease (covid- ) occurred on the diamond princess cruise ship making an international journey, which led to quarantine of the ship at yokohama port, japan. a suspected covid- case was defined as a passenger or crew member who developed a fever or respiratory symptoms, and a confirmed covid- case had laboratory-confirmation of severe acute respiratory syndrome coronavirus (sars-cov- ) infection. between and february , individuals were tested for sars-cov- and were positive ( passengers (median age: years; interquartile range (iqr): – ; males: %) and crew (median age: years; iqr: – . ; males: %). other than the hong kong-related index case, symptom onset for the earliest confirmed case was january, days after the cruise ship left port. attack rates among passengers were similar across the decks, while beverage ( . %, / ) and food service staff ( . %, / ) were most affected. attack rates tended to increase with age. a comprehensive outbreak response was implemented, including surveillance, provision of essential medical care, food and medicine delivery, isolation, infection prevention and control, sampling and disembarkation. an outbreak of coronavirus disease (covid- ) occurred on the diamond princess cruise ship making an international journey, which led to quarantine of the ship at yokohama port, japan. a suspected covid- case was defined as a passenger or crew member who developed a fever or respiratory symptoms, and a confirmed covid- case had laboratory-confirmation of severe acute respiratory syndrome coronavirus (sars-cov- ) infection. between and february , individuals were tested for sars-cov- and were positive ( passengers (median age: years; interquartile range (iqr): - ; males: %) and crew (median age: years; iqr: - . ; males: %). other than the hong kong-related index case, symptom onset for the earliest confirmed case was january, days after the cruise ship left port. attack rates among passengers were similar across the decks, while beverage ( . %, / ) and food service staff ( . %, / ) were most affected. attack rates tended to increase with age. a comprehensive outbreak response was implemented, including surveillance, provision of essential medical care, food and medicine delivery, isolation, infection prevention and control, sampling and disembarkation. on january , chinese health officials announced detection of a case with novel coronavirus disease in wuhan, china [ , ] . as the outbreak expanded in wuhan, there was spillover of covid- cases outside the country. the first covid- cases outside of china were reported in thailand on january [ ] . now, all world health organization (who) regions have domestic cases and the number of cases has been increasing in many countries in europe and the americas [ ] . japan detected its first covid- case on january , a japanese resident who travelled to wuhan for winter vacation and returned to japan for work [ ] . there were subsequent reports of new cases in japan both with and without travel history to wuhan [ ] . the reason for the high speed of covid- expansion is still under debate, but transmission from asymptomatic cases or pre-symptomatic is suggested [ ] . for other betacoronaviruses, such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), the primary mode of transmission is considered to be through droplets or direct contact of the mucous membranes with infectious respiratory droplets or fomites [ , ] . the primary mode of transmission for sars-cov- is also considered to be via droplets, along with contribution from contact [ ] . however, sars-cov has exhibited efficient transmission, leading to a higherthan expected number of cases (super-spreading event) [ ] , with possible airborne transmission being observed under particular conditions [ ] . here, we report our preliminary findings from the investigation of a covid- outbreak on an international cruise ship, the diamond princess, at yokohama port, japan up to february . the findings from this descriptive study may facilitate increased understanding of covid- and could contribute to public health responses on how to address cruise-related covid- outbreak challenges. a case of covid- related to the internationally-operated diamond princess cruise ship was reported to the ministry of health, labour and welfare (mhlw), japan on february by health officials in hong kong [ ] . japan was notified by hong kong authorities on february that the hong kong-related covid- case (index case) had arrived in japan on january and boarded the cruise ship on january at yokohama, japan. the case participated in a tour of kagoshima, japan on january during a shore-based stop and disembarked the ship in hong kong on january. the case had a cough starting on january, developed a fever on january and was diagnosed with covid- at a hospital in hong kong. the cruise-ship implemented a quarantine on february, after which laboratory-confirmed cases of covid- were identified on board on february. the cruise ship had , passengers and , crew members onboard from more than countries. there were eight decks for passenger cabins and eight decks for crew cabins. the ship contained a seven-bed medical clinic staffed by doctors and nurses. after the cruise ship left yokohama port, japan on * january, it visited hong kong on january, vietnam on january, taiwan on january and okinawa, japan on february, returning to yokohama port on february. we defined a confirmed case as a passenger or crew member with positive rt-pcr test result for sars-cov- (box). we defined a suspected case as a passenger or a crew member who had a fever ≥ . °c or respiratory symptoms during the investigation period or identified during the review of the ship's medical clinic records. an asymptomatic case was defined as a person without symptoms at the time of investigation. we defined a close contact as someone who joined the kagoshima tour with the index case, who shared a cabin with a confirmed case or who shared the same cabin with a suspected case. starting on february, quarantine officers took oropharyngeal samples from suspected cases and close contacts. samples were tested for sars-cov- using a rt-pcr test according to the national standard [ ]. testing was conducted by local public health laboratories in yokohama and neighbouring areas, and quarantine laboratories at yokohama, narita, and tokyo. as at february, we changed the testing strategy and only tested suspected cases, which had been increasing in number. along with the change in testing strategy, starting from february, a hotline for self-identified febrile passengers and crew was set up and a fever clinic was launched alongside the ship's medical centre. following a call to the hotline, doctors from japan's disaster medical assistance team (dmat) and ministry of defence went to the cabin of the individual to assess their condition. after evaluation, an oropharyngeal swab was taken from suspected cases for rt-pcr testing. definitions of confirmed and suspected cases, and close contacts in diamond princess cruise ship covid- outbreak, yokohama port, japan, january- february confirmed case: passengers or crew who tested positive for severe acute respiratory syndrome coronavirus (sars-cov- ) by rt-pcr suspected case: passengers or crew who had a fever ≥ . °c or respiratory symptoms close contact: -passengers or crew who joined a tour of kagoshima, japan -passengers or crew who shared the same cabin with a confirmed case -passengers or crew who shared the same cabin with a suspected case a a the third definition was only used until february , when the operation was strained by increasing number of suspected and confirmed cases. data sources during investigation of diamond princess cruise ship covid- outbreak, yokohama port, japan, january- february medical consultation records at the ship's medical centre january- february data from the medical consultation records between january, when the ship started the cruise, and february when the temporary fever clinic was set up on the ship. reviewed for assessing suspected cases. all rt-pcr test results were collected from quarantine laboratories. used to collect demographic information, as well as symptoms and symptom onset date via paper-based questionnaire with consent form. fever hotline and medical records of on-board fever clinic - february the log of fever hotline and medical charts from the fever clinic were reviewed for assessing suspected cases. we collected all suspected and confirmed cases' general information, clinical symptoms, rt-pcr test results and contact information from the multiple sources described in table . we merged the data to create a line list for the period of to february. we described the epidemiology of the suspected cases and confirmed cases. we calculated the test positivity rate among suspected cases according to the date of the sample. the attack rate was calculated based on the passenger and crew data provided by the cruise ship. we used excel to conduct all the analysis. this report was exempt from the requirement for institutional ethics review since it was a public health investigation approved by the japanese infectious disease law and quarantine law. a total of samples, representing individuals, were tested for sars-cov- between and february. of these, ( %) were suspected cases, ( %) were close contacts, two were individuals tested before emergency disembarkation for medical reasons, three were family members of these individuals and the remainder (n = , %) were individuals without information about symptoms and contacts. of the total samples tested, ( %) were positive. among the confirmed cases, ( %) met the suspected case definition before testing while ( %) shared a cabin with a confirmed case ( among the confirmed cases with information about symptoms at the time of sampling available, were asymptomatic ( %); including the two individuals who disembarked because of emergency medical indication. among symptomatic cases (n = ), the most common symptom was fever ≥ . °c (n = , %), followed by cough (n = , %). three cases mentioned diarrhoea as a symptom, which was less common than sore throat. the earliest date of symptom onset among the confirmed cases onboard was january ( figure a ). the number of confirmed cases remained low for approximately weeks after, and then showed a notable increase. symptom onset date of the first crew member case was february, days after that of the first passenger case. the epidemic curve of suspected cases indicated that passengers and crew members were exhibiting symptoms from the beginning of the cruise and the number of suspected cases gradually increased daily ( figure b) . the cumulative incidence of suspected cases between january and february, i.e. prior to quarantine on february, was . per , passengers and . per , crew members between january and february. test positivity by testing date among suspected cases was % ( / ) from to february and increased to % ( / ) on february for passengers whereas it was one of eight on to february and nine of on february for crew members (table , figure ). the attack rate was highest among passengers to years of age, but this is based on small numbers. the attack rates by age group among passengers were lowest in the to years group and increased with age thereafter (table ) . attack rates among crew also tended to increase with age. the attack rate of date of symptom onset ( ) j a n j a n j a n j a n j a n j a n j a n j a n j a n j a n j a n j a n after notification of the case from hong kong, the mhlw began a quarantine on the cruise ship at yokohama port on february. the emergency operating centre (eoc) was set up on february, and on this date, all passengers were also isolated in their cabins and internal air re-circulation was stopped to reduce the possible risk of airborne transmission. crew members continued to work, following guidance from the ship's standard health precautions, and were isolated when individual crew members were considered to be infectious after assessment by the cruise ship's medical staff. on february, a temporary on-site fever clinic was launched to address the increasing number of febrile passengers and crew members by japanese medical staff, while the ship's medical clinic focused on other patients, those with non-covid- -related symptoms, on the cruise ship. respiratory samples were taken daily from suspected cases and were shipped to the quarantine laboratories for testing once or twice a day. the laboratory results came the following day and all confirmed cases were disembarked and transferred to designated hospitals within a day. mhlw provided medicine, surgical/n masks, gloves, alcohol-based hand rub, and basic knowledge of coronavirus infection and infection prevention to passengers (e.g. hand hygiene and cough etiquette). it also provided training on infection prevention and control to the crew. quarantine personnel wore surgical masks, except during meals at the eoc on the cruise ship, with added eye protection, long-sleeve gowns and gloves when taking samples. mhlw decided to disembark all the asymptomatic passengers and crew members, and those with symptoms who tested negative after days of quarantine. because of the mass testing workload, mhlw prioritised testing populations, with individuals over years of age prioritised for sampling before disembarkation. the outbreak response operation was complex and we worked closely with the captain, managers and crew of the cruise ship to make decisions for the next steps in response efforts. the primary challenge identified was food preparation and delivery to quarantined passengers. because passengers needed to be isolated, we requested the crew to continue to work, so meals were prepared and delivered by crew members under the guidance of infection prevention and control staff. another challenge was the language barrier which made it difficult to conduct effective isolation measures because of the delay in providing instruction; however, collaboration with crew members worked efficiently. this report details the early phase of the outbreak investigation on a cruise ship quarantined in yokohama port that followed the confirmation of a disembarked passenger having covid- . this event required a large-scale quarantine that we had not experienced before, with a large number of international passengers and crew members further adding to the public health challenge. we believe that the findings from our experience are useful to respond to a similar covid- event in an international cruise ship such as that quarantined at california in march [ ] or nagasaki in april [ ] . the earliest date of symptom onset for a confirmed case was january, which was only days after the start of the voyage and which suggests that the case could have already been infected before the voyage. the outbreak of covid- had already been reported in wuhan by mid-january, and so it was also possible that more than one covid- case might have been on board the cruise ship. it is also reasonable to infer that the index case became infectious while onboard the cruise ship. the epidemic curve of symptomatic cases showed that passengers and crew members with fever and/or respiratory illness increased immediately after the start of the cruise. there were several well-attended cocktail and wine parties during the cruise where large numbers of passengers gathered at one place for several hours. even though passengers had no symptoms when they attended the party, possible transmission of sars-cov- from asymptomatic or pre-symptomatic cases has been reported [ , ] . attendance at these parties could explain why passenger cases were scattered across all the decks with no discernible pattern. also, further, continued exposure to the virus can be assumed to have ended when passengers were isolated in their cabins, unless a cabin mate was already infected. some confirmed cases in cabin mates were identified soon after the start of isolation on february, suggesting that they were exposed to the virus before the isolation began. the effectiveness of isolation measures should be evaluated at least weeks after the initiation of isolation, since cases identified later need to be counted. the results suggest that the transmission dynamics among crew was different from those among passengers. among passengers, the highest attack rate was observed in the to years of age group, and this might be because of frequent contacts among young people, which could be a driving force of the disease's spread. the only interaction between crew and passengers was during crew work shifts, as crew members spent most of their time in a relatively fixed place in their crew quarters. the attack rate among food service workers was higher than among other occupations among the crew (table ) , which supports the hypothesis that the disease spread through cocktail and wine parties. the possibility of airborne transmission of sars-cov- could not be ruled out at that time [ ] and internal air re-circulation was stopped on the cruise ship. our findings suggest that sars-cov- transmission among close contacts (droplet and direct contact) as opposed to airborne transmission, was primarily responsible for covid- 's spread on the ship. passengers and crew aged over years were more likely to develop symptoms, especially individuals over years of age. because older people were at higher risk of developing severe infections [ ] , they were the population to be prioritised for testing and disembarkation. another high-risk population for poor health outcomes were those with a comorbidity; however, it took additional time to check for comorbidities among passengers on the cruise ship. we detected asymptomatic cases ( %) among confirmed cases, mainly among passengers who joined a bus tour in kagoshima. it is important to note that being asymptomatic at one point of time does not exclude the possibility of developing symptoms later on [ , ] . the two cases who disembarked the ship because of another medical indication were positive for covid- but asymptomatic at the time of disembarkation. this finding led us to recommend that all passengers be tested regardless of their symptoms. the recent finding that the viral load of sars-cov- in asymptomatic cases was reported to be similar to that in symptomatic cases supports our judgment [ ] . this poses the challenge for contact tracing and isolation measures that are usually considered after symptom onset. the increasing test positivity rate among the crew was a worrying sign even though most were based on small numbers. test positivity is used as a surrogate of incidence [ ] , and the incidence among the crew was considered to be high. because the crew were vital to continue ship's operations and the provision of food to more than , individuals onboard the ship, stopping their contact with others would have limited vital services to passengers and cruise ship operations. the decision to request that the crew continue ship's operations was a highly political one. the strength of this study includes the availability of a classic cohort and information from all passengers and crew onboard the ship. two main limitations of this report are sampling and reporting biases. under the emergency response for a novel infectious disease, it was difficult to test the entire target population because of limited testing capacity. as a result, we changed our strategy to test cabin mates of positive cases only if they met the criteria of suspected cases. also, crew had to continue their daily routine along with tasks related to the quarantine, and fever may not have been reported comprehensively. finally, we tried to identify the incubation period by evaluating cabin mates of the confirmed cases. however, it was impossible to distinguish the source of the infection as being the cabin mate or an exposure before the isolation. we summarised the epidemiology of an early phase of a covid- outbreak on a cruise ship. the outbreak started shortly after the start of the journey of the cruise ship and among passengers, it seemed to be controlled by the isolation in their cabin. the decision to continue to have crew working was a political one aiming to ensure the provision of ongoing passenger services and ship operations during quarantine at a time when there was a concurrent need to limit further transmission on board the ship. a quarantine operation on a large cruise ship requires a comprehensive outbreak response including a high-level of collaboration with crew members. a mistake in the date the cruise ship left yokohama port, japan was corrected on june , upon request of the authors. we thank all the staff working on the response to the covid- outbreak on the cruise ship, including the ship's crew, officials from the ministry of health, labour and welfare, including quarantine officers and quarantine/public health laboratory staff, officials from the ministry of defence, members of japan's disaster medical assistance team (dmat) and dr masataro norizuki at the national center for global health and medicine. we also thank local public health laboratories for pcr testing. we appreciate ms anita samuel for her kind review and proofreading. none declared. clinical features of patients infected with novel coronavirus in wuhan a novel coronavirus from patients with pneumonia in china journey of a thai taxi driver and novel coronavirus situation report - coronavirus disease (covid- ) the first case of novel coronavirus infection in japan. tokyo: ministry of health, labour and welfare national institute of infectious diseases (niid). [descriptive epidemiology of confirmed -novel coronavirus ( -ncov) cases in japan evidence supporting transmission of severe acute respiratory syndrome coronavirus while presymptomatic or asymptomatic the severe acute respiratory syndrome middle east respiratory syndrome aerosol and surface stability of sars-cov- as compared with sars-cov- guideline for isolation precautions: preventing transmission of infectious agents in health care settings the government of the hong kong special administrative region. chp investigates additional case of novel coronavirus infection. hong kong: the government of the hong kong special administrative region public health responses to covid- outbreaks on cruise ships -worldwide the outbreak situation of the prefecture in nagasaki-shi of new coronavirus infectious disease transmission of -ncov infection from an asymptomatic contact in germany presumed asymptomatic carrier transmission of covid- world health organization (who) clinical characteristics of coronavirus disease in china evidence of sars-cov- infection in returning travelers from wuhan, china sars-cov- viral load in upper respiratory specimens of infected patients malaria burden through routine reporting: relationship between incidence and test positivity rates ty, hk, kk conducted and analysed the study and drafted the manuscript. ms and tw reviewed the manuscript and provided technical advice. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- -ikptxviu authors: van diep, nguyen; norimine, junzo; sueyoshi, masuo; lan, nguyen thi; hirai, takuya; yamaguchi, ryoji title: us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between and date: - - journal: springerplus doi: . /s - - -z sha: doc_id: cord_uid: ikptxviu since late , outbreaks of porcine epidemic diarrhea virus (pedv) have reemerged in japan. in the present study, we observed a high detection rate of pedv, with . % ( / ) of diarrhea samples (suckling, weaned, and sows) and . % ( / ) of farms experiencing acute diarrhea found to be positive for pedv by reverse transcription pcr. sequencing and phylogenic analyses of the partial spike gene and orf of pedv demonstrated that all prevailing japanese pedv isolates belonged to novel genotypes that differed from previously reported strains and the two pedv vaccine strains currently being used in japan. sequence and phylogenetic analysis revealed prevailing pedv isolates in japan had the greatest genetic similarity to us isolates and were not vaccine-related. unlike vaccine strains, all prevailing field pedv isolates in japan were found to have a number of amino acid differences in the neutralizing epitope domain, coe, which may affect antigenicity and vaccine efficacy. the present study indicates recent pedv isolates may have been introduced into japan from overseas and highlights the urgent requirement of novel vaccines for controlling pedv outbreaks in japan. porcine epidemic diarrhea (ped) is a highly contagious and devastating viral enteric disease characterized by vomiting, acute onset of severe watery diarrhea, and dehydration. ped has a high infectivity and a particularly significant mortality in piglets (pensaert and de bouck ) . the porcine epidemic diarrhea virus (pedv), an enveloped, single-stranded rna virus belonging to the alphacoronavirus genus of the coronaviridae family, is responsible for ped. the pedv genome is approximately kb in length and is composed of seven open reading frames (orf) that encode four structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n), and three major non-structural proteins, including replicases a and b, and orf (song and park ) . of the structural protein, the pedv s protein plays a pivotal role in regulating interactions with specific host cell receptors to mediate viral attachment and entry. moreover, the s protein is associated with the induction of host neutralizing antibodies, growth adaptation in vitro, and attenuation of virulence in vivo (song and park ) . thus, study of the s glycoprotein has been essential in understanding the genetic relationships between pedv strains, the epidemiological status of pedv in the field, and the development of vaccines (song and park ; chen et al. ) ; (temeeyasen et al. ) . in addition to the s glycoprotein gene, the orf gene has received a large amount of attention in the aspect of pedv virulence. orf gene plays a role in encoding an ion channel protein (wang et al. ) and it has been suggested to be an important determinant for virulence of this virus (song and park ) . the virulence of ped can be reduced by altering the orf gene through cell culture adaptation (park et al. ) , and variation in orf was reported to be associated with viral attenuation in the natural host (song et al. ) . also, vaccine-derived isolates with unique continuous deletions of and orf nucleotides have been confirmed (chen et al. ; park et al. ) . therefore, these unique deletions in the orf gene can be used to differentiate between field and attenuated vaccine strains. moreover, orf gene variation may represent a useful tool in molecular epidemiological studies of pedv (park et al. (park et al. , song et al. ; chen et al. ) . in japan, the first outbreak of ped-like disease was reported in late and early (kusanagi et al. ; takahashi et al. ) , and was followed by pandemics between late and (sueyoshi et al. ; tsuda ) . afterwards, there have been sporadic ped outbreaks in intervals of several years. since late , numerous diarrhea epidemics, suspected to be caused by ped, have occurred in pigs throughout japan. these epidemics were characterized by severe diarrhea, dehydration, and vomiting in pigs of all ages. mortality rates were particularly high among suckling pigs. up to the end of august , more than , of , , pigs from infected farms have died of ped in japan based from the report of ministry of agriculture, forestry and fisheries (maff) (http://www.maff.go.jp). however, there have been few studies investigating the re-emergence of pedv in japan. this study aimed to evaluate the genetic characteristics and molecular epidemiology of the emergent japanese pedv isolates using genome analysis and phylogenetic analysis of the partial s gene and orf . a total of . % ( of ) of samples (suckling, weaned, and sows) from pig farms ( . %) experiencing acute diarrhea in six prefectures were found to be positive for pedv by rt-pcr. pedv-positive samples were identified from the following prefectures: miyazaki (n = ), kagoshima (n = ), aichi (n = ), akita (n = ), hokkaido = ), and aomori (n = ). to investigate the heterogeneity of the recent japanese isolates and their genetic relationship with modified live vaccines, in addition to pedv vaccine strains (p -v and -p c ) used in japan, representative isolates were selected for sequencing of the partial s gene and full orf gene. the partial s gene, including the co- k equivalent (coe) domain, of pedv samples from pedvinfected farms were amplified, purified, and sequenced. the partial s sequences were aligned at nucleotides - (amino acids - ) of the full s gene. identical nucleotide sequences were distinguished and excluded, resulting in the identification of individual sequences from the total of field pedv isolates (table ) . however, sequencing revealed high genetic variation between nucleotides and (amino acid residues - ). a total of nucleotide substitutions were detected, leading to amino acid changes, within the partial s gene (fig. ) . the coe domain (amino acids - ) of the s protein consists of aa and contains epitopes that are capable of inducing pedv-neutralizing antibodies (chang et al. ) . compared to the two vaccine strains (p- v and -p c ), all japanese field strains had different amino acids at positions (a → s), (t → s), and (g → s) within the coe domain. furthermore, differences in amino acids were found at the following sites within the coe domain of the s protein: , and (l → f) as shown in fig. . to investigate the heterogeneity of prevailing pedv strains in japan, phylogenetic tree of partial s genes of pedv field strains and two vaccine strains were constructed together with previously reported japanese pedv strains (nk, kh, mk, p- ) and reference strains from other countries available in genbank. consistent with previous reports (park et al. ; puranaveja et al. ; temeeyasen et al. ), phylogenetic tree based on partial s gene sequences in this study demonstrated that pedv strains can be divided into three groups: g , g , and g . group g can be further divided into subgroups: g - , g - and g - ( fig. ) . notably, all field pedv isolates circulating in japan were found to cluster closest to isolates from the usa (usa/ iowa/ / , oh , iowa , mn, and ia , usa/colorado/ , isu - -ia-homogenate), and us strain-like pedv from south korean (knu- , knu- - , knu- , knu- ) collected from to ); they were found cluster within the same subgroup g - (fig. ) . further, isolate jm- clustered with s indel strains (oh , iow , knu- ), forming a distinct minor branch within the cluster. on the other hand, vaccine strains p -v and strain nk clustered within group g , while the vaccine strain -p c and old strains mk, kh, and p- belonged to subgroup g - . these results demonstrated distantly genetic relationships between field isolates and vaccine strains, in addition to previously reported pedv strains in japan. pairwise alignment of field japanese pedv isolates demonstrated high nucleotide and amino acid sequence identity between strains ( . - . and . - %, respectively). notably, field pedv isolates collected from prefectures in this study had identical partial s gene sequence ( % nucleotide homology). japanese to investigate the genetic relationship between recent japanese field isolates, and modified live vaccine strains and reference strains, the nucleotide sequences of the orf genes of recent pedv isolates and vaccine samples (p -v and -p c ) were sequenced and analyzed (table ) . sequencing data revealed the orf genes of all pedv samples were bp in length and (table ). sequence analysis revealed that the orf genes of the japanese field isolates were relatively well-conserved. only point substitutions were observed at nt , , , , and , with only the substitution at nt resulting in a non-synonymous substitution of t to i at residue . phylogenetic analyses revealed that, based on the orf gene, all pedv isolates could be divided into three groups namely: g , g , and g (fig. ) . notably, all japanese field isolates clustered closely with us isolates and recent south korea isolates, forming a separate subcluster within g . on the other hand, the vaccine strains, p -v and -p c , which have been used to prevent pedv infection in japan, were found within g , which are therefore genetically distant from prevailing field isolates. dna sequence homology of . - % was observed between orf genes of recent japanese isolates that had the highest dna identity ( . - %) with the commonly observed on ped-vaccinated farms, leading to the loss of high numbers of pigs. as a result, the genetic characteristics and origins of prevailing pedvs in japan, the efficacy of pedv vaccines being used in japan in protecting previously well pigs from prevailing pedvs, and the genetic differences between vaccine strains and field pedv isolates remain critically important questions that have yet to be fully elucidated. we therefore performed this study to address these important issues regarding ped. in this study, . % ( of ) of pig farms in six prefectures (miyazaki, kagoshima, aichi, hokkaido, aomori, and akita) were confirmed as infected with pedv, and . % ( of ) of samples were found to be positive for pedv. this result demonstrates a high prevalence of pedv infection in japanese pig herds. the pedv spike glycoprotein has a high degree of variability and contains several epitopes (chang et al. ; sun et al. ). among these epitope sites, the coe domain (aa - ) is an important region capable of inducing pedv-neutralizing antibodies (chang et al. ) . in comparison with vaccine strains (p- v and p c ) commonly used in japan, all the field isolates were found to have - different residues in the coe domain, particularly at these positions ( , , and ). notably, the amino acid at these three sites were all serine, one of few major amino acids that are capable of generating new o-linked glycosylation or phosphorylation. netphos . server (http://www.cbs.dtu.dk/services/ netphos) and netphosbac . server (http://www.cbs. dtu.dk/services/netphosbac- . ) were used for prediction of phosphorylation site. the result showed that phosphorylation was generated from serine residues at the position and of the field japanese isolates. on the other hand, using bepipred . server (http://www. cbs.dtu.dk/services/bepipred) to predict the location of linear b cell epitopes, no remarkable difference between the field pedv isolates and the vaccine strains were found. therefore, further research is needed to determine whether these amino acid differences may affect the antigenicity of prevailing pedv isolates and consequently influence the efficacy of the vaccines currently used on japanese pig farms. in may , pedv was detected for the first time in the united states. subsequently, us strain-like pedvs were reported in south korea in late , and germany in may (hanke et al. ) . the result of phylogenetic and genetic analysis of the partial s gene demonstrated that prevailing japanese pedv isolates had been previously unreported in japan, shared the greatest similarity with us strainlike strains, and may have been introduced into japan via unknown routes. to date, two distinct pedv strain types have been identified in us: the highly virulent us pedv (us prototypes) (stevenson et al. ) and the s indel pedv variant, which contains insertions and deletions in the n-terminal region of the s protein, reported to cause milder disease in the field (vlasova et al. ; . interestingly, isolate jm- was grouped in the same cluster as the s indel variant (oh , iow , knu- ) with % nucleotide identity observed between these strains. clinical signs recorded on pedv-infected farms demonstrated that only piglets (out of a total of pigs on the farm) had disease manifesting as diarrhea, with piglets dying at the time of ped onset. this finding suggests the japanese field isolate jm- may, in fact, be the s indel pedv variant. this pedv variant is prevalent and associated with low morbidity and mortality in ped outbreaks in japan, although more extensive genome sequencing is required to clarify this finding. orf is an accessory gene thought to influence virulence and cell culture adaptation and has been used as a viral target in attempts to reduce pedv virulence. generally, orf has utility as a valuable tool in the study of pedv molecular epidemiology and for differentiating between field and vaccine-derived isolates. our study revealed that the orf gene of the japanese vaccine strains, p -v and p c , have unique deletions ( and nt, respectively) that can lead to reading frame-shift and coding of truncated polypeptides. this genetic characteristic can be used to differentiate between field and attenuated-derived vaccine pedv. moreover, the finding of nt deletion in orf gene of p -v in this study is different from the result of a previous study (park et al. ) in which authors reported that p -v have nt deletion in orf gene. this difference may be due to the genetic variation of p -v during the time of the study. the results of the present study demonstrated that no deletion were observed in the japanese field isolates, suggesting that they are not vaccine-related. phylogenetic analysis further demonstrated that japanese field isolates had greatest genetic similarity with us strains. this study demonstrated a high detection rate of pedv on pig farms in japan. all recent japanese pedv isolates were found to have previously unreported genotypes that differed from japanese strains prior to as well as vaccine strains currently being used in japan and had the greatest genetic similarity with us isolates, as compared with other countries. these findings suggest that prevailing pedv isolates may have been introduced into japan from overseas. the distant genetic relationship and amino acid differences in the neutralizing epitope coe domain between recent pedv isolates and the vaccine strains may be responsible for unsuccessful ped control in japan. therefore, the development of new vaccines with greater protection against pedv outbreaks in japan is required. a total of samples were collected from suckling pigs, weaned pigs, and sows at pig farms (farrow-to-finish and farrow-to-wean) experiencing acute diarrhea from six prefectures from north to south of japan between december and october . the number of samples from each prefecture was as follows: miyazaki (n = ), kagoshima (n = ), aichi (n = ), akita (n = ), hokkaido (n = ), aomori (n = ). one to fecal samples, intestinal samples, or intestinal contents, were obtained from each outbreak of diarrhea. fecal samples were taken from animals showing signs of diarrhea at the time of collection. samples of intestine and intestinal content were collected from animals that have died due to severe diarrhea within h. all the samples were temporarily preserved in ice boxes at time of collection, kept in icebox containing dry ice during transportation, and stored at freezer (− °c) when it arrived at the laboratory of veterinary pathology, univeristy of miyazaki, using cryogenic freezing systems. samples of two vaccine strains, p- v (produced by nisseiken co., ltd) and -p c (produced by kaketsuken co., ltd), were collected from commercial vaccine bottles used in pig farms in japan. specimens from sick pigs were homogenized and diluted five times in dulbecco's modified eagle's medium with a low concentration of glucose. samples were then centrifuged at rpm for min at °c. supernatants were stored and subsequently used for rna extraction. for each pedv sample, total rna was extracted from to µl aliquots of supernatant using reliaprep ™ rna cell miniprep kits (promega corpoation, wi, usa) in accordance with the manufacturer's instructions. the presence of pedv in samples was detected by reverse transcription polymerase chain reaction (rt-pcr) using a previously published primer pair (kim et al. ) . briefly, nucleotide strands of these primers are ′-ttctgagtcacgaacagcca- ′ (p , forward), ′-catatgcagcctgctctgaa- ′ (p , reverse). the size of amplified product was bp. the amplified genomic region of the s gene contains the neutralizing epitope region, co- k equivalent (coe) (chang et al. ) . one tube rt-pcr reaction was performed using accessquick ™ rt-pcr system kits (promega corpoation, wi, usa). exactly, µl of rna template was mixed with a reaction mixture, which contained . µl of accessquicktm master mix ( ×), . µl of each specific primer ( µm), and . µl of amv reverse transcriptase ( u/µl). then, µl of nuclease-free water was added to reach the total volume reaction of µl. the rt-pcr reaction were done using takara pcr thermal cycler (japan). following a reverse transcription step of °c for min and an incubation step of °c for min, cycles were performed as follows: °c for s, °c for s, and °c for min. cycles were followed by a terminal min extension step at °c. the last stage was preserving the pcr products at °c. the rt-pcr products were visualized by electrophoresis in a . % agarose gel containing ethidium bromide. a primer pair was designed for amplifying the full orf gene of pedv with the following sequences: forward primer (orf -f), ′-gtcctagacttcaaccttac-gaag- ′; and reverse primer (orf -r), ′-aactac-tagaccattatcattcac- ′. the predicted size of the orf pcr product was bp. for the application of the partial s gene and the orf gene of pedv, rt was first performed using random primers and oligodt primers from reverse transcription system kits (promega, madison, wi, usa). complimentary dna was immediately used to amplify the partial s gene (primer pair p / p ) and orf gene (primer pair orf -f/orf /r) using gotaq ® green master mix kits (promega, madison, wi, usa) under the following condition: denaturation at °c for min; cycles of denaturation at °c for s, annealing at °c for s, and extension at °c for min. pcr products were purified using fastgene gel/pcr extraction kits (nippon genetics co., ltd, japan), according to the protocol of the commercial kit's instruction. all sequencing reactions were carried out in duplicate and sequences were determined in both direction with bigdye ® terminator v . cycle sequencing kits and an abi prism xl genetic analyzers (applied biosystems, ca, usa). the resultant nucleotide sequences were deposited in genbank under the following accession numbers: kt -kt . nucleotide and deduced amino acid sequences were edited, aligned (muscle algorithm), and analyzed using bioedit version . . and molecular evolutionary genetics analysis (mega) software version . (tamura et al. ) . phylogenetic trees based on the nucleotide sequences of the partial s gene and orf gene were constructed with maximum likelihood method using hasegawa-kishino-yano substitution model with discrete gamma distribution, and bootstrap tests of replicates in the mega v. program. identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian, china comparison of porcine epidemic diarrhea viruses from germany and the united states differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate outbreak-related porcine epidemic diarrhea virus strains similar to us strains full-genome sequence analysis of a variant strain of porcine epidemic diarrhea virus in south korea sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea cloning and further sequence analysis of the orf gene of wild-and attenuated-type porcine epidemic diarrhea viruses molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea a new coronavirus-like particle associated with diarrhea in swine chinese-like strain of porcine epidemic diarrhea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences an immunohistochemical investigation of porcine epidemic diarrhoea identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan mega : molecular evolutionary genetics analysis version . genetic diversity of orf and spike genes of porcine epidemic diarrhea virus in thailand porcine epidemic diarrhea: its diagnosis and control distinct characteristics and complex evolution of pedv strains pedv orf encodes an ion channel protein and regulates virus production the work was conducted with funding from the university of miyazaki. the authors declare that they have no competing interests. there is no human or animal subject involved in the experiment.received: june accepted: november authors' contributions ry, ms, and th collected the samples and epidemiological information as well as pathology findings. nvd and ry designed, conducted molecular tests, and did sequencings. nvd, ry, jn and ntl designed, interpreted the sequencing findings and wrote the paper. all authors prepared the manuscript. all authors read and approved the final manuscript.author details laboratory of veterinary pathology, department of veterinary, faculty of agriculture, university of miyazaki, - gakuenkibanadai-nishi, miyazaki - , japan. faculty of veterinary medicine, vietnam national university of agriculture, hanoi, vietnam. key: cord- - sfv p authors: azuma, kenichi; yanagi, u; kagi, naoki; kim, hoon; ogata, masayuki; hayashi, motoya title: environmental factors involved in sars-cov- transmission: effect and role of indoor environmental quality in the strategy for covid- infection control date: - - journal: environ health prev med doi: . /s - - - sha: doc_id: cord_uid: sfv p the severe acute respiratory syndrome coronavirus (sars-cov- ), a new zoonotic agent that emerged in december , causes coronavirus disease (covid- ). this infection can be spread by asymptomatic, presymptomatic, and symptomatic carriers. sars-cov- spreads primarily via respiratory droplets during close person-to-person contact in a closed space, especially a building. this article summarizes the environmental factors involved in sars-cov- transmission, including a strategy to prevent sars-cov- transmission in a building environment. sars-cov- can persist on surfaces of fomites for at least days depending on the conditions. if sars-cov- is aerosolized intentionally, it is stable for at least several hours. sars-cov- is inactivated rapidly on surfaces with sunlight. close-contact aerosol transmission through smaller aerosolized particles is likely to be combined with respiratory droplets and contact transmission in a confined, crowded, and poorly ventilated indoor environment, as suggested by some cluster cases. although evidence of the effect of aerosol transmission is limited and uncertainty remains, adequate preventive measures to control indoor environmental quality are required, based on a precautionary approach, because covid- has caused serious global damages to public health, community, and the social economy. the expert panel for covid- in japan has focused on the “ cs,” namely, “closed spaces with poor ventilation,” “crowded spaces with many people,” and “close contact.” in addition, the ministry of health, labour and welfare of japan has been recommending adequate ventilation in all closed spaces in accordance with the existing standards of the law for maintenance of sanitation in buildings as one of the initial political actions to prevent the spread of covid- . however, specific standards for indoor environmental quality control have not been recommended and many scientific uncertainties remain regarding the infection dynamics and mode of sars-cov- transmission in closed indoor spaces. further research and evaluation are required regarding the effect and role of indoor environmental quality control, especially ventilation. in late december , a cluster of severe pneumonia cases emerged in humans in wuhan, hubei province, china [ , ] . the causative pathogen was identified as a novel coronavirus that was named the severe acute respiratory syndrome coronavirus (sars-cov- ) [ , ] . the disease rapidly spread internationally, raising global public health concerns, and was subsequently termed coronavirus disease [ , ] . the most common clinical manifestations of patients with covid- are fever, cough, shortness of breath, and fatigue. some patients have also shown radiographic ground-glass lung changes and eventually died of acute respiratory distress syndrome (ards) [ , ] . the world health organization (who) declared covid- as a global pandemic on march , [ ] . sars-cov- is mainly transmitted human-to-human through close contact, respiratory droplets, fomites, and contaminated surfaces [ ] [ ] [ ] [ ] . the who adapted a m social distancing policy, based primarily on the assumption that the virus is transmitted through largely isolated droplets within this range [ ] . however, the possibility of airborne transmission through airborne particles with diameters smaller than μm has been suggested [ ] . several factors are involved in the transmission of sars-cov- between individuals, including the environment in buildings and human behavior [ ] [ ] [ ] [ ] [ ] [ ] . similar to the transmission routes of other respiratory viruses, such as influenza or human coronavirus [ ] [ ] [ ] , possible exposure pathways for covid- infection by the sars-cov- are finger contact with virus-contaminated surfaces (fomites) and subsequent finger contact with the facial membranes; inhalation of the virus carried in airborne particles (inhalable or respirable particles) exhaled from cough or vocalization; and droplet spray, the direct projection of the virus carried in particles exhaled from cough or vocalization onto the facial membranes. therefore, environmental factors in buildings, including temperature, humidity, stability on fomites, and ventilation and filtering systems, such as in public places, healthcare settings, restaurants, hotels, recreation facilities, or residential houses where people are close together, could have a significant influence on the infection. adequate control of these environmental factors and proper human behavior in accordance with these environmental conditions play a significant role in preventing the spread of covid- [ ] . as most people spend more than % of their daily lives inside buildings, it is essential to understand the potential transmission dynamics of sars-cov- inside a building, the spatial dynamics, and the building operational factors that potentially promote and mitigate the transmission of sars-cov- and the spread of covid- . this article aimed to review the effect of environmental factors in buildings, spatial dynamics, and building operational factors. in addition, a strategy to prevent sars-cov- transmission in building environments based on indoor environmental quality control recommended by the japanese ministries is also summarized. the characteristics of environmental air quality and environmental surfaces contaminated by the virus are important factors that determine the infectivity retention and extent and speed of the spread of the virus. the long-time persistence of these environmental factors influences the spread of covid- [ ] . the sars-cov- genetic material, ribonucleic acid (rna), was detected in the rooms of both symptomatic and asymptomatic cruise ship passengers up to days after the cabins were vacated [ ] . the sars-cov- rna was detected on the surfaces of the floor around the toilet, in the bedroom, bed pillow, phone, table, television remote control, chair arm, toilet flush button, toilet seat, and other items in the cruise ship [ ] . although the infectiousness of those materials is not known, the environment around the covid- cases was contaminated extensively with sars-cov- during the covid- outbreak on the cruise ship. the persistence of sars-cov- in aerosols in different environmental conditions has been reported. the results are summarized in table . van doremalen et al. compared the survival rate and half-life of sars-cov- and sars-cov- within h of aerosolization at a temperature of °c- °c and a relative humidity (rh) of %. both viruses were detectable after h of aerosolization and the median half-lives were . and . h for sars-cov- and sars-cov- , respectively [ ] . these results showed that the stability of sars-cov- was similar to that of sars-cov- . smither et al. reported that sars-cov- is more stable at a medium rh of - % (decay rate, . %/min) compared with a higher rh of - % (decay rate, . %/min) in tissue culture media (tcm), whereas the converse was observed in artificial saliva, with a decay rate of . % per minute at a medium rh and . % per minute at a higher rh [ ] . the results of the half-life obtained with tcm at a medium rh were similar to those described above [ ] . although the infectious dose in humans is not known, sars-cov- may be able to remain viable and infectious in aerosols for hours (depending on the inoculum shed), if the virus is produced within smallparticle aerosols. sars-cov- is susceptible to heat treatment but can persist for at least days at refrigerated temperatures of °c. with an increase in the incubation temperature, the time for virus inactivation was reduced dramatically to days at °c and to min at °c [ ] . sars-cov- demonstrates tendencies similar to those of sars-cov- [ ] . in simulated saliva on a stainless steel surface, sars-cov- exhibits negligible decay over min in darkness but loses % of its infectivity every . - . min, depending on the intensity of simulated ultraviolet (uv) b radiation levels, when exposed to simulated sunlight representative of the summer solstice at °n latitude at sea level on a clear day [ ] . these data indicate that sunlight may inactivate sars-cov- rapidly on surfaces, suggesting that persistence and subsequently exposure risk may vary significantly between indoor and outdoor environments. experimental studies using sars-cov- aerosols produced from artificial saliva found that simulated sunlight inactivates the virus rapidly [ ] . in dark conditions, the half-life of aerosolized sars-cov- is approximately min in simulated saliva. the high-intensity sunlightsimulated summer was found to show a % reduction in infectious concentration after min. even with the low-intensity sunlight-simulated late winter or early fall, a % reduction was observed at min. however, relative humidity ( , , , and %) had no significant effect on the survival of the aerosolized virus. these data indicate that sunlight is useful in mitigation strategies to minimize the potential for aerosol transmission. stability on surfaces of fomites sars-cov- can persist on plastic, stainless steel, and glass surfaces between and days at room temperature (table ) [ , ] . the persistence of sars-cov- on those surfaces was similar to that of sars-cov- [ , , ] . the survivability of these viruses on metal surfaces differed according to the type of metal. both sars-cov- and sars-cov- survived for shorter periods on copper ( and h, respectively) than on stainless steel surfaces [ ] . the antimicrobial properties of copper and copper alloy have been reported against various viruses [ , ] . several mechanisms have been proposed regarding copper-induced cellular toxicity to the virus. reactive oxygen species generated by free copper ions cause the cells to commit metabolic suicide. copper ions can induce protein destabilization on the virus. copper ions have a direct effect on virus inactivation by causing aggregation of virus particles [ ] . this might explain the short survival of sars-cov- and sars-cov- on copper surfaces compared with other metal surfaces. studies have reported that copper alloys (≥ % copper) reduced the surface microorganisms when incorporated into various hospital furnishings and fittings [ , ] . the use of copper in combination with optimal infection-prevention strategies may further reduce the risk of patients and healthcare workers acquiring covid- infection in healthcare environments. sars-cov- showed variable persistence on different porous surfaces, such as paper, cardboard, wood, cloth, and mask. sars-cov- survived on the inner and outer layers of surgical facemasks for and days, respectively [ ] . in other words, the infectious virus can be recovered from a surgical mask after days ( °c, % rh). sars-cov- survived for day on cardboard, wood, and cloth [ , ] . it survived for days on banknote paper [ ] . however, the virus survived for only min on paper and tissue paper, with complete decay after h [ ] . under the same conditions, sars-cov- survived for a longer time ( day) than sars-cov- , which survived for only h [ ] . interestingly, the stability of sars-cov- was enhanced when present with bovine serum albumin, which [ ] is used commonly to represent sources of protein found in human sputum [ ] . conversely, sars-cov- decayed more rapidly when either the humidity or the temperature was increased, but the droplet volume and surface type (stainless steel, plastic, or nitrile glove) did not impact the decay rate significantly. at room temperature ( °c), the virus half-life ranged from . to . h depending on the relative humidity but was reduced to . - . h when the temperature was increased to °c [ ] . these findings suggest that a potential for fomite transmission may persist for hours to days in indoor environments and that the survivability of fomites is affected by temperature and relative humidity, as well as by the presence of protein found in human sputum. in summary, virus survival decreases with an increase in temperature. maintaining temperatures above °c for more than min usually inactivate most viruses [ ] . sars-cov- is also susceptible to heat treatment. persistence of sars-cov- on dry inanimate surfaces was range for - days. the persistence of influenza virus, rhinovirus, and norovirus was reported at - days, h − days, and h − days, respectively [ ] . thus, sars-cov- can remain infectious on the surfaces compared with the influenza virus. however, the persistence of sars-cov- was significantly low on copper as compared with other surfaces such as plastics, stainless steel, glass, and fabrics. in addition, sunlight can rapidly inactivate sars-cov- . these findings droplet and contact transmission have accounted for the main routes of infection related to covid- . in addition, the who announced the possibility of aerosol infection in specific circumstances, such as endotracheal intubation, bronchoscopy, etc. [ ] . on the other hand, some researchers have claimed a risk of short-and medium-range person-to-person distance in aerosol transmission, but evidently, airborne transmission (longrange distance) has not been acknowledged [ ] . nicas et al. estimated that the relative contributions of four influenza virus exposure pathways, namely ( ) virus-contaminated hand contact with facial mucous membranes, ( ) inhalation of respirable cough particles, ( ) inhalation of inspirable cough particles, and ( ) the spray of cough droplets onto facial mucosa, account for , , . , and % risk of influenza infection, respectively [ ] . the transmission routes for these cough particles can be categorized as "droplet transmission," where droplets (> μm diameter, traveling < m) containing viable viruses make contact with the nose, mouth, eyes, or upper respiratory tract; and "airborne transmission" where droplet nuclei (< μm diameter, which can travel > m) are inhaled by susceptible individuals [ ] . the who has stated that airborne transmission is different from droplet transmission as it refers to the presence of microbes within droplet nuclei, which are generally considered to be particles smaller than μm in diameter, can remain in the air for long periods of time and can be transmitted to others over distances greater than m [ ] . from the view of this statement, the covid- virus can be transmitted by direct contact via infected people and indirect contact via surfaces in the immediate environment or with objects used by the infected person, not by airborne transmission through small airborne particles. recently, researchers insisted on the potential risk of indoor airborne transmission of sars-cov- and the importance of sufficient and effective ventilation, particle filtration, and air sterilization as infection control measures inside buildings [ ] . the who recognized the potential risks of the airborne spread of covid- [ ] . however, a recent experimental study using transgenic mice indicated that sars-cov- could be experimentally transmitted among mice by close contact, through respiratory droplets, but is hardly transmitted through exposure to airborne particles [ ] . a recent study also indicated that the role of airborne particles as carriers of the virus diffusion is not evident [ ] . in discussing the airborne transmission of sars-cov- , it is important to understand the characteristics of aerosol particles emitted by cough and speech in indoor environments. this section describes briefly the size distribution of droplets, the emission rate of droplets by cough and speech, and the resuspension of particles deposited on floor surfaces. sars-cov- has a diameter of - nm, with characteristic spikes ranging from to nm [ ] . johnson et al. determined an aerosol droplet size distribution ranging from nm to mm, generated by breathing, speech, and voluntary coughing by using the expired droplet investigation system with an aerodynamic particle sizer (aps) and a droplet deposition analysis (dda) [ ] . in the case of speech, three different droplet size distribution modes were identified, with median diameters at . , . , and μm. in the case of voluntary coughing, the modes were located at . , . , and μm. therefore, a wide range of droplet sizes is emitted by speaking and coughing. the key point in this study was that small droplets can be emitted not only by speaking and coughing but also through breathing. individuals infected by sars-cov- without symptoms can also transmit the infection to others. the emission by natural breathing without coughing or sneezing is also important to understand the transmission from infected individuals without symptoms. asadi et al. placed aps in a laminar flow hood to characterize the number and size distribution of particles emitted by individual human volunteers while performing various vocalizations and breathing activities [ ] . the results showed that the rate of particle emission during normal human speech is correlated positively with the loudness (amplitude) of vocalization, ranging from approximately to particles per second ( . to particles/cm ) for low to high wavelengths, regardless of the language spoken (english, spanish, mandarin, or arabic). the results also indicated that the droplets could be emitted not only by speech but also by singing a song, cheering loudly for sports games, and so on. rahmani et al. reviewed the methods for the sampling and detection of coronaviruses, especially sars-cov- . most of the samplers used, such as polytetrafluoroethylene filters, gelatin filters, and cyclones, showed a suitable performance to trap sars-cov and middle east respiratory syndrome (mers)-cov, followed by polymerase chain reaction (pcr) analysis [ ] . some studies reported detecting sars-cov- from patients' rooms in hospitals, although it seems difficult to discriminate whether these were airborne or transmitted through respiratory droplets because sampling conditions (i.e., the patient's distance from the sampler, patient's activities, coughing and sneezing during sampling time, etc.) can affect the results. liu et al. investigated airborne sars-cov- by measuring viral rna in aerosols in two different hospitals in wuhan during the covid- outbreak in february and march [ ] . the unique point in this study was to investigate the size distributions of airborne sars-cov- droplets. aerosol samples were collected using a miniature cascade impactor (sioutas impactor, skc) that could separate aerosols into five ranges (> . , . - . , . - . , and . - . μm on -mm filter substrates and - . μm on -mm filters) and determined through the quantification of their genetic material (rna). sars-cov- aerosols were found to include mainly two size ranges, one in the submicrometer region (d p . - . μm) and the other in the supermicrometer region (d p > . μm), in isolation wards of ventilated patient rooms and medical staff areas. the authors hypothesized that the source of the submicrometer virus-laden aerosols may be resuspension from the surface of the protective apparel worn by medical staff while they are removing the equipment. the resuspension occurs largely because of the particle surface properties, human activities, environmental conditions, and so on. resuspension occurs mainly as a result of human activities, especially walking in indoor spaces. qian et al. reviewed particle resuspension owing to human walking in indoor environments [ ] . from the results presented, resuspension is an important source compared with other indoor sources, such as cooking and stoves, and resuspension increases with particle size in the range of . - μm. many researchers proposed resuspension terms, such as a resuspension rate coefficient (h − ), a resuspension fraction (-), an emission rate (mg/h), a resuspension factor (m − ), and a resuspension emission factor (mg/mg). rosati et al. experimentally investigated the characterization of the size distribution of resuspended particle matter from the carpet during walking events [ ] . the resuspension emission factor by particle size was defined as the resuspension emission rate by particle load on the carpet by particle size. the emission factors were approximately − to − of particle diameter to μm. therefore, a small portion of particles deposited on the floor can be resuspended in indoor air. the resuspension is progressively more likely to occur by larger particles than by smaller ones. although there are a few studies on bioaerosols, and especially virus particles, bioaerosol resuspension is clearly not the same as infectious virus resuspension. it is important for airborne transmission to understand the droplet diameter emitted from patients. previous studies indicated that an aerosol droplet size distribution was ranged from nm to mm, generated not only by speaking and coughing but also through breathing. from the field measurements in the hospital, sars-cov- aerosols could be detected to include mainly two size ranges, . - . μm and larger than . μm, in isolation wards of ventilated patient rooms and medical staff areas. moreover, the resuspension is progressively more likely to occur by larger particles than by smaller ones, but bioaerosol resuspension cannot be clearly the same as infectious virus resuspension. the number of secondary infections of covid- varies widely, and most outbreaks of many secondary infections occur under common indoor environmental factors [ , ] . given the lack of data to define environmental control measures against covid- based on quantitative analysis, it is important to understand the conditions common to outbreak cases and to take measures to minimize the indoor environmental factors that promote infection. in this section, we summarize the cases of outbreaks in which indoor environmental factors, including human behavior, are believed to have facilitated infection, as well as the environmental survey of sars-cov- . on january , , a covid- outbreak occurred in a restaurant in guangzhou, china, infecting ten people in three families. the minimum length of time during which the index-infected person was present with the secondary infected person was min, and the possibility of contact infection was considered to be low based on in-store camera recordings. fifty percent (five of ten) of those at the same table as the infected person were found to be infected within the next days. at the adjacent leeward table, % (three of four) were infected. two of the seven people at the windward table were infected. other diners who were located away from the airflow around the infected tables, as well as the staff, were not infected. lu et al. concluded that the airflow from the air-conditioners promoted droplet infection and recommended that distances between people are maintained and ventilation improved [ ] . in this case, li et al. conducted a detailed investigation of the indoor environment by measuring the ventilation rate and conducting a numerical thermo-fluid analysis. as a result, they found that only the ventilation fan in the restroom was in operation. the ventilation fan on the wall of the restaurant was sealed and not in operation, and the ventilation rate ranged from . to . air changes per hour (ach) and . to . l/s per person. they suggested that poor ventilation may have been the primary cause of the spread of the infection by aerosol transmission [ ] . ninety-four people tested positive for the virus on one floor of the call center, where employees worked. [ , ] . meat-processing plants have emerged as hotspots for sars-cov- around the world. an outbreak of sars-cov- at germany's largest meat-processing facility resulted in more than confirmed infections during tests conducted a month after the initial outbreak. guenther et al. conducted a causal investigation into the may outbreak. it was suggested that the infected person, who was the first example of a supplemental case, transmitted the virus to a colleague more than m away over consecutive working days. the low temperature in an environment with the low intake of outside air and air circulation through the airconditioning system in the hall, combined with the high physical workload of workers with heavy breathing, have been suggested as factors that enabled transmission over distances greater than m by virus-containing aerosol particles. they stated that under these circumstances, distances of . - m are not sufficient to prevent infection and that the wearing of masks, improved ventilation and airflow, and the installation of filtering devices are necessary to reduce the risk of infection [ ] . these outbreaks strongly suggest that in a confined, crowded, and poorly ventilated environment where conversations, loud vocalizations, and heavy breathing take place, sars-cov- can spread through the air at a distance of m or more and may result in a large number of secondary infections. the who defines droplets and droplet nuclei as respiratory aerosols more than μm in diameter and the residue of dried respiratory aerosols up to μm in diameter, produced by the evaporation of droplets coughed or sneezed into the atmosphere or aerosolized infective material, respectively [ ] . based on field measurement results at wuhan hospitals during the covid- outbreak, liu et al. reported that the peak concentration of sars-cov- aerosols appears in two distinct size ranges: at the submicron scale with dominant aerodynamic diameters between . and . μm; and at the supermicron scale with diameters greater than . μm [ ] . the main sources of sars-cov- aerosols are coughs and sneezes by infected persons. the capacity for droplets to travel long distances in airflow is determined largely by their size. most communicable respiratory infections are transmitted via large droplets over short distances or contact with contaminated surfaces. large droplets (diameter, > μm) tend to settle quickly from the air, and thus the risk of pathogen transmission is limited to individuals in close proximity to the saliva droplet source. small droplets (diameter, ≤ μm) may be involved in short-range transmission (i.e., when the distance between individuals is less than m) and are likely to evaporate into droplet nuclei (diameter, < μm) in favorable environments, making them candidates for long-distance aerosol transmission [ ] . the terminal settling velocity of a particle increases rapidly with its size, as it is proportional to the square of particle diameter. the terminal settling velocity of a particle up to μm in size is less than . cm/s ( . m/s) [ ] , and because the particle diameter decreases as a result of the evaporation of the droplets during settling, droplets will remain suspended for a longer time indoors based on the relative indoor humidity [ ] . therefore, aerosols up to μm in diameter are carried easily over long distances (final inlet air) by the indoor airflow generated by air conditioning or ventilation equipment. field measurements show that the largest and average velocities in occupant space are . and . m/s, respectively [ , ] . it is possible to control aerosols containing a virus with a proper indoor airflow plan. regarding the indoor airflow plan, there are different types of ventilation systems such as piston flow ventilation and mixing-type ventilation. when the infected individual's position can be fixed, piston flow ventilation or push-pull ventilation is applicable. however, as the positions of infected people cannot be pinpointed in general environments, mixing-type ventilation is more effective because it dilutes the infectious aerosols to decrease their concentration in the air. to describe the relationship between the air exchange rate and the probability of infection, the wells-riley model was used: this model is based on the assumption that the air in the room is well mixed, leading to a uniform concentration of bioaerosols throughout the space. to estimate the probability of infection in a room, the quantum generation rate must be determined. a quantum represents the minimum dose that can cause infection in the host [ ] , and the quantum generation rate is the number of quanta produced per hour per infectious individual. table provides the values of quantum generation rates reported for different infectious aerosols [ ] . dai and zhao estimated the quantum generation rate of covid- by fitting the relationships between known rates and r (the basic reproduction number) of the infectious agents listed in table [ ] . the results indicate that the quantum generation rate for covid- ranges from to h − . figure shows the schematics of the two typical heating, ventilation, and air-conditioning (hvac) systems used in japanese office buildings. system a is the centralized hvac system (cs) and system b is the individual hvac system (is). the ventilation rates of the cs are generally approximately ach for outdoor air and ach for return air. moreover, the cs normally uses an air filter with a collection efficiency approximately equivalent to a minimum efficiency reporting value (merv) of , indicating a removal efficiency of % of droplets from human respiration activities, most of which are less than - μm in diameter [ ] [ ] [ ] . in this case, the equivalent change rate is approximately . ach (= ach + ach × %). figure shows the probability of infection plotted against the equivalent air change rate (hourly rate of room ventilation with clean air) based on eq. ( ). the prediction conditions are shown as i = person, p = . m /(hour⋅person), t = h, room floor area = m , ceiling height = . m. the higher the equivalent air change rate (the room ventilation rate with clean air/ room volume), the lower the probability of infection. furthermore, the cs shows a lower probability of infections than the is because of the larger amount of clean air. the use of highly efficient particle filtration in centralized hvac systems reduces the airborne load of infectious particles [ , ] . the collection efficiency of air filters for suspended particles by particle size is given in table . in japanese office buildings, medium-efficiency air filters (merv - in table ) are typically used, whereas high-efficiency particulate air filters (hepa: . % or higher particle collection efficiency for particles sized . μm at the rated airflow volume) are used for rooms such as hospital operating rooms that demand high air cleanliness. it is known that ultraviolet germicidal irradiation (uvgi) systems are used for air and surface disinfection. investigations of the bactericidal effect of sunlight in the late th century planted the seed of air disinfection by uv radiation. the first to nurture this seed was wells, who both discovered the spread of airborne infection by droplet nuclei and demonstrated the ability of uvgi to prevent such a spread. with modern concerns regarding multi-and extensive drug-resistant tuberculosis, bioterrorism, influenza pandemics, and severe acute respiratory syndrome, interest in uvgi continues to grow [ ] . the centers for disease control and prevention (cdc) [ ] and the american society of heating, refrigerating and air-conditioning engineers (ashrae) [ ] recommend the application of uvgi to fight airborne diseases. microbes are uniquely vulnerable to light of wavelengths at or near . nm because the maximum absorption wavelength of a deoxyribonucleic acid (dna) molecule is nm. the chemical compound pyrimidine in a dna base strongly absorbs uv light. after irradiation, the dna sequence where two pyrimidines link can form pyrimidine dimers. these dimers can change the dna double-helix structure and interfere with dna duplication, as well as lead to the destruction of the replication ability of cells and thus render the cells noninfectious [ , ] . regarding the sterilization or inactive performance of uvc, the population st of microbes exposed to any biocidal factor is described by the characteristic logarithmic decay equation: where k = the standard decay-rate constant (cm /mw⋅s), i = the intensity of uvgi (mw/cm ), and t = the exposure time (s). the standard decay-rate constant of the influenza a virus has been reported as . cm /μw⋅s [ ] [ ] [ ] . recently, upper-room uvgi and in-duct uvgi have been the two primary applications of uvgi air disinfection. in-duct uvgi is designed to disinfect air as it passes through the hvac system before it is recirculated or exhausted, and it has been also suggested to reduce nonspecific building-related illnesses [ , ] . in summary, since small droplets (diameter, ≤ μm) are likely to evaporate into droplet nuclei (diameter, < μm) in favorable environments, and the terminal settling velocity of a particle up to μm in size is lower than . cm/s ( . m/s), it is possible to control aerosols containing a virus with a proper indoor airflow plan and sufficient ventilation rates. moreover, filtration is effective in the reduction of aerosol concentration. ventilation and filtration can contribute to the reduction of indoor infection probability. on the other hand, it is well-known that uvgi is an effective measure against microbes. the who, the cdc, and the ashrae recommend the application of uvgi to fight airborne diseases. the application in japan will be expected. it is important to decide the adequate measures against a new infectious agent by analyzing actual infection cases and to execute them as soon as possible. as of february , , the ministry of health, labour and welfare (mhlw) covid- response team examined a total of cases among clusters and investigated who acquired infection from whom. all clusters were associated with close contact in indoor environments, including fitness gyms, a restaurant boat on a river, a club with live music, healthcare facilities, and a snow festival where there were eating spaces in tents with minimal ventilation rate [ ] . therefore, the mhlw published a document titled "prevention of the covid- clusters" abbreviation: sars-cov severe acute respiratory syndrome coronavirus fig. traditional japanese office building hvac systems: a a centralized hvac system; and b a centralized ventilation system with an individual air-conditioning system on march , [ ] , showing the need for adequate ventilation in buildings because a ventilation standard for infection control has not been established in general buildings in japan and the characteristics of indoor spaces where the clusters occurred might include poor ventilation and crowding. therefore, the " cs," namely, "closed spaces with poor ventilation," "crowded spaces with many people," and "close contact," such as from intimate conversations, loud cheering, singing, or exercise within a short distance from other individuals, were proposed as important factors that result in a covid- cluster [ , ] . the mhlw published "ventilation to improve "closed spaces with poor ventilation" in commercial facilities" on march , , based on the analysis by the mhlw covid- response team concerning the actual ventilation state against the standards in the law for maintenance of sanitation in buildings, and recommended adequate ventilation rates in accordance with the standards of the law and the increase in the ventilation rates by adjusting the ventilation systems and opening the windows [ ] . furthermore, this recommendation was added in the "measures to prevent the large-scale spread of covid- in workplaces" on march , [ ] . on april , , a document titled "maintenance of air-conditioning and ventilation systems in specific buildings" was published and adequate ventilation was requested again [ ] . moreover, on april , , the managers of commercial facilities were informed of the "methods of ventilation for improving "closed spaces with poor ventilation," and the guidelines of ventilation measures in both specific buildings and other buildings were provided [ ] . in these documents, the required ventilation rate of approximately m /h per person was recommended [ ] . when this ventilation rate was determined, the results of studies on tuberculosis [ ] [ ] [ ] and measles [ ] , which are infectious diseases known to be spread by airborne transmission, were considered [ ] . the ventilation rates needed are indicated not per room but per person and if adequate ventilation is not secured in the room, the number of persons should be limited to avoid overcrowding, which may be a factor in clusters of infected individuals. the ventilation rates needed to prevent sars-cov- transmission have not been reported; thus, opening windows has also been recommended. however, it was difficult to recommend the specific methods because the conditions of the rooms (e.g., the number of windows, and the width and direction of the windows) are different. in the expert meeting on novel coronavirus disease control on may , a "new lifestyle" for the long-term fight against covid- was suggested, comprising three basic measures: social distancing, wearing face masks, and washing hands [ ] . since the middle of april, researchers on building hygiene have collected recent scientific findings and discussed the ventilation measures towards summer [ ] . during the rainy season and summer, opening the windows may create a poor indoor environment with insufficient air-conditioner cooling and dehumidification, resulting in increased risks of heatstroke, insomnia, and allergies owing to mold and mites. after discussions, the researchers produced a document titled "indoor environmental measures in summer against covid- : the suggestion from the researchers on building hygiene" on may , and provided it to the expert meeting members on novel coronavirus disease control and headquarters of the mhlw [ ] . from the present evidence, it is difficult to indicate specific standard values, such as ventilation rates. thus, ventilation and air-conditioning measures recommended under some conditions were shown, see table . on may , , the ministry of the environment and the mhlw served local governments a notice of "action for prevention of heatstroke in ," which requested that they take action to prevent heatstroke under the spread of covid- [ ] . the headquarters of covid- prevention in the mhlw considered expert opinions, scientific literature, international standards, and laws and regulations in japan in the search for effective methods, and published a document titled "ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke" on june , [ ] . in addition, it published the document titled "methods of ventilation for improving "closed spaces with poor ventilation" with prevention of heatstroke for owners using air-conditioning system without ventilation in commercial facilities" on june , [ ] . the document shows points to remember while opening windows for ventilation, in using air cleaners, and so on. furthermore, in the "q&a for the public," information was provided on home air conditioners and ventilation during the hottest season. it was indicated that air conditioners should be used to prevent heatstroke, but that general air conditioners do not have a ventilatory function; thus, the whole air of a room should be ventilated twice or more per hour and the use of a -h mechanical ventilation system is effective. the society of heating, air-conditioning and sanitary engineering of japan started a special committee against covid- and published a document titled "is covid- spread by air-conditioning or ventilation?-experts' opinions" on june , . in the document, the relationship between airconditioning or ventilation and the risk of infection, the possibility of the spread of covid- through air conditioners, the infection risk by inadequate ventilation and filters, and the prevention of heatstroke with infection control are discussed and the following remarks were obtained [ ] : in japanese buildings, which are designed and managed in accordance with the law for maintenance of sanitation in buildings, considering the performance of ventilation and filters, the risk of the spread of covid- in a room through airconditioning systems is believed to be very low. however, in the case of buildings without the general-performance filters in air conditioners, commercial package-type air conditioners, or a fan coil unit (i.e., fcu), another ventilation method should be added. securing ventilation rates per person may prevent droplet infection through airflow. in hospitals and clinics where individuals can wear face masks all the time, the droplet from infected individuals can be prevented. when adequate ventilation rates are secured, it is necessary to use air conditioners to prevent heatstroke in summer. in hospitals and clinics where individuals are believed to be infected, the airflow from air conditioners should not be directed to any person. considering the fact that in a poorly ventilated room, an individual was infected when spending time with an infected person for less than an hour, not intermittent but continuous ventilation is desirable. in cases where natural ventilation is available, it is desirable to secure ventilation rates by opening the windows and so on if an adequate room temperature is secured. however, in the buildings designed and managed in accordance with the law for maintenance of sanitation in buildings, natural ventilation may cause a bad influence on air balance and may be beyond the standards of the law. thus, it is desirable to consult experts. an industry-classified guideline for the prevention of the spread of covid- was made based on the proposal by the experts' meeting on may , . as of writing this article, approximately guidelines were available on the website of the cabinet secretariat of the japanese government [ ] . the items concerning building environments are cleaning, disinfection, ventilation, room density, etc., and guidelines are shown according to the characteristics of each industry. regarding ventilation, the specific rates or times are not indicated because of the various conditions of ventilation systems or windows. since pneumonia caused by sars-cov- was confirmed in december , the mechanism of infection in an indoor environment has not been clarified. it is important to understand infection mechanism through various studies such as investigation of infection cluster spaces and experiments on the relationship between such environmental factors as temperature and humidity and the infectiousness of the virus. based on these studies, further adequate strategy for the infection risk management in an indoor environment is required. in environmental stabilities of sars-cov- , this virus can persist on the fomite surfaces, such as plastic or metal, between and days in an indoor environment such as a building. sars-cov- in aerosols is also stable for at least several hours if the virus is produced within small-particle aerosols. sars-cov- is mainly transmitted between humans through close contact and respiratory droplets, including fomite transmission. however, close-contact aerosol transmission through smaller aerosolized particles is likely to be combined with respiratory droplet and contact transmission in a confined, crowded, and poorly ventilated indoor environment, as some cluster cases have suggested. therefore, adequate preventive measures to control indoor environmental quality are required. the risk of closecontact aerosol transmission can be reduced sufficiently by taking measures such as ensuring adequate ventilation in accordance with the number of individuals in a room and wearing masks to reduce the aerosols emitted into the indoor air. filtration and uvgi systems have also been developed. however, indoor environmental quality control measures such as ventilation alone cannot prevent exposure from an infected person at a short distance or through environmental surfaces; thus, adequate preventive measures against respiratory droplet and contact transmission, such as disinfection of fomites, hand hygiene, uncrowded spacing, and the wearing of masks, must be taken at appropriate times and places. in japan, epidemiological links to confirmed covid- cases showed that clusters of cases occurred in fitness gyms, a restaurant boat on a river, and a club with live music in february . based on these findings, the expert panel for covid- focused on the " cs," namely, "closed spaces with poor ventilation," "crowded spaces with many people," and "close contact." in addition, the mhlw has recommended adequate ventilation in all closed spaces in accordance with the existing standard of the law for maintenance of sanitation in buildings. although specific ventilation standards for the control of sars-cov- transmission cannot be derived from the existing scientific evidence, covid- has caused serious global damage to public health, community, and the social economy. the united nations conference on environment and development adopted the rio declaration on environment and development in [ ] . table recommendations on the ventilation and air-conditioning measures to prevent covid- infection every indoor space • enough ventilation is necessary to prevent covid- infection. • opening windows is effective for ventilation and opening them wide and for a longer time is desirable. • in summer, air conditioners are necessary to prevent health risks such as heatstroke, etc. • general air conditioners do not function as ventilators, so mechanical ventilation and opening windows are necessary. • when opening windows, it is necessary to prevent harmful insects and animals from coming in. spaces in which air conditioning and ventilation systems are installed • it is necessary to check the systems and to confirm enough ventilation rates are secured. • it is necessary to limit the number of persons in a room or shorten the time they are inside secure enough ventilation rates per person. • it is necessary to investigate what the building is used for, how often it is used, what kind of airconditioning and ventilation system is used in the building when taking such measures as the better control of air-conditioning and ventilation systems, or the use of air cleaners, and the use of humidifiers in winter. principle states: "in order to protect the environment, the precautionary approach shall be widely applied by states according to their capabilities. where there are threats of serious or irreversible damage, lack of full scientific certainty shall not be used as a reason for postponing cost-effective measures to prevent environmental degradation." this principle was also applied to the threats to human health in the public health domain [ ] [ ] [ ] . based on principle , the implementation of practical precautionary measures that took into account technical feasibility, adequate control measures and strategies, and social, economic, and cultural conditions is essential to protect the public from sars-cov- transmission. however, the implementation of an approach based on the precautionary principle should start with a scientific evaluation, as complete as possible, and where possible, identifying at each stage the degree of scientific uncertainty [ ] . therefore, further research and evaluation are required to evaluate the effect and role of indoor environmental quality control, especially ventilation. in this paper, we summarize the effect and role of environmental factors in buildings, spatial dynamics, building operational factors, and a strategy to prevent sars-cov- transmission in a building environment. most transmission has occurred in indoor environments in a closed space, especially inside a building. although ventilation based on the existing regulation has been recommended by the mhlw as one of the initial political actions to prevent the spread of this novel infectious disease, the specific standard has not been recommended. to protect public health from sars-cov- transmission, further research on infection dynamics and the mode of infection in sars-cov- transmission in closed spaces, investigations on indoor environments in the infected cluster spaces, experiments on the influences of indoor environmental conditions on covid- infection, and the effects of ventilation are urgently required. in addition, the development of control measures, guidelines, and strategies to maintain indoor environmental quality at an adequate level are required. a new 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public health actions the precautionary principle, public health, and public health nursing the precautionary principle: protecting public health, the environment and the future of our children. copenhagen: world health organization regional office for europe communication from the commission on the precautionary principle. brussels: commission of the european communities not applicable. the six authors are justifiably credited with authorship, according to the authorship criteria. in detail: ka-conception, design, acquisition, and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval is given; uy and mh-conception, acquisition, and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval given; nk, hk, and mo-acquisition and interpretation of data, drafting of the manuscript, critical revision of the manuscript, final approval given. no funder supported this work. ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -fzijbney authors: nemoto, manabu; oue, yasuhiro; higuchi, tohru; kinoshita, yuta; bannai, hiroshi; tsujimura, koji; yamanaka, takashi; kondo, takashi title: low prevalence of equine coronavirus in foals in the largest thoroughbred horse breeding region of japan, – date: - - journal: acta vet scand doi: . /s - - - sha: doc_id: cord_uid: fzijbney background: equine coronavirus (ecov) is considered to be a diarrheic pathogen in foals. in central kentucky in the united states, it has been shown that approximately % of thoroughbred foals are infected with ecov and thus it is considered widely prevalent. in contrast, the epidemiology of ecov and its relationship to diarrhea in foals are poorly understood in japan. we investigated ecov in rectal swabs collected from thoroughbred foals in japan. results: we collected rectal swabs from diarrheic foals in the hidaka district of hokkaido, the largest thoroughbred horse breeding region in japan, between and . in addition, rectal swabs were collected from healthy foals in . these samples were tested by reverse transcription loop-mediated isothermal amplification and a real-time reverse transcription-polymerase chain reaction. all samples collected from diarrheic foals were negative, and only three samples ( . %) collected from healthy foals were positive for ecov. compared with central kentucky, ecov is not prevalent among thoroughbred foals in the hidaka district of hokkaido. conclusion: ecov is not prevalent and was not related to diarrhea in thoroughbred foals in the hidaka district of hokkaido between and . certain pathogens are common causes of diarrhea in foals, equine rotavirus being the most common [ ] . equine coronavirus (ecov) is also a diarrheic pathogen in foals [ ] . recently, several ecov outbreaks in adult horses occurred in the united states [ ] and japan [ ] [ ] [ ] . fever, anorexia, lethargy, leukopenia and digestive disorders were observed, and these clinical signs were reproduced in an experimental challenge study [ ] . equine coronavirus was also detected in fecal samples of diarrheic foals in the united states [ , ] , but there have been no reports of an ecov outbreak in foals. slovis et al. [ ] reported that approximately % of healthy and diarrheic thoroughbred foals in central kentucky in the united states were infected with ecov, using a real-time reverse transcription-polymerase chain reaction (rt-pcr) assay. these results indicate that ecov is prevalent among thoroughbred foals in central kentucky. in japan, ecov outbreaks had previously occurred only in the draft racehorse population. draft racehorses include french percheron, breton and belgian horses, with body weights around kg. outbreaks of ecov had not been reported in the japanese thoroughbred horse population, and its epidemiology is poorly understood. it is also unclear whether ecov is related to diarrhea in japanese foals. therefore, we investigated ecov using molecular diagnostic methods on rectal swabs collected from thoroughbred foals in the hidaka district open access *correspondence: nemoto_manabu@equinst.go.jp epizootic research center, equine research institute, japan racing association, - shiba, shimotsuke, tochigi - , japan full list of author information is available at the end of the article of hokkaido, which is the largest thoroughbred horse breeding region in japan. between and , we collected rectal swabs from diarrheic foals aged days to months in the hidaka district of hokkaido by using bd bbl cul-tureswab ez (becton, dickinson and company, fukushima, japan). rectal swabs of twenty-two and four diarrheic foals were collected twice and three times, respectively. multiple samples were collected with - days intervals (average . days). we collected samples from farms in , samples from farms in and samples from farms in . rectal swabs from diarrheic foals were stored at − °c in the veterinary clinic until transport. they were transported to the diagnostic laboratory at around − °c, and after arrival they were kept at − °c until use. additionally, rectal swabs were collected from healthy foals on farms in the same region in by using bd bbl cultureswab plus (becton, dickinson and company). healthy foals were between and days of age. rectal swabs from healthy foals were stored and transported at °c for several days. after arrival, they were kept at − °c until use. these samples were immersed in maintenance medium [ ] or phosphate buffered saline. viral rna was extracted from samples with magna pure lc total nucleic acid isolation kit (roche diagnostics, mannheim, germany). reverse transcription loop-mediated isothermal amplification (rt-lamp) and real-time rt-pcr assays were selected because these molecular methods have high sensitivity for ecov [ ] . the rt-lamp reaction was performed using a primer set described previously [ ] and a loopamp rna amplification kit (eiken chemical, tokyo, japan) according to the manufacturer's instructions. calcein, a fluorescent detection reagent (eiken chemical, tokyo, japan), was added to the reaction mixture for visual detection. the mixtures were incubated at °c for min and then heated at °c for min to terminate the reaction. real-time rt-pcr assay was conducted using a primer set described previously (ecov- f, ecov- r and ecov- p) [ ] and taqman fast virus -step master mix (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. thermal cycling was performed as [ ] : °c for min and °c for s, followed by cycles at °c for s and °c for s. for sequence and phylogenetic analysis of the nucleocapsid (n) gene, rt-pcr was performed using the primer set ecov-nf ( ′-tcaggcatggacaccgcattgtt- ′) and ecov-nr ( ′-ccaggtgccgacataaggttcat- ′) [ ] using pri-mescript ii high fidelity one step rt-pcr kit (takara bio, otsu, japan). rt-pcr products were directly sequenced commercially by fasmac (atsugi, japan). sequence analysis was performed using the blast and clustalw programs, and vector nti advance software (invitrogen, carlsbad, ca, usa). phylogenetic analysis of nucleotide sequences was conducted with mega software version . [ ] . a phylogenetic tree was constructed based on nucleotide sequences using the neighbor-joining method. statistical analysis of the tree was performed with the bootstrap test ( replicates) for multiple alignments. [ , ] , obi-hiro [ ] , tokachi [ ] and obihiro - [ ] phylogenetic analysis was performed for the nucleotide sequences of the n gene (fig. ) . phylogenetic analysis showed that hidaka-no. / and hidaka-no. / are closely related to the obihiro - and tokachi strains, respectively. in this study, all diarrheic samples were negative for ecov. this indicates that ecov is not a causative agent of diarrhea in thoroughbred foals in hidaka district of hokkaido. using rectal swabs collected from healthy foals, only three samples ( . %) were positive for ecov. rectal swabs from healthy foals preserved at worse condition than that of diarrheic foals as described above. however, ecov was only detected in rectal swabs from healthy foals, and therefore it is unclear whether storage condition influenced the result in this study. compared with central kentucky, ecov is not prevalent among thoroughbred foals in hidaka district of hokkaido, but some outbreaks have occurred in draft racehorses. one reason for the lack of ecov positive samples in thoroughbred foals may be that they do not usually have contact with draft horses. these results suggest that the prevalence of ecov varies greatly depending on region. to further show this, it is necessary to investigate ecov in other countries as well. in sequence and phylogenetic analyses of the n gene, there is little difference between hidaka-no. / and obihiro - . obihiro - was isolated in the ecov outbreak in march [ ] , and therefore the hidaka in conclusion, this study shows that ecov is not prevalent and was not related to diarrhea in thoroughbred foals in the hidaka district of hokkaido between and . infectious agents detected in the feces of diarrheic foals: a retrospective study of cases viral diarrhea emerging outbreaks associated with equine coronavirus in adult horses prevalence of disease with inference of equine coronavirus infection among horses stabled in a draft-horse racecourse isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc strain epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in experimental inoculation of equine coronavirus into japanese draft horses neonatal enterocolitis associated with coronavirus infection in a foal: a case report characterization of a coronavirus isolated from a diarrheic foal infectious agents associated with diarrhoea in neonatal foals in central kentucky: a comprehensive molecular study virucidal effect of commercially available disinfectants on equine group a rotavirus rapid detection of equine coronavirus by reverse transcription loopmediated isothermal amplification mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods balasuriya ub. genomic characterization of equine coronavirus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we are grateful to all of the equine practitioners of the hidaka agriculture mutual aid association for collecting rectal swabs and to mr. akira kokubun, ms. kazue arakawa, ms. akiko suganuma and ms. kaoru makabe for invaluable technical assistance. mn outlined the design of the study, performed the experiments and drafted the manuscript. yo participated in the design of the study and interpretation of the data. th and yk carried out the clinical observations of horses and the sample collections. hb, kt, ty and tk participated in interpretation of the data and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -xoqrc xo authors: miyaji, kazuki; suzuki, aki; shimakura, hidekatsu; takase, yukari; kiuchi, akio; fujimura, masato; kurita, goro; tsujimoto, hajime; sakaguchi, masahiro title: large-scale survey of adverse reactions to canine non-rabies combined vaccines in japan date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: xoqrc xo canine non-rabies combined vaccines are widely used to protect animals from infectious agents, and also play an important role in public health. we performed a large-scale survey to investigate vaccine-associated adverse events (vaaes), including anaphylaxis, in japan by distributing questionnaires on vaaes to veterinary hospitals from april , through may , . valid responses were obtained for , vaccinated dogs at animal hospitals; we obtained vaaes information for last vaccinated dogs in each veterinary hospital. we found that of the , , dogs showed vaaes. of the dogs, death was observed in , anaphylaxis in , dermatological signs in , gastrointestinal signs in , and other signs in . onset of vaaes was mostly observed within h after vaccination (n = , . %). in this study, anaphylaxis events occurred within min after vaccination, and about half of these events occurred within min (n = , . %). furthermore, where anaphylaxis was reported, additional information to support the diagnosis was obtained by reinvestigation. our resurvey of dogs with anaphylaxis yielded responses on dogs; of these demonstrated collapse ( . %), demonstrated cyanosis ( . %), and both signs occurred in ( . %). higher rates of animal vaaes, anaphylaxis, and death were found in japan than in other countries. further investigations, including survey studies, will be necessary to elucidate the interaction between death and vaccination and the risk factors for vaaes, and thus develop safer vaccines. moreover, it may also be necessary to continually update the data of vaaes. canine non-rabies combined vaccines, containing zoonotic pathogen leptospira, are widely used in small animal veterinary medicine. vaccination is aimed at fig. . the questionnaire form used for the investigation of vaccine-associated adverse events (vaaes). the questionnaire was distributed by the japan small animal veterinary association to veterinary hospitals in japan from april , through may , . et al., roth, ) occasionally observed in humans and dogs after vaccination, and which sometimes causes death (brooks, ; sakaguchi et al., ) . according to large epidemiological studies on canine vaaes, the incidences of anaphylaxis were . / , vaccinated dogs in the united kingdom (gaskell et al., ) and . / , vaccinated dogs in the united states (moore et al., ) . based on a survey of a small number ( ) of japanese veterinary hospitals, the anaphylaxis rate in japan appeared to be somewhat higher, at . % ( / vaccinated dogs) (fujimura, ) . however, to date, no large-scale investigation had been carried out on vaaes associated with canine non-rabies combined vaccine in japan. moreover, although incidences of anaphylaxis were provided, none of the previous studies (gaskell et al., ; moore et al., ; fujimura, ) considered important details, such as concurrent symptomatic states. here, we report the results of a large-scale survey of vaaes in japan, based on diagnosis made by experienced veterinarians in their practices. our results reveal useful information on vaaes, including critical detail on anaphylaxis and death caused by canine non-rabies combined vaccines. it is useful to record vaccine reactions routinely, and report vaaes to specific agents such as local governments in veterinary medicine as well as human medicine. the questionnaires about adverse reactions to canine non-rabies combined vaccines were distributed by the japan small animal veterinary association to veterinary hospitals in japan from april , through may , . the questionnaires not only recorded standard information: date of birth, breed, sex and neuter status, weight, and date of vaccination, but also included important factors associated with adverse reactions, such as type of vaccination, signs, and time of their onset since vaccination (fig. ) . practicing veterinarians diagnosed adverse reactions and classified them into groups according to clinical signs: death, anaphylaxis, dermatological signs (swelling of face, pruritus, urticaria, flush, and erythema), gastro-intestinal signs (vomiting and diarrhea), and other signs (including hypodynamia and anorexia). in cases of anaphylaxis, additional information was obtained by reinvestigation to support the diagnoses. to define the population, each responding veterinarian also stated the number of affected cases per last vaccinated dogs. the following non-rabies combined vaccines for dogs are commonly used in japan: combined live vaccines composed of canine parvovirus, canine distemper virus, canine adenovirus type , and canine parainfluenza virus (group ); adding live or inactivated coronavirus to group (group ); adding inactivated leptospira to group (group ); adding inactivated coronavirus and leptospira to group (group ); adding live coronavirus and inactivated leptospira to group (group ). duramune mx (kyoritsu seiyaku corporation, tokyo, japan; fort dodge animal health, iowa, usa), eurican (merial animal health, lyon, france), and nobivac dhppi (intervet, boxmeer, netherlands) were classified as group ; canine- ii (kyoto biken laboratories, kyoto, japan), duramune dx , and vanguard plus /cv (pfizer animal health, new york, usa) were classified as group ; canine- , eurican , and nobivac dhppi+l as group ; duramune dx and vanguard plus /cv-l as group ; and canine- (ii) as group . sex and neuter status were analyzed as categorical data. continuous variables of age and weight were converted to categorical variables because nonlinear trends were detected in the model-fitting process. dogs were grouped on the basis of age at date of vaccination as follows: - months, months to . years, . - . years, . - . years, . - . years, . - . years, and > . years. weight was converted from continuous to categorical data of - kg, - kg, and > kg. the p values for anaphylaxis in total vaaes were evaluated using a multivariate unconditional logistic regression model. the variants included sex and neuter status, weight, age, and vaccine group. the model was assessed for significance by use of the le cessie-van houwelingen test. a value of p < . was considered significant. statistical analysis was performed with r version . . (www.r-project.org/). valid responses were obtained for , vaccinated dogs, from animal hospitals; of these, dogs were diagnosed with vaae ( . / , vaccinated dogs). unfortunately, the detailed information regarding dogs with no adverse events could not be obtained in the present study. of the dogs, anaphylaxis was observed in , dermatological signs in , gastrointestinal signs in , and other signs in (table ) . a single death ( . / , vaccinated dogs) was reported within a few days after vaccination. in decreasing order of frequency, ( . %) miniature dachshunds, ( . %) chihuahuas, ( . %) mixedbreeds, and ( . %) toy poodles were reported to have vaaes in the present survey; other breeds were involved less frequently. the population with vaaes included ( . %) sexually intact males, ( . %) sexually intact females, ( . %) castrated males, ( . %) spayed females, and ( . %) unknown. almost half these dogs (n = , . %) were between the ages of and months. dogs in the weight category of - kg had the highest frequency of adverse reactions (n = , . %). most adverse events (n = , . %) were observed within h after vaccination (fig. ) . more detailed information about vaccine-associated anaphylaxis is provided in tables and , as well as fig. . miniature dachshunds (n = ) accounted for approximately % of the anaphylaxis cases. anaphylaxis occurred in four miniature schnauzers out of a population of the eight with vaaes. of dogs diagnosed with anaphylaxis, ( . %) were sexually intact males, ( . %) were intact females, ( . %) were castrated males, and ( . %) were spayed females. according to age and weight, the greatest frequency of anaphylaxis was recorded among dogs aged - months (n = , . %) and among dogs weighting less than kg (n = , . %), respectively. a multivariate logistic regression model, including sex and neuter status, weight, and age satisfied requirements for goodness of fit (p = . ). in the final model, there was no significant relationship between anaphylaxis and the factors of sex and neuter status (p = . ), weight (p = . ), age (p = . ), or vaccine group (p = . ). all cases of anaphylaxis occurred within min after vaccinations, and about half occurred within min (n = , . %) (fig. ) . when we attempted to resurvey the dogs that experienced anaphylaxis, responses were received for of these dogs (recovery ratio = . %). as shown in table , collapse and cyanosis were observed in almost all of the dogs, with ( . %) demonstrating collapse, ( . %) demonstrating cyanosis, and ( . %) showing both signs. a single death (chihuahua, intact female, months, . kg), occurring h after vaccination, was reported in our questionnaire. the risks involved in vaccination of dogs were highlighted by previous large epidemiological studies (gaskell et al., ; moore et al., ) ; however, surveys on vaaes had only been performed on a small scale in japan (ohmori et al., (ohmori et al., , a fujimura, ) . here, we present the results of a large-scale investigation of vaaes at japanese veterinary hospitals. we found a vaae rate of . / , vaccinated dogs, which is much higher than the rates reported in the united kingdom ( . / , vaccinated dogs) (gaskell et al., ) and the united states ( . / , vaccinated dogs) (moore et al., ) . previous reports suggested that small breed dogs (< kg), especially dachshunds, were more prone to vaaes than larger dogs (gaskell et al., ; moore et al., ; moore and hogenesch, ) . according to the japan kennel club (http://www.jkc.or.jp), miniature dachshund, chihuahua, and toy poodle are the most popular breeds in this country, and account for just over half of the total registrations in the club ( . %). this popular breed bias and the proposed breed susceptibility may contribute to higher vaaes rate in japan than in other countries. factors known to cause vaccine reactions include the primary vaccine agent or antigen, adjuvants, preservatives, stabilizers, and residues from tissue culture used in vaccine production (hogenesch et al., ; roth, ; georgitis and fasano, ) . our previous study reported large amounts of bovine serum albumin (bsa) and bovine igg contents in canine vaccines (ohmori et al., b) . furthermore, we found ige reactivity against fetal calf serum (fcs) components in dogs with allergic reactions after vaccination, suggesting that most of these reactions might be caused by fcs components derived from the culture media used to produce vaccines (ohmori et al., b (ohmori et al., , . many vaccines that are commonly used in japan are imported from abroad, although a few are made in japan. multilateral studies are necessary to clarify the relationship between vaaes and vaccine by-products, and to ascertain whether certain vaccines are more likely to give rise to vaaes. among the many clinical signs of adverse reactions to vaccines, anaphylaxis is the most dramatic (roth, ) . in the present investigation, the incidence of anaphylaxis ( . / , vaccinated dogs) in dogs was remarkably higher than those reported in previous studies (gaskell et al., ; moore et al., ; moore and hogenesch, ) . the dogs showing anaphylaxis in the present study included many small breeds such as miniature dachshund and miniature schnauzer. this implies that a breed predisposition may play a key role in anaphylaxis after vaccination of dogs, just as in total vaaes, and that genetic factors may be involved in these clinical signs. moore et al. ( ) have indicated that the risk of vaaes was significantly increased for small and neutered dogs, highest for dogs approximately - years old, and least for dogs ≥ years of age. unfortunately, as the information regarding dogs with no adverse events could not be obtained in the present study, the odds ratios of vaaes could not be estimated; however, we did estimate the p values for anaphylaxis according to adverse reactions after vaccine administrations using a logistic regression model. additionally, we were not able to analyze the relationship between breed and anaphylaxis due to small sample numbers for many breeds. however, no significant differences were observed in the anaphylaxis risk by sex and neuter status, weight, and age; thus, our data seem to show the same tendency of potential risk factors for vaaes in the results of moore et al. ( ) . the results of the present study suggest that various factors such as weight and age could predict the potential risk of adverse reactions, including anaphylaxis after vaccination, which may allow practicing veterinarians to allay the anxiety of at least a few owners regarding vaccination. as only a single death was reported in this investigation, a direct association between death and vaccine administration was not clear; however, as an incidence of . / , vaccinated dogs was reported in the united state (moore et al., ) , the mortality rate also appeared to be increased in japan. this case did not show anaphylactic signs, although other clinical sign was observed. further extensive surveys are needed to uncover the predictive factors and clinical signs that precede such outcomes, in order to prevent such unfortunate events. moreover, to clarify the interaction between death and vaccination, comparison with randomized unvaccinated dogs may be effective. we present data regarding vaaes, and in particular, on anaphylaxis, based on a large-scale study in japanese veterinary hospitals. higher rates of vaaes, anaphylaxis, and death were observed in japan compared to other countries. further international studies aimed at obtaining similar information will be necessary to elucidate the risk factors, including breed predisposition, the quality of vaccine products, and prior sensitization with variable allergens, for vaaes, with a view to developing safer vaccines. in addition, as our study was not current, it may be also be necessary to continually update the data of vaaes. adverse reactions to canine and feline vaccines a survey on the adverse reactions to combined vaccines in a small group of veterinary hospitals ( hospitals) veterinary products committee working group report on feline and canine vaccination allergenic components of vaccines and avoidance of vaccination-related adverse events vaccine-induced autoimmunity in the dog adverse events diagnosed within three days of vaccine administration in dogs adverse vaccinal events in dogs and cats a retrospective study on adverse reactions to canine vaccines in japan suspected allergic reactions after vaccination in dogs in japan ige reactivity to vaccine components in dogs that developed immediate-type allergic reactions after vaccination immunoblot analysis for ige-reactive components of fetal calf serum in dogs that developed allergic reactions after non-rabies vaccination mechanistic bases for adverse vaccine reactions and vaccine failures ige antibody to gelatin in children with immediate-type reactions to measles and mumps vaccines minimum estimated incidence in japan of anaphylaxis to live virus vaccines including gelatin the authors would like to thank all staff of participating veterinary hospitals for their kind assistance in responding to the questionnaires. we also thank the japan small animal veterinary association for its support. we thank dr. sakae inouye in otsuma women's university for critical review of this manuscript. this research was partially supported by the promotion and mutual aid corporation for private schools of japan, grant-in-aid for matching fund subsidy for private university, and by a project grant awarded by the azabu university research services division. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord- -pq u kh authors: kim, jewoo; lee, choong-ki title: role of tourism price in attracting international tourists: the case of japanese inbound tourism from south korea date: - - journal: journal of destination marketing & management doi: . /j.jdmm. . . sha: doc_id: cord_uid: pq u kh abstract tourism price has been extensively used to predict tourism demand. however, there is no agreement on the proper indicators of its components. use of different price indicators may be the reason for researchers’ apparently inconsistent results. the purpose of this study was to identify proper price indicators for the demand model of japanese inbound tourism from south korea. after comparing six models, each with different price indicators, the model with relative price and exchange rate but without transport cost was identified as the best model in which relative price, exchange rate, and per capita income were found to be significant. international tourism has experienced sustained expansion over the past six decades, substantially contributing to the world economy. international tourist arrivals reached the unprecedented milestone of billion in and generated us$ billion in expenditures (world tourism organization, ) . accordingly, many researchers have conducted studies on international tourism flows and their major determinants (carey, ; chatziantoniou, filis, eeckels, & apostolakis, ; de vita & kyaw, ; garín-muñoz, ; law, goh, & pine, ; lee, ; wang, ) . tourism demand forecasting is important for effective use of limited resources in both private and public sectors (lee, var, & blaine, ; song & witt, ) . since many tourism products such as airline seats and hotel rooms are perishable, efficient planning based on accurate estimation of demand is critical for successful tourism businesses. an inability to meet demand often leads to business failure in the tourism industry (song & witt, ) . for the public sector, tourism forecasting provides a basis for planning investments in tourism infrastructure such as airports and highways (lee, song, & mjelde, ) . since such large-scale infrastructure projects require considerable public funds over the long term, the expected return on investment (roi) should be spelled out in the planning stages, and roi is in large part determined by forecasting tourism demand. accordingly, accurate demand estimation is essential when appraising investment plans' economic feasibility (song & witt, ) . recognizing the importance of tourism demand forecasting, the econometric approach has been widely adopted to predict tourism demand (song & li, ) . in econometric studies of tourism demand, per capita income, tourism price, promotional efforts, and external shocks have been identified as important determinants of tourism demand (li, song, & witt, ; lim, ; song & li, ) . however, results regarding the effects of price variables (e.g.relative prices, exchange rates, transport cost) on international tourism demand vary widely. some inconsistent results may be attributable to use of different variables as a proxy for the same tourism price factor. this inconsistent use of price variables suggests that more research needs to be done to identify the price variables that best represent international tourism price. given that previous empirical studies yielding the inconclusive results on price effect have been conducted in the context of different countries, proper price variables may be different from destination to destination. indeed, tourists' responses to changes in explanatory variables are country-specific and therefore the variables' elasticity varies by destinations (crouch, ; dwyer, forsyth, & rao; gil-pareja, llorca-vivero, & martínez-serrano, ) . moreover, recent changes in economic circumstances such as currency depreciation resulting from unconventional monetary policy (us, eurozone, japan), shifts in exchange rate policy (switzerland, singapore, china), and drastic drops in oil prices triggered by expanded supplies of shale gas may substantially influence the cost of travel to the countries changing international tourism demand. thus, choosing the proper price variables for the tourist destinations examined could be critical to the accurate estimation of tourism demand for those destinations. the aim of this study is to address the selection of proper tourism price variables in identifying underlying factors in tourism demand http model. specifically, this study estimates and compares several demand models of japanese inbound tourism from south korea (hereafter korea), each of which includes different price variables. japanese inbound tourism from korea was chosen for the following reasons: ( ) korea is the largest tourism market for japan; and ( ) price variables have a clear effect on tourism demand, as shown by the fact that the japanese yen (jpy) has depreciated against the korean won (krw) from the fourth quarter of to the third quarter of . the findings of this study contribute to a better understanding of price variables when forecasting international tourism demand for a specific country. in addition, because the japanese government intends to increase its annual tourist arrivals to million by when japan hosts the tokyo olympics (ong, ) , identifying determinants of tourism demand for japan is of great importance for both policymakers and tourism practitioners. tourism demand studies fall into two categories: qualitative and quantitative (peng, song, & witt, ) . quantitative demand studies are dominant in the tourism demand literature (song & turner, ) and two forecasting approaches are the most commonly used: timeseries and econometric (song & li, ) . the time-series approach is useful in that estimation procedures are relatively simple and only one data series is needed for estimation (peng et al., ) . in this approach, demand forecasting is performed by analyzing the patterns of past demand movements and then, from that, predicting future movements (song & li, ) . on the other hand, the econometric approach predicts tourism demand based on the causal relationship between dependent (e.g. tourists) and explanatory (e.g. income) variables with sound theoretical basis (peng et al., ) . the empirical usefulness of this approach lies in identifying which factors contribute most to tourism demand song, witt, and li, ). in econometric tourism demand studies, several economic factors have been found to be significant determinants of international tourism demand (prideaux, ) . based on classic economic demand theory (i.e. the higher the prices of goods and services, the lower the demand for those products), tourism price has been commonly used in demand models as a primary determinant (hui & yuen, ; uzama, ). according to crouch ( ) , witt and witt ( ) , and webber ( ), tourism price consists of living cost plus transport cost. living cost, in turn, has two components: prices of tourism goods and services in the destination country and exchange rates. in general, prices of tourism goods and services have a negative relationship with tourism demand. the relationship may be sensitive to changes in domestic tourism prices in the origin country and therefore many demand models include prices of destination tourism products relative to the origin country in order to consider the cross-price effect (loeb, ; tan, mccahon, & miller, ) . because price information of tourism goods and services is generally unavailable, consumer price index (cpi) has commonly been used as a proxy for relative prices (akis, ; lee, ; morley, ; muchpondwa & pumhidzai, ) . furthermore, actual prices of a destination's tourism products have not been found to be clearly superior to cpi in accounting for tourism demand (martin & witt, ) . although cpi is most commonly used for relative prices (akis, ) , gil-pareja et al. ( ) included purchasing power parity in their demand model instead of cpi and garín-muñoz ( ) developed her own tourism price index. many studies have proved that tourism demand is significantly responsive to changes in relative prices (eilat & einav, ; hiemstra & wong, ; patsouratis, frangouli, & anastasopoulos, ; qu & lam, ; webber, ) . for example, webber ( ) identified the determinants of tourism demand for australia in eight tourism markets using engle and granger's procedure and johansen's procedure. the results showed that relative prices had a significantly negative effect on tourist arrivals from six of the eight countries, all but malaysia and the u.k. hiemstra and wong ( ) investigated the outbound tourism from seven major countries to hong kong between and . in the study, autoregressive estimation was employed for seven tourism demand models and relative prices were included only in the demand model for australia-hong kong. the results of the study provided clear evidence of a significant effect of relative prices on tourist arrivals from australia to hong kong. on the other hand, muchpondwa and pimhidzai ( ) found no link between relative prices and tourism demand. lee et al. ( ) showed that the significant effects of relative prices on international tourism demand varied depending on the origin country to country indicating mixed results. exchange rates, the other indicator of living cost, have also been frequently examined in tourism demand studies (e.g., de vita & kyaw, ; di matteo & di matteo, ; dritsakis & gialetaki, ; edward, ; wang, ; yap, ) . when the currency of a country devalues, its tourism becomes more price competitive and therefore travel demand for the country is likely to increase (de vita & kyaw, ) . conversely, as the value of a country's currency rises, the decreased price competitiveness of its tourism results in a reduction in inbound tourism (de vita, ) . however, this view has not always been borne out in international tourism markets. some researchers (e.g. di matteo & di matteo, ; eilat & einav, ; hiemstra & wong, ; roselló-villalonga, aguiló-pérez, & riera, ; wang, ) have found evidence that exchange rates have a significant effect on tourism demand, while others (e.g. hui & yuen, ; muchapondwa & pimhidzai, ; webber, ) failed to find such evidence. in addition, lee ( ) developed several demand models for inbound tourism to korea and the results of exchange rate were inconsistent. these results indicate that the effects of exchange rates are asymmetric across countries and even across different markets in a single country. it has generally been held that relative prices and exchange rates should be considered in tourism demand models, but it remains controversial whether these two components of living cost should be examined separately or combined as effective relative prices (durbarry & sinclair, ; gray, ; tan et al., ) . according to song and witt ( ) , price changes in a destination country can be calibrated by movements in exchange rate. therefore, the destination price level that potential foreign tourists pay attention to is relative prices adjusted by exchange rates. based on this argument, many researchers (e.g. chang, khamkaew, & mcaleer, ; divisekera, ; garín-muñoz, ; kliman, ) convert relative prices into the currency of an origin country in tourism demand equations. numerous studies (e.g. de vita, ; divisekera, ; durbarry & sinclair, ; hiemstra & wong, ) have argued for the significance of effective relative prices. tan et al. ( ) concluded that effective relative prices were a better measure of living costs for malaysian and indonesian tourism. in contrast, eilat and einav ( ) and o'hagan and harrison ( ) argue that relative prices and exchange rates should enter separately into tourism demand models because it is highly likely that tourists will have more up-to-date information on exchange rates than on relative prices. accordingly, they are more likely to use exchange rates to estimate cost of travel to the destination. moreover, it has been noted that responsiveness of tourists to exchange rates is distinctive from their responsiveness to changes in relative prices (gray, ; lee, ; muchapondwa & pimhidzai, ) . with this in mind, many researchers (e.g. de vita & kyaw, ; di matteo & di matteo, ; eilat & einav, ; edward, ; wang, ) have included exchange rates as a separate variable in their tourism demand equations. transport cost is also a component of tourism price. as transport cost accounts for a large proportion of total travel cost, an increase in transport cost tends to negatively affect destination choice with a subsequent decline in tourism flows (covington, thunberg, & jauregui, ; divisekera, ) . airfares, oil prices, or distance have been typically used as proxies for transport cost (durbarry & sinclair, ; kulendran & witt, ; song & witt, ) . kulendran and witt ( ) compared the forecasting performance of cointegration and least-squares regression models, indicating that transport cost represented by airfares significantly influenced outbound tourism flows from the u.k. nelson, dickey, and smith ( ) conducted time-series and cross-section analyses to investigate tourism from the us mainland to hawaii. airfare was found to be a significant determinant of tourism demand for hawaii. since transport cost changes as a function of fuel costs and geographical distance, oil prices and distance between two countries have been added to demand models as a proxy for transport cost (hiemstra & wong, ) . for example, wang ( ) employed the autoregressive distributed lag model to identify the determinants of taiwan's inbound tourism, particularly focusing on the effect of disastrous events. the study reported that oil prices had a significantly negative effect on taiwan tourism indicating that foreign tourists are less likely to visit taiwan as transport cost rises. de vita ( ) used a distance variable to approximate transport cost through a gravity-type tourism demand equation and found distance was significant on tourism demand for countries. the popular use of gravity models in tourism demand studies has allowed extensive investigation of the explanatory power of distance. such studies include eryiğit, kotil and eryiğit ( ) , park and jang ( ) , and uysal and crompton ( ) . however, oil prices and distance may be inappropriate for representing transport costs since neither variable considers the other, but both are main elements of the fuel cost function. airfares are also limited due to complex fare structures and lack of data availability (song & witt, ; webber, ) . as one contributor to the increasing trend in international tourism, japanese inbound tourism has increased from . million arrivals in to . million arrivals in (japan national tourist organization, ). despite occasional crisis events (e.g. the / terrorist attacks, the outbreak of severe acute respiratory syndrome (sars), the meltdown of the fukushima nuclear power plant), japanese inbound tourism continues to grow. from to , the annual growth rate was . % on average and the figure has accelerated in recent three years averaging . % along with considerable depreciation of the currency ( . %). as a result, japan has the sixth largest international tourism share among asian countries (world tourism organization, ) . three east asian countries -china, korea, and taiwanaccounted for approximately % of japan's inbound tourism market in terms of number of tourist arrivals (japan national tourist organization, ). among these countries, korea has been the biggest market for japan for the last two decades. korean tourists to japan has increased from . million in to . million in (see fig. ) and their share of the japanese inbound tourism market was between . % and . % (japan national tourist organization, ). despite the importance of korea in the japanese tourism industry, few studies have examined the determinants of the korean demand for japan tourism. there has been extensive exchange between korea and japan and in various areas such as economy, culture, politics, and diplomacy because of their geographic proximity. the major presence of korea in japanese tourism may be a result of the strong ties established in the various areas (lee, song, & bendle, ) . moreover, due to geographic proximity and the strong ties between the two countries, koreans are likely to be aware of japanese economic conditions and thus reasonably responsive to changes in japanese tourism price. accordingly, the impact of the different price variables of japan tourism, whether positive or negative, can be readily captured in relation to korea. this study developed tourism demand models from korea to japan between the first quarter of and the fourth quarter of . the dependent variable (rtour t ) was the number of korean tourists divided by the total population of korea. the use of population as a deflator was done to control for the increase in korean tourists caused merely by population growth. based on classic demand theory in which demand is a function of product price and individual income, two economic variables influencing demand for international tourism were added to the models: per capita income and tourism price. as the most important determinant of international tourism, per capita income is a measure of individual spending power. an increase in individual income is likely to lead to greater spending power creating tourism demand. this variable was calculated as the gross domestic product (gdp) of korea divided by its population and then converted into real per capita income (rincome t ) by dividing per capita income by korean cpi. to investigate the price effect on japanese inbound tourism from korea, this study used relative prices, effective relative prices, exchange rates, oil prices, and jet-fuel prices. for the selection of adequate price variables, this study developed and compared six demand models with different price variables as follows: the first model included relative prices and exchange rates separately to measure living cost in japan. relative price (rprc t ) was calculated as the ratio of japanese cpi to korea cpi and exchange rate (exc t ) was measured as the value of jpy per krw. the second model added oil prices as a proxy for transport cost. real oil price (roil t ) was computed by dividing crude oil prices per gallon by korean cpi. the third model used jet-fuel prices as a proxy for transport cost instead of oil prices. despite the importance of distance in transport cost functions, oil price variable does not reflect the distance between two countries. in addition, crude oil may have different price movement from the jet fuel actually used by aircraft. thus, this study developed a variable of jet-fuel prices as new proxy for transport cost, since it takes into account both distance and actual fuel price. real jetfuel price (rjet t ) was calculated as (jet-fuel prices per gallon×(distance/average fuel efficiency per seat))/korean cpi. in the fourth model, a combined variable, effective relative price (rprc t ), was used to represent living cost. effective relative price (erprc t ) was calculated by adjusting relative price (rprc t ) with exchange rate (exc t ). the fifth model added real oil price (roil t ) to the explanatory variables used in the fourth model. in the sixth model, real jet-fuel price (rjet t ) replaced real oil price (roil t ) used in the fifth model. in addition to the economic variables, five binary dummy variables were included in our models to take into account the effect of special events. to account for the impacts of radiation risk in fukushima and the visa-free entry (vfe) program, two dummy variables (dm nuclear , dm vfe ) were added. since nuclear leakage is a continuing concern and the vfe program is still in place, these dummy variables were coded as one after their starting date ( were added to control for seasonal effects. the base group was the fourth quarter. the expected signs for the variables were: rexc t , rincome t , dm vfe , dm season > and rprc t , erprc t , roil t , rjet t , dm wcup , dm sars , dm fcrisis , dm nuclear < . as in most previous tourism demand studies, our models used a double log-linear function. the double log specification allows researchers to interpret the coefficients as elasticity. preliminarily, serial correlation of the six models was assessed using the durbin-watson test and breusch-godfrey test. because both of the tests found serial correlation in all models, this study employed prais-winsten estimation, one type of feasible generalized least squares (fgls), to estimate the six demand models. the study period was from the first quarter of to the third quarter of . quarterly number of korean tourist arrivals was obtained from the japan national tourist organization ( ). for computation of explanatory variables, economic indices were acquired from the databases of the organization for economic co-operation and development ( ) table . while pearson correlation analysis was performed for continuous variables, the pointbiserial correlation method was applied to the pairs of continuous and binary variables. phi correlation was used when both variables had binary values. point-biserial correlation and phi correlation are equivalent to pearson correlation (gould & rogers, ; revelle, ) . the results showed that none of the explanatory variables were highly correlated indicating that the estimated models did not have multicollinearity problems which can reduce the overall data fit of a model and increase the variance of coefficients, decreasing the model's statistical power (hair, black, babin, & anderson, ) . table summarizes the results of model estimation. the results showed that all six models explained more than % of the variance in korean tourist arrivals to japan. in terms of r , models , , and which treated relative price (rprc t ) and exchange rate (exc t ) separately appeared to provide a better fit to the data than models , , and which used effective relative price (erprc t ). model and model or were nested because model could be obtained from model or . model and model or were nested in the same way. model (or model ) was the restricted model for the full models of models and (or models and ). based on these relationships, rigorous model selection analysis was performed. using a partial f-test, we checked whether the restricted models without a transport cost variable showed a significant difference in overall model fit to data compared to the full models. the partial f-test results revealed no significant differences in overall fit between all nested model pairs (see table ). this indicates that the removal of transport cost variables from the full models does not significantly change the explained variance in japanese inbound tourism from korea. in terms of model parsimony, model was better than models and , and model was better than models and . since a partial f-test can be used only for nested models, this study employed akaike information criterion (aic) to compare nested and un-nested models together (burnham & anderson, ; cavanaugh, ) . the results of aic presented in table showed that the models using relative price (rprc t ) and exchange rate (exc t ) had relatively lower aic than the models with effective relative price (erprc t ). among the models using relative price (rprc t ) and exchange rate (exc t ), the aic of model was the lowest, suggesting that model offers the best fit to the data, having the smallest information loss. since model selection analysis identified model as the best model, this study presents the coefficient estimation results from model . the living cost variables represented by relative price (rprc t ) and exchange rate (exc t ) accounted for a significant amount of the changes in japanese inbound tourism from korea. the coefficients show that a % decrease in relative price (rprc t ) results in a . % increase in japanese inbound tourism from korea and that a % depreciation in jpy (or a % appreciation in krw) leads to a . % increase in japanese inbound tourism from south korea. real per capita income (rincome t ) also had a significant impact on international tourism demand to japan from korea: a % increase in koreans' individual income results in a . % increase in tourism demand for japan. the coefficients of dummy variables for the special events had the expected signs except for the dummy for world cup. however, only the dummies for the financial crisis (dm fcrisis ) and the fukushima nuclear disaster (dm nuclear ) had a significant effect on japanese inbound tourism from korea. seasonal dummy variables were also significant. specifically, the dummies for the first and the third quarters were significantly positive, implying that korean tourists visit japan mainly in summer and winter. this study compared six tourism demand models, each with different price variables, to identify proper price variables affecting japanese inbound tourism from korea. partial f-test based on the nested (or hierarchical) relationship of demand models and aic analysis were used for model comparison. the results of this study showed that separate inclusion of relative prices and exchange rates was more effective in accounting for the changes in japanese inbound tourism from korea than a price variable combining these two price indicators. model comparison results also showed that exclusion of a transport cost variable did not decrease the explanatory power of the models. this study has several theoretical, methodological, and practical implications. in terms of theory, the results confirm the view that exchange rates should be treated separately in tourism demand models because exchange rates are often used for destination selection apart from relative prices (muchapondwa & pimhidzai, ; o'hagan & harrison, ; witt & witt, ) . some researchers (gray, ; lee, ) argue that the price level actually recognized in origin note. rtour t is the dependent variable for all six models. *p < . ; **p < . . results of partial f-tests. note. model is the restricted model for the full models of models and , and model is for models and . countries is highly dependent on exchange rates and therefore foreign tourists' responses to changes in exchange rates are very different from their responses to relative prices. this view is supported by the argument that when potential tourists cannot obtain updated information on tourism prices, they tend to use the exchange rates, which is readily available to the public (muchapondwa & pimhidzai, ; webber, ) . considering this, it is notable that exchange rates are an important consideration in destination selection even though koreans may be well aware of the prices of japanese tourism products due to the two countries' proximity and sustained active relationship. this suggests that it is not easy, even between closely associated countries, for foreign consumers to gain knowledge of destination prices or accurately update their existing price knowledge over time. either case may lead them to use the current exchange rates in their travel decisions. thus, this study's significant contribution is to extend existing tourism demand literature by empirically identifying two price variables used for tourism destination choice in the korea-to-japan context. in the identified model, per capita income was significant on japanese tourism demand from korea. this result is consistent with previous studies (e.g. garín-muñoz, ; lee, ; webber, ) which found this variable to be an important determinant of tourism demand. two separate price variables were also significant determinants. the significant results for relative prices, exchange rates, and per capita income support the application of the classic demand theory to japanese inbound tourism. the results also indicate that the demand for japan tourism by koreans can be effectively estimated without considering transport cost. this implies that changes in transport cost do not affect japanese inbound tourism from korea. two different measures of transport cost, oil price and jet-fuel price, were indeed found to be unrelated to changes in japanese inbound tourism from korea in the full models. the results suggest that tourists planning short-haul international travel are less likely to take into account the cost of transport to their destination. as a result, caution should be exercised in including transport cost in modeling the demand for shorthaul international travel. in addition to the theoretical implications previously discussed, this study has important methodological implications. in an attempt to identify proper price variables, this study demonstrated model comparison methods for nested models (partial f-test and aic) and for unnested models (aic) (bowerman & o'connell, ; burnham & anderson, ) . by developing new measures of transport cost which consider actual aircraft fuel prices and the distance between two countries, this study tried to address the limitations of using oil price and distance as a sole proxy for transport cost. practical implications are also evident from the results of this study. this study found evidence for the significance of per capita income. in other words, outbound tourism from korea to japan is sensitive to movements in the korean economy. this suggests that economic growth in korea generates a significant increase in the korean demand for tourism to japan. although destination countries have limited ability to influence per capita income of inbound tourists, since it is mainly affected by economic conditions in origin countries, destination countries can still take advantage of this characteristic. for example, when japanese inbound tourism from korea shrinks due to a local or global recession, japan's tourism industry could counter this loss by reallocating their promotional resources to tourism markets experiencing an economic boom or less economy-sensitive markets. to implement this strategy, japan would need to monitor economic conditions in korea and its other major tourism markets such as china, taiwan, and the us. as discussed earlier, relative prices and exchange rates had a significant effect on japanese inbound tourism demand from korea. combined with the results for other explanatory variables in the most appropriate model, this shows that relative prices contribute the most to japan's inbound tourism in terms of elasticity and indicates that prices of tourism products are the most important factor in the choice of destination by tourists. given that prices of tourism products are manageable by destination country or tourism industry to a certain extent, and the importance of price in attracting international tourists to japan, a solid pricing strategy is critical for the japanese tourism industry to maintain destination competitiveness. some researchers (e.g. bowen, ; griffin, shea & weaver, ; papadopoulos, ) have emphasized the importance of the price competitiveness of japanese tourism products with those of neighboring countries such as china and korea, which provide relatively cheap tourism products. for price competitiveness, the relentless efforts of the private sector, including cost reduction, new technology adoption, and research and development, should be made on a continuous basis (blake, sinclair, & soria, ; dwyer & kim, ) . in the same vein, government is of great importance in providing sustainable price-competitive tourism products. the recent jump in tourist arrivals to japan, for example, was triggered by a sharp decline in the value of jpy resulting from japanese prime minister shinzo abe's economic policy known as 'abenomics' (ong, ) . this indicates that government economic policy is likely to influence tourism demand by altering economic conditions, which are responsible for the prices of tourism products (edward, ; eilat & einav, ) . furthermore, governments could offer tax credits for international tourists' expenditures (dimanche, ) and tax incentives to tourism firms which undertake extensive overseas marketing campaigns, hold international meetings, and renovate or replace their facilities (blake & sinclair, ; mckehee & kim, ) . great care must therefore be taken in formulating tourism and economic policies in order to promote international tourism through price competitiveness and stability. as with many other tourism demand studies, this study is not without limitations. first, the relatively small subject pair and sample observations may limit the generalizability of the results. second, although none of the explanatory variables could be excluded due to multicollinearity, the variables may not be totally free from the multicollinearity problem given that significant correlations exist between the variables. one way to overcome the first limitation would be to use panel data. examining the relationship between different price variables and tourism arrivals in the context of multiple tourism markets would improve the generalizability of the variable selection results. for the second limitation, it would be worthwhile employing dynamic factor analysis, a time-series extension of factor analysis proposed by geweke ( ) . by applying factor analysis to multivariate time series data, dynamic factor analysis can address the correlation between explanatory variables 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arrivals tourism statistics a quantitative analysis of canadian overseas tourism cointegration versus least squares regression modeling tourism demand: a decision rules based approach major determinants of international tourism demand for south korea: inclusion of marketing variable the impact of visa-free entry on outbound tourism: a case study of south korean travelers visiting japan the forecasting of international expo tourism using quantitative and qualitative techniques determinants of inbound tourist expenditures recent developments in econometric modeling and forecasting a meta analysis review of international tourism demand international travel to the united states: an econometric evaluation substitute prices in models of tourism demand motivation for agri-tourism entrepreneurship the use of cpi for tourism prices in demand modelling modelling international tourism demand for zimbabwe estimating time series and cross section tourism demand models: mainland united states to hawaii data market shares of us tourist expenditure in europe: an econometric analysis japan market update: abenomics year on opportunities in tourism. international enterprise singapore a conceptual tourism marketing planning model: part an extended gravity model: applying destination competitiveness competition in tourism among the mediterranean countries demand modeling and forecasting factors affecting bilateral tourism flows a travel demand model for mainland chinese tourists to hong kong point-biserial correlation modelling tourism demand dynamics tourism demand modeling and forecasting: a review of recent research international handbook on the economics of tourism http:// dx tourism demand modelling and forecasting econometric approaches uk: routledge forecasting international tourist flows to macau the advanced econometrics of tourism demand london: routledge resources oxford handbook on economic forecasting http:// dx modeling tourist flows to indonesia and malaysia tourism highlights bureau of transportation statistics determinants of demand for international tourist flows to turkey marketing japan's travel and tourism industry to international tourists the impact of crisis events and macroeconomic activity on taiwan's international inbound tourism demand. tourism management outdoor adventure tourism: a review of research approaches appraising an econometric forecasting model forecasting tourism demand: a review of empirical research modelling the spillover effects of exchange rates on australia's inbound tourism growth key: cord- -ihh q f authors: nan title: posters p - p date: - - journal: eur biophys j doi: . /s - - -x sha: doc_id: cord_uid: ihh q f nan in order to understand mrna stability, we proceed to the studie of life time model system composed of the regb ribonuclease and the ribosomal protein s . the t bacteriophage life cycle is modulated by regb which mediate speci c mrna degradations. regb cleaved the mrna at the middle of the translation initiation region (shine-dalgarno sd). studies had shown that regb activity is enhanced with addition of s which is normaly required for the translation of mrna in case of unusual sd regions. s is considered as a key factor of translation's inititiation. composed of six similar domains (f -f ), it interacts with the ribosome by his f -f domains and with the mrna by f -f . we had shown that the f fragments got the same properties than the whole protein. many global architectures had been published (linear or globular conformation). then we try to understand what is the catalysis way of s in the regb activation and in rna recognition. we study the global conformation of f fragment. for that, we use nmr to determinate the domains interfaces. at rst we made the nmr backbone assignement of n/ c/ h labeled fragments (f -f ). then we analysed hsqc overlapping of each fragments in order to identify interfaces residues. the residues identi cation on s homologous model allowed us to determinate interaction region between domains. currently we study of s -rna interactions to determinate if the ligand conformation could change the interaction region and involve conformationals changes. translocation of amino acids from the membrane interface to the interior: theory and experimiment c. aisenbrey , e. goormaghtigh , j.-m. ruysshaert , b. bechinger ulp/cnrs, chemistry, rue blaise pascal, strasbourg, université libre, campus plaine cp / , brussels the interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total re ection fourier transform infra red (atr-ftir) and oriented solid-state nmr spectroscopies. related peptides have previously been shown to exhibit antibiotic and dna transfection activities. the -residue lah x and lah x peptides were designed in such a manner to form amphipathic helical structures in membrane environments. four histidines and four/six variable amino acids x constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. the dichroic ratio of atr-ftir spectra, or the orientation-dependent n solid-state nmr chemical shift have been used to follow the ph-dependent transition from in-plane to transmembrane alignments upon increase in ph. a theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the gibbs free energy of transition. the novel concept provides access to the free energy changes associated with the amino acids x incorporated into an extended -helix and in the context of phospholipid bilayers. for the peptides of the lah x series the gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: l < a i < s f < t g < v w << y. here we present a novel solid-state nmr approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes. the method works with transmembrane or in-plane oriented peptides that have been labelled with , , - h -alanine and n-leucine at two selected sites. proton-decoupled n and h solid-state nmr spectroscopy at sample orientations of the membrane normal parallel to the magnetic eld direction have been used to characterize the tilt and rotational pitch angle of these peptides in considerable detail. the same samples when inserted into the magnetic eld at degrees tilted alignments provide valuable information on the rotational diffusion constants in membranes and thereby of the association and size of peptide complexes within the membrane environments. whereas monomeric transmembrane peptides exhibit spectral averaging and well-de ned resonances, larger complexes are characterized by broad spectral line shapes. in particular the deuterium line shape is sensitive to association of a few transmembrane helices. in contrast, the formation of much larger complexes affects the n chemical shift spectrum. aisenbrey ch. & bechinger, b. biochemistry , - ( ) a meccano set approach of joining trpzip a water soluble -hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide p. chetal, v. chauhan, d. sahal international centre for genetic engineering & biotechnology,new delhi ,india a residues long, water soluble, monomeric -hairpin peptide "trpzip" [cochran et al ( ) pnas , - ], stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine ( f) containing helical octapeptide. circular dichroism studies of this residues long peptide, "trpzipalpha" (ac-gewtwddatktwtwte-gggg-fal fal fa-nh ) in water have revealed the presence of both the -hairpin and the helical conformations. this is the rst instance where a f containing peptide has been found to display a helical fold in water. the uorescence emission wavelengths of tryptophan in ac-g-w-g-nh , trpzip and trpzipalpha were . , . and . nm respectively. the uorescence quantum yield of trpzip was . fold higher than trpzipalpha suggesting that proximal interactions between the -hairpin and the helix caused the quenching of tryptophan uorescence in the former by the fs in the latter. the molar ellipticity of the far uv couplet characteristic of trpzip was reduced in trpzipalpha and the cd based thermal melting temperatures at nm were c (trpzip) and c (trpzipalpha). a concentration dependent variable temperature cd study in water showed that in trpzipalpha, increasing temperature is detrimental to the -hairpin, but it augments the helical motif by intermolecular oligomerization. our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. structural-based differential stability in the yoeb-yefm toxin-antitoxin module i. cherny, e. gazit department of molecular microbiology and biotechnology, faculty of life sciences, tel aviv university, tel aviv , israel the speci c physiological role of natively unfolded proteins is only recently beginning to be explored. a notable case in which natively unfolded state appears to have physiological signi cance is the e. coli yoeb-yefm toxin-antitoxin (ta) module. a crucial element in proper functioning of ta systems requires physiological instability of the antitoxin in contrast to the stable pro le of its toxin partner. we have shown that yefm antitoxin is a natively unfolded protein, lacking secondary structure even at low temperatures. in contrast, its toxin partner has a well-folded conformation at physiological temperatures. we suggest that the structural-based differential thermodynamic stability between the two components is the cause for their differential physiological stability, since structural instability of the antitoxin exposes it to cellular quality-control machinery. we further revealed that yefm and yoeb interact and form tight complex and determined it stoichiometry. a potential use of ta systems is as novel antibacterial targets. indeed, we identi ed homologous yefm-yoeb systems in a large number of bacteria including major pathogens. we aim to design peptides capable of interfering with the yefm-yoeb interaction, thus releasing the toxin to execute its detrimental function. for this purpose, we identi ed a short linear determinant within yefm that is involved in yoeb interaction. this peptide motif will be optimized for development of antibacterial lead molecules. a. chatterjee, a. prabhu, a. ghosh-roy, r. v. hosur tata institute of fundamental research, mumbai, india- dynein light chain (ddlc ), a member of the cytoplasmic motor assembly exists as a monomer or a dimer (functional form) under different experimental conditions. here we report the unfolding characteristics of the monomeric ddlc at ph , due to urea and guanidine hydrochloride, by various biophysical techniques. it is observed that the unfolding pathways due to the two denaturants have many differences. urea unfolding seems to be two state, while guanidine unfolding is more complex. the nmr experiments carried out at low denaturant concentrations have enabled detailed characterization of the structure and dynamics of the near native excited states of the protein. these are similar to the native state in structure, except for the small extensions of the helices in the nterminal half of the protein. however, the local stabilities of the and -strands are perturbed and this occurs differently in the two denaturants. in the guanidine case the entire multi-stranded -sheet in the c-terminal half is destabilized. in either case the motional characteristics, seem to suggest the presence of a nite population of the dimer in the excited state ensemble. these states are suggested to be likely intermediates in the momoner-dimer transition, and their characterization here thus provides clues to the molecular mechanism of the transition. it is also envisaged that the near native excited states could play regulatory roles in the functioning of the protein. kinetic bottlenecks identi cation in different folding models f. cecconi , c. guardiani , r. livi infm -istituto di sistemi complessi -cnr, italy, centro interdipartimentale per lo studio delle dinamich complesse, università di firenze, italy, dipartimento di sica, università di firenze, italy the ww domains are a family of fast folding, compact, modular domains featuring a triple-stranded, antiparallel beta-sheet. the ww domain of the pin protein, due to the availability of a complete picture of the residues involved in thermodynamic stability and in the formation of the transition state, in particular, represents an excellent benchmark to test computational methods. the objective of the present work is to identify the kinetic bottlenecks in the folding process through md unfolding simulations at increasing temperatures. the kinetic bottlenecks are related to the establishment of contacts requiring the overcoming of a large entropy barrier and acting as a nucleus for the creation of further contacts. the key sites are therefore those involved in contacts showing a dramatic decrease in fractional occupation near the speci c heat peak. the technique was applied to the go model and to a model based on the knowledge of secondary structure, providing in both cases a picture of the folding process consistent with the experimental data. evidence is also shown that while the go model allows a more accurate prediction of the native structure, the folding pathway is better described by the other model. the protein talin plays a key role in coupling the integrin cell adhesion molecules to the actin cytoskeleton and in integrin activation. the globular head of talin, which binds -integrins, is linked to a rod containing an actin-binding site and binding sites for the protein vinculin, which regulates the dynamic properties of cell-matrix junctions. we have determined the structure of three domains which contain vinculin binding sites (vbss) and shown that each of these are made up of helical bundles. the structures of complexes between vinculin and peptides corresponding to the vbss show that the residues which interact with vinculin are buried in the hydrophobic core of the helical bundles of the talin domains. nmr studies of the interaction of one of these domains with vinculin shows that it involves a major structural change in the talin fragment, including unfolding of one of its four helices, to make the vbs accessible. while the observation of folding of unstructured regions of a protein on interaction with a 'partner' is quite common, this kind of major unfolding to permit a protein-protein interaction is much less common. ways in which it may be regulated will be discussed. conformational changes of eye lens proteins studied by combined saxs and high pressure s. finet , f. skouri-panet , a. tardieu european synchrotron radiation facility, rue horowitz, bp , grenoble france, institut de minéralogie et de physique des milieux condensés, rue de lourmel, paris france, protéines: biochimie structurale et fonctionnelle, case , quai st-bernard, paris france -, -and -crystallins are the main components of vertebrate eye lenses, with exceptional structural and associative properties. the crystallins are known to be exceptionally stable in vivo since they have to last the lifetime of the organism. they therefore represent an extreme case of stability versus unfolding and aggregation. these proteins are mainly beta strands. -crystallins are kda monomers (from to % sequence identity), and -crystallins are large hetero-oligomers of about kda. -crystallins are molecular chaperone; they belong to the ubiquitous superfamily of small heat shock proteins, shsps. here, the conformation and the stability of -and -crystallins were investigated by small angle x-ray scattering (saxs) and high pressure, depending upon temperature and ph. at room temperature, -crystallins have shown a partially reversible change in size from to kb, and this effect was enhanced by the combination of temperature and pressure. in the case of -crystallins and in the pressure range up to kb, the pressure was combined with temperature and ph. the results depend upon the different itself. structural studies on pore-forming peptides s. m. ennaceur , d. bown , j. m. sanderson chemistry department, university of durham, biology department, university of durham the resistance of pathological microorganisms to conventional antibiotic drugs has created a need for new antibacterial agents. biologically active antimicrobial peptides that act as primary defense agents in a large variety of species are thought to have the potential as precursors for a new range of drugs from antibiotics to cancer treatments. this study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: we have successfully synthesised model membrane peptides with a beta-sheet structural motif and have used a wide range of techniques to analyse their interactions with phospholipid (pc) membranes. the synthetic peptides were very hydrophobic and only soluble in uorinated alcohols such as hfip and to a lesser extent tfe. we found hfip to have a very strong af nity for pc membranes and carried out a series of experiments to investigate this af nity. binding of hfip to pc membranes was found to be reversible and we exploited this property in d crystal trials of our synthetic peptides. we over expressed the c-terminal domain of brka, a gram negative autotransporter protein, which forms a beta-barrel channel in the outer membrane (omp), for comparison with our model peptides. we performed d crystal trials on the omp and imaged the resulting protein arrays by stem and afm. a protein spontaneously folds into a unique native structure in physiological conditions. this process accompanies a huge loss of the conformational entropy (ce). our major concern is to specify the factor that can compete with the ce loss. the previous discussions concerning protein folding have been focused on contributions to the free energy of folding from the interaction potentials in a system. a view lacking in earlier studies is that the folding is critically in uenced by the translational movement of water molecules. when solute particles contact each other in a solvent, the excluded volumes for the solvent molecules overlap, and the total volume available to their translational movement increases. this leads to a gain in the translational entropy (te) of the solvent. this type of te effect should be much stronger in protein folding where the tight packing of the side chains occurs. an elaborate statistical-mechanical theory is employed to analyze the te of water in which a peptide or a protein molecule is immersed. it is shown that the te gain upon folding is large enough to compete with the ce loss. when water is replaced by another solvent whose molecular size is larger, the te gain decreases to a remarkable extent. we suggest that the entropic loss accompanying the self-assembly and the formation of ordered structures in a living system is compensated mostly by the te gain of water, highlighting an aspect of the crucial importance of water in sustaining life. domain ii of ribosome recycling factor is required for disassembly of the post-termination complex p. guo, l. zhang, y. feng, g. jing institute of biophysics, chinese academy of sciences, national laboratory of biomacromolecules, beijing, china ribosome recycling factor (rrf) consists of two domains and, in concert with elongation factor ef-g, triggers dissociation of the post-termination ribosomal complex. however, the exact function of the individual domains of rrf remains unclear. to clarify this, two rrf chimeras, ecodi/ttedii and ttedi/ecodii, were created by exchanging the rrf domains between the proteins from escherichia coli and thermoanaerobacter tengcongensis. the ribosome recycling activity of the rrf chimeras was compared with their wild-type rrfs by using in vivo and in vitro activity assays. the experiments show that like wild-type tterrf, the ecodi/ttedii chimera fails to complement the rrf ts phenotype of e.coli lj (frr ts ) strain and has no polysome breakdown activity. however, under the same conditions, the ttedi/ecodii chimera complements the rrf ts phenotype and has polysome breakdown activity equivalent to that of wild-type ecorrf. the results indicate that domain ii of rrf is the functional domain that is mainly responsible for the disassembly of the post-termination ribosomal complex, and the speci c interaction between rrf and ef-g on the ribosomes mainly depends on the interaction between domain ii of rrf and ef-g; while domain i of rrf is the main contributor for binding ribosomes and maintaining the stability of the rrf molecule. this study provides direct genetic and biochemical evidence for the assignment of individual functions of rrf domains. self-assembly of natural somatostatin into liquid crystalline nano brils w the natural neuropeptide somatostatin- is a cyclic tetradecapeptide hormone, with broad inhibitory effects on both endocrine and exocrine secretions. we report the self-assembly of somatostatin in solution, into stable liquid crystalline nano brils, based on the neuropeptide bioactive backbone conformation. the system was studied as a function of peptide concentration, milieu composition and time, using optical and electron microscopy, x-ray scattering, vibrational spectroscopy and sec/rp-hplc. in pure water, the formation of twisted nano brils (around nm wide and a few microns long) was characterized. their structure relies on the native somatostatin -hairpin and on intermolecular antiparallel -sheets networks. the nano brils were observed to laterally associate further with increased concentration and time, as well as to generate hexagonal phases. increase in ionic strength (sodium chloride, phosphate) was found to signi cantly favor the self-association process. the soft conditions of formation of the somatostatin nano brils support biological relevance, for instance to the biological mechanism of storage of the neuropeptide hormone. unraveling the physical origin of the structure of fully denatured ubiquitin s. golic grdadolnik, f. avbelj national institute of chemistry, hajdrihova , ljubljana, slovenia the structure and dynamics characterization of non-native states of proteins is crucial for understanding the mechanism of protein folding. recently many experimental studies have shown variations of conformational propensity and exibility along the backbone chain of fully denatured proteins. it has been supposed that areas of residual structure may serve as initiation sites of protein folding. however, the physical origin of these variations is still unclear. we analyze the structure of fully urea-denatured ubiquitin. the experimental veri cation of conformational propensities of protein backbone is obtained through structurally dependent nmr parameters. although the secondary structure of ubiquitin under strong denatured conditions is not detectable and no correlation with the native overall topology is found, the variations of nmr parameters along the backbone follow the secondary structure elements of its native state. we show that these variations are in accord with the recently developed electrostatic screening model of denatured proteins ( ). in this model, the backbone conformations of residues in unfolded protein are determined by local backbone electrostatic interactions and their screening by backbone solvation. many virulence factors from gram-negative bacteria are autotransporter proteins. the nal step of autotransporter secretion is c nterminal threading through the outer membrane (om), followed by folding. this process requires neither atp hydrolysis nor a proton gradient. pertactin, an autotransporter from bordetella pertussis and the largest -helix structure solved to date, folds much more slowly than expected based on size and native state topology, yet folding intermediates are not aggregation-prone. equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the c-terminal half of the protein. examination of the pertactin crystal structure does not reveal the origin of the enhanced c-terminal stability. yet sequence analysis reveals that, despite size and sequence diversity, all autotransporters are predicted to fold into parallel -helices, suggesting this structure may be important for secretion. for example, slow folding in vivo could prevent premature folding of in the periplasm prior to the assembly of the om porin. moreover, extra stability in the c-terminal rungs of the -helix may serve as a template for the formation of the native protein during secretion, and formation of the growing template may contribute to the energy-independent translocation mechanism. coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. thermal and functional properties of e.coli outer membrane protein-receptor fhua fhua is e.coli outer membrane protein, which transports iron into the bacterial cell and also serves as receptor for several phages. in order to get more deep information about structural properties of fhua, we've studied its thermal properties by means of calorimetry. we've also investigated the interaction between t , ira phages and directly fhua by means of viscometry. the calorimetric result of heat denaturation of membrane protein fhua and next deconvolution of the recorded calorimetric curve with two transitions has shown that in a chosen conditions the structure of fhua consists of domains. though both t and ira phages grow on the common bacterial strain (e.coli k ho ), expressing fhua the results of viscometric investigation show that under direct interaction of phages with fhua the receptor activity of protein revealed only for t phage. therefore, we conclude that other than fhua protein serves as receptor for ira phage. it should be mentioned that the phage dna ejection process induced by receptor was observed for the rst time by us in an incessant regime. electron spray ionization mass spectroscopy (esi-ms) is a powerful tool for the investigation of the protein folding or proteins non-covalent interactions in solution since charge state distributions (csds) in esi-ms are affected by the conformational state and mass relates on the association state. we used this tecnique to inquire at different ph and different conditions the dimerization process of the porcine and bovine -lactoglobulins that share a high sequences similarity and close d structures. dimerization oflactoglobulins is reversible and involves both electrostatic and hydrophobic interactions. it was possible to detect simultaneously both the monomeric and dimeric form of the proteins in solution, pointing out the different dimerization behaviour of the two isoforms. we assessed the maximal stability of the dimeric structure at ph for the porcine protein and ph for the bovine one. moreover we showed that bovine lactoglobulin has a stronger dissociation costant than the porcine protein. further we showed that it is possible to modulate the dimerization equilibrium of the bovine isoform at ph both increasing temperature and adding methanol without inducing denaturation of the protein. a possible novel method of protein structure prediction; a. ikehata division of structural bioinformatics science of biological spramolecular systems graduate school of integrated sciences mechanism of protein folding has been mysterious since an nsen's dogma was sugested.here i would like to propose a possible novel method of protein structure prediction; origami method. the method comes from a protein backbone property of uctuation and the residue hydrophobicity. l. marsagishvili , m. shpagina , z. podlubnaya institute of theoretical and experimental biophysics ras, pushchino state university amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. despite the different nature of proteins-precursors their amyloids have common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speci c binding to congo red and thyo avin-t. amyloid deposits are observed in different diseases such as myositis, myocarditis, cardiomyopathies and others. we showed that sarcomeric cytoskeletal proteins of titin family (x-, c-, h-proteins) of rabbit skeletal muscles are capable to form amyloid brils in vitro. these proteins already contain % of beta-sheet structure necessary for formation of amyloids. the amyloid nature of their brils was conrmed by electron, polarization and uorescence microscopy. as x-, c-, h-proteins form amyloid brils easily in vitro, there is a danger of fast growth their amyloid deposits in vivo. taking into account common properties of amyloids formed by different proteins, our results clear the ways for conducting by amyloidogenesis in human organs and tissues. work is supported by rfbr grants - - , "universities of russia" . . and program of the presidium ras "fundamental sciences for medicine". probing the folding capacity and residual structures in -and -residue fragments of staphylococcal nuclease d. liu, x. wang, y. feng, l. shan, j. wang national laboratory of biomacromolecules, institute of biophysics, chinese academy of sciences n-terminal fragments of staphylococcal nuclease (snase) with different chain lengths were used as a model system in the folding study. the detailed characterization of conformational states of - and - residues snase fragments (snase and snase ) and their v w and g w mutants can provide valuable information on the development of conformations in the folding of snase fragments of increasing chain lengths in vitro. in this study, the presence of retained capacity for folding and residual structures in snase and snase is detected by cd, uorescence, ftir, and nmr spectroscopy. snase is represented as an ensemble of interconverting conformations. the uctuating nascent helix-and âsheet-like structures, localized in regions of a -a and t -v , respectively are transiently populated in snase . the native-like tertiary conformations are obtained for g w and v w and for snase in the presence of . m tmao. analysis of the results of such studies indicate that folding of snase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. thermal disruption of a spanning network of hydration water and conformational changes of elastin a. krukau , i. brovchenko , a. oleinikova , a. geiger international max-planck research school in chemical biology, otto-hahn str. , d- , germany, physical chemistry, university of dortmund, otto-hahn str. , d- , germany hydration water strongly in uences the structural and dynamical properties of biomolecules. the existence of a spanning hydrogenbonded network formed by the hydration water enables the function of biomolecules at low hydration levels. we can expect that the formation/disruption of the spanning network formed by the hydration water in solution also affects crucially the protein properties. we present the rst computer simulation study of the thermal disruption of a spanning network formed by the hydration water of a biomolecule (elastin-like peptide). this process obeys the laws of d percolation transition, similarly to the formation of a spanning water network with increasing hydration level [ ]. the spanning water network transforms into an ensemble of small water clusters with increasing temperature: it is still permanent at k and exists with probability % at k. in the same temperature interval, the conformation of the peptide changes noticeably: its radius of gyration increases sharply (by %) at about k. these two phenomena may be related to the "inverse temperature transition" at about k, where an elastin solution separates into two phases. in our simulations, the displacement of hydration water by the addition of a denaturant (urea) or by other peptide molecules causes an even stronger increase of the radius of gyration (up to % lipidic cubic phases formed by distinct water and lipid volumes provide bicontinuous d bilayer matrices that have speci c and controllable water channel sizes and large surface areas. these systems have proven to be also valuable as membrane mimetic structures, as promising matrices for controlled-release and delivery of proteins, vitamins and small drugs in pharmacological applications, and they offer a d lipid matrix for successful crystallization of membrane proteins which do not easily crystallize in bulk solution. the present study is directed towards a better understanding of the interplay between curved cubic lipid phases and the protein entrapped within their aqueous channel structures. as model systems, we have chosen a cubic ia d phase formed by an uncharged lipid, monoolein, and incorporated different proteins, such as cytochrome c, -chymotrypsin and insulin, in its narrow water channels. we show that the protein secondary structure and unfolding behaviour may be in uenced by the con nement and, vice versa, the topology of the lipid matrix may change as a function of protein size and concentration. in fact, even new cubic lipid structures may be formed that are not known in pure lipid systems. furthermore, we compare the aggregation scenario of insulin in bulk solution and in the narrow water channels of the cubic lipid matrix and discuss the differences found in terms of the geometrical limitations imposed by the con nement. after endocytosis, i.e. at acidic ph, the t domain inserts in the membrane of the target cell and helps the translocation of the catalytic domain into the cytoplasm. therefore, the t domain has a key role in the strategy of internalization of the toxin. the study of the interaction of the t domain with membranes and its ph dependence is important for a better understanding of the diphtheria toxin translocation mechanism. at least, two steps can be distinguished during the membrane insertion of the t domain. the rst step involves hydrophobic interactions with the membrane and is related to the ph-induced stabilization in a molten-globule state. in the second step, electrostatic interactions are preponderant and the ph-sensitivity comes from changes of the balance between repulsive and attractive electrostatic interactions. the role of the n-terminal part of the t domain in the second step has been investigated by studying peptides corresponding to the amphiphilic helices found in this part of the domain. the results are correlated with those obtained with a single trp mutant probing the n-terminal region of the whole domain. the translocation mechanism will be discussed in view of the physico-chemical properties of the peptides. in complex systems with many degrees of freedom such as peptides and proteins there exist a huge number of local-minimumenergy state. one way to overcome this multiple-minima problem is to perform a simulation in a generalized ensemble where each state is weighted by a non-boltzmann probability weight factor so that a random walk in potential energy space may be realized (for reviews, see refs. [ ] [ ] [ ] xas spectroscopy results show that there are two different structures of the metal binding site in the a peptide according to whether they are complexed with cu or zn ions. while the geometry around copper is suggestive of an intra-peptide binding with three histidine residues bound to the metal, the zinc site geometry is compatible with an inter-peptide aggregation mode. this result reinforces the hypothesis that assigns opposite physiological roles to the two metals, with zinc favouring and copper blocking peptides aggregation and consequent plaque formation. effect of pressure on the conformation of proteins. a molecular dynamics simulation of lysozyme a. n. mccarthy, r. grigera instituto de física de líquidos y sistemas biológicos (iflysib), conicet-unlp-cic, university of la plata, argentina the effect of pressure on the structure and dynamics of lisozyme was studied by md computer simulation at bar ( pa) and kbar using gromacs package. all-atoms (ff gmx) force eld were used for the minimization process and for all the md simulation and kept all protein bond lengths constrained (lincs algorithm). water molecules (spc/e model) were constrained using the settle algorithm. for the electrostatic forces we applied the reaction field method. lennard-jones interactions were calculated within a cut-off radius of . nm. the results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. the studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. from the analysis of secondary structures along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. the difference in solvent accessed surface (sas) with pressure shows a clear inversion of the hydrophilic/hydrophobic sas ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions. direct observation of mini-protein folding using uorescence correlation spectroscopy h. neuweiler, s. doose, m. sauer applied laser physics & laser spectroscopy, university of bielefeld, bielefeld, germany the "trp-cage" motif represents the smallest and one of the fastest folding mini-proteins known to date. the globular fold is characterized by a hydrophobic core burying a single tryptophan (trp) residue. here, we report on the direct observation of trp-cage folding kinetics using uorescence correlation spectroscopy (fcs). our method is based on the selective uorescence quenching of oxazine dyes by trp which becomes ef cient only upon contact formation between the dye and the indole moiety of trp. by sitespeci cally labeling the dye to trp-cage, temporal uorescence uctuations of the dye-peptide conjugate, caused by intramolecular contact formation between dye and trp, directly report on conformational dynamics and folding transitions of the peptide chain. in order to measure uorescence uctuations directly in solution we used fcs on a confocal uorescence microscope setup. fcs allows us to reveal conformational dynamics with nanosecond timeresolution, under thermodynamic equilibrium conditions, and in highly dilute solutions (i.e. at nano-molar sample concentrations). our method con rms microsecond folding kinetics of the trp-cage motif, previously estimated with non-equilibrium temperature-jump techniques. we further investigated stability and folding rates under denaturing conditions and at various temperatures, giving further insight into structural transitions during the folding process. identi cation and mutagenesis of a region of tnt required for the stability of tnt-tni coiled-coil evolutionarily conserved heptad hydrophobic repeat (hr) domains present in troponin subunits tnt and tni are involved in alpha helical coiled-coil formation. using recombinant peptides from fast-skeletal tnt and tni, we examined the contributions of amino acid residues within these hr domains as well as anking these domains, to the stability of the coiled-coil interaction. a series of tnt fragments were tested for their ability to form coiledcoil with tni hr domain. we show that the tnt region - , although remains outside of the coiled-coil domain, is absolutely required for the stability of the coiled-coil. interestingly, the region tnt - contains few absolutely conserved residues that are potential candidate for ionic interaction, as predicted by molecular modeling. using single point mutants we show that among all the conserved residues, residue lysine is most important in stabilizing the coiled-coil interaction, whereas others play accessory role. we propose that the lysine initiates the stabilization of the coiled-coil interaction and then the other residues acts in a zipper like fashion. magnesium promotes conformational switching of ca sensor s. mukherjee, k. v. r. chary tata institute of fundamental research the importance of mg , one of the most abundant metal ion in the cell cytoplasm, relating to the calcium sensor mechanism is demonstrated. in this study it has been shown that the amino acid long calcium binding protein from entamoeba histolytica (ehcabp) can exist in three different forms namely, the calcium-free (apo) form, the magnesium bound (apo-mg) form and the calcium bound (holo) form. these three forms have been characterized using chromatographic, calorimetric as well as various spectroscopic techniques. there is a radical difference in the stability between the calcium free and ca bound forms. the calcium free form has molten globule like characteristics. mg stabilizes the closed conformation of the apo form, where the hydrophobic core remains buried. the presence of mg signi cantly alters the calcium binding cooperativity thereby increasing the cooperativity of the conformational switching between the open and closed conformation which is an important aspect of such regulatory proteins. a structural model for the molten-globular form of apo-ehcabp and its equilibrium folding towards completely folded holo state in presence and absence of mg will be presented. intermediate states of formin binding protein ww domain: explored by replica-exchange simulation y. mu school of biological science, nanyang technological university, singapore ww domain of formin-binding protein (fbp) is a model system for beta strand folding study. although it is small, only amino residues in total, the folding kinetics of fbp ww domain proved to be biphasic. an extensive molecular dynamics-monte carlo hybrid method, called replica-exchange simulation strategy is employed to study the folding/unfolding of the fbp ww domain in explicit water model. begin with randomly chosen conformations from high temperature unfolding trajectory distributed in replicas ( different temperatures covering from k to k), the simulation lasts nanoseconds in each replica. in the end an interesting distribution of conformations shapes up. we nd that there are three distinctive subgroups, one being the unfolded conformations with rmsd averaging around Å , one being native conformations with rmsd . Å, more interestingly, the third group having rmsd Å, an intermediate folding ensemble. by checking the intermediate ensemble in detail, we nd that it is quite heterogeneous and the heterogeneity mainly comes from the exibility of the c-terminal loop region. our ndings provide a microscopic picture of folding kinetics of the ww domain: the stable intermediate states with mis-registered hydrogen bonds on the c-terminal beta strand make this peptide folding as a three-state folding model rather than a usual two-state model. h. rezaei, f. eghiaian, j. perez, y. quenet, y. choiset, t. haertle, j. grosclaude institut national de la recherche agronomique, france in pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. to get better insight into the molecular mechanisms of prpc/prpsc conversion, we studied the kinetic pathway of heat induced amyloidogenesis of the full length recombinant ovine prp (arq) at ph . . according to the size exclusion chromatography experiments, three sets of oligomers were generated from the partial unfolding of the monomer. the effect of concentration on the oligomerization kinetics was different for the three species obsreved suggesting that they are generated from distinct kinetic pathways. limited proteolysis and peptide analysis of the two best separated peaks showed a difference in the accessibility of the c-terminal domain of these two oligomers, and allowed the identi cation of regions undergoing a structural change during the conversion process. the analysis of correlations between oligomer populations as well as numerical kinetic modeling led us to propose a multi-step kinetic pathway describing the evolution of each species as a function of time. the existence of at least three distinct oligomerization pathways on one hand and the differences in the accessibility of the two puri ed oligomers on the other hand re ect the structural plasticity of the prp protein. studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. the conformational transition from the monomeric cellular prion protein prp c in -helical structure into the aggregated -sheet-rich multimer prp sc is supposed to be responsible for the so-called prion diseases. it is commonly admitted that the recombinant prp could serve as model of conversion of the normal prion protein prp c . fundamentally, the interaction of proteins with surfaces either uid or solid involves both protein binding and unfolding. our goal in studying protein adsorption is to determine the nature and the amplitude of the structural changes occurring during non-speci c adsorption. the protein-clay interaction depends on several parameters such as protein hydration, net charge, charge distribution on the protein surface. ftir spectroscopy is well-suited to probe structural changes of proteins at a molecular level at aqueous/solid interfaces. the conformational states of the full-length ovine prp adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pd range . - , using ftir spectroscopy. during the intoxication of a cell, the diphtheria toxin binds to a cell surface receptor, is internalized and reaches the endosome. the translocation (t) domain from the toxin interacts with the membrane of the endosome in response to the acidic ph found in this compartment. this process drives then the passage of the catalytic domain of the toxin through the membrane into the cytoplasm. the interaction of the t domain with the membrane involves at least two steps. in the rst step occurring at ph , t adopts a molten globule conformation, which is able to bind super cially to the membrane through hydrophobic interactions by the c-terminal region (helices th and th ). in a second step occurring between ph and , penetration into the membrane involves electrostatic interactions. this step leads to a functional inserted state. trp was mutated to phe in order to use trp located in helix th as a probe of its behavior during the interaction with the membrane as a function of ph. we found that the second step is correlated with the reorganization of the n terminal region in the membrane and is controlled by electrostatic interactions. peptide conformational search using generalized simulated annealing method p. g. pascutti , f. p. agostini , c. osthoff , k. c. mundim , m. a. moret ibccf -ufrj -brazil, lncc -brazil, iq-unb -brazil, ceppev -fundação visconde de cairú / df-uefs -brazil the three-dimensional structure of proteins is mainly determined by the sequence of amino acids, making possible the development of ab initio methods for peptides and protein structure prediction. we proposed a stochastic method based on a classical force field and the generalized simulated annealing (gsa), which utilizes tsallis generalization of boltzmann-gibbs statistics. we have applied this method for peptide conformational search and as a complement for comparative modeling of proteins, searching the conformation for loops and mismatched sequence alignments. the gsa ef ciency depends on the right choice of the parameters involved in the conformational calculation: the qv parameter which de nes a function for visiting the molecular energy surface; and the qa parameter de ning an acceptance probability, both according with tsallis statistics. to avoid the conformational trapping in local energy minima we introduced a new parameter in this work, qt, to control the temperature decreasing. to investigate the qv, qa and qt best parameters set, we used the -alanine and -alanine peptides, which have a known alpha-helical structure in low dielectric environment. the global minimum energy occurs for the alpha-helix folded structure, and was found for qv ranging from . to . , qa from . to . , and qt from . to . . we observed also that convergence values for qv decrease while for qa and qt increase for folded structures. p. s. santiago, É. v. de almeida, m. tabak instituto de quimica de são carlos-usp cp são carlos sp brasil extracellular hbgp is a giant hemoglobin, similar to other annelid hemoglobins, having a molecular weigth of . mda. the effect of ctac in the oligomeric protein structure was assessed by optical absorption and emission spectroscopies. optical absorption spectra of hbgp . mg/ml as a function of ctac concentration from up to . mm evidentiate changes both in soret band at nm, and at nm associated to light scattering which appears at low ctac concentration. below mm of surfactant extensive light scattering occurs together with signi cant shifts of max of soret band from nm to around nm, which is probably due to oxidation of the original oxyhbgp. light scattering reaches a maximum value disappearing for higher ctac concentrations. fluorescence spectra show a signi cant increase in intensity ( fold) upon titration with ctac. this is consistent with the dissociation of the oligomer with signi cant reduction of intrinsic quenching of tryptophan uorescence due to the heme groups. similar data were obtained at protein concentration of mg/ml. in this case a signi cant increase of light scattering is observed with protein precipitation at a narrow ctac range followed by re-disolution at higher ctac concentration. differently from anionic sds surfactant, cationic one induces protein aggregation. support: cnpq and fapesp. in situ formation of silk protein nano-particles studied by small angle x-ray scattering m. w. roessle , c. riekel , d. i. svergun european molecular biology laboratory; outstation hamburg/germany, european synchrotron radiation facility; grenoble/france silk threads from the mulberry silkworm bombyx mori are used for the production of textiles since centuries. in modern applications silk proteins are chosen because of their good tensile strength, their high biocompatibility as well as of the resorbability for the human body. however, the processing of silk by the silkworm is barely understood. the silk proteins are stored as a solution in the glands of the silkworm and processed during the spinning into a co-block polymer like ber. in a rst step the random coil silk proteins are transformed into molecules with beta-sheet subdomains, which provide a protein-protein interface for the ber assembly. this transformation can be mimicked by a rheometer applying a shear force to the silk protein solution. applying combined small/wide angle x-ray scattering (saxs/waxs) transient formed silk nanoparticles upon increasing shear force were found. further details of these macromolecules were derived by xing the transient state with chemicals such as polyethylene oxide (peo). the resulting data can be analyzed in detail by saxs data evaluation software and low resolution models of the found nanoparticles were derived. moreover, the internal structure of the particles was explored as well as suggestions for the silk processing of the silkworm could be made. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. e. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. unfolding of the extrinsic proteins of photosystem ii ( kda, kda and kda) induced by pressure unfolding of the three extrinsic proteins of spinach photosystem ii induced by pressure has been systematically investigated. thermodynamic equilibrium studies indicated that these proteins are very sensitive to pressure. at o c all the proteins show a reversible unfolding transition by about mpa for kda, mpa for kda and mpa for kda. the ph and temperature dependence of pressure unfolding of these proteins were explored. the stabilization effect of reagents sucrose etc on the proteins was found to associate not only with the increase in the unfolding free energy, but also with the reduction of the absolute value of v u . pressure-jump studies of unfolding of kda protein revealed a negative activation volume for unfolding and a positive activation volume for refolding, indicating that in terms of system volume, the transition state lies between the folded and unfolded states. comparison of temperature dependence of v u # , v f # and v u indicated that the thermal expansivities of the transition state and the unfolded state are similar and larger than that of the folded state. aggregation-prone intermediate protein structures on the refolding pathway l. smeller , j. fidy , k. heremans semmelweis university dept. biophysics and radiation biology, budapest, hungary, department of chemistry, katholieke universiteit leuven, belgium the folding of the polypeptide chain into a native conformation can be studied by experimental systems, where the environmental parameters causing the denatured state can be easily and fast eliminated. one of such parameters is the high hydrostatic pressure. refolding of the unfolded protein can be studied after decompression. the refolding pathway can contain several intermediate states. on the other hand, the destabilization of the native proteins can populate conformations, where the polypeptide chain is not completely folded. these metastable conformations can easily aggregate. deposition of insoluble protein aggregates plays a crucial role in the conformational diseases (parkinson, alzheimer's disease, amyloidozis, etc.) stability and conformation of the above mentioned metastable conformations were investigated in case of myoglobin, apo-horseradish peroxidase, lipoxygenase. fluorescence and infrared spectroscopy and light scattering experiments were used to explore not only the structure but also the aggregation af nity of the intermediates in case of all the above mentioned proteins a well de ned temperature range could be determined, where the metastable not completely folded structures were populated considerably during the refolding process. these intermediate conformations were significantly more aggregation prone, than the native conformers existing in the same temperature range. aggregation processes in beta-amyloid peptides: effects of molecular chaperons a. sgarbossa, d. buselli, f. lenci cnr istituto bio sica, pisa, italy several neurodegenerative pathologies, like parkinson's, hungtington's and alzheimer's diseases, are related to the formation of small peptides aggregates, which amyloid brils originate from. understanding the molecular mechanisms responsible for these processes can, therefore, contribute to clarify the origin and, hopefully, to control the development of the afore mentioned diseases. here we report the results of an in vitro study aiming to affect the aggregation kinetic of - and - beta-amyloid peptides by means of an endogenous chaperone-like protein (alpha-crystallin) and an exogenous polycyclic aromatic pigment (hypericin) that can perturb the aggregation process through stacking interactions with the peptides aromatic residues. because of the well known problems in getting reproducible and reliable results, particular attention has been devoted to carefully check the preparation procedures of the samples. the effects of both alpha-crystallin and hypericin on the self-assembly process have been examined at different times of the aggregation kinetics. the results are discussed in relation with the involvement of different molecular structures in the amyloid brillation phenomenon. remodelling the folding of thioredoxin by removal of the c-terminal helix j. santos, j. m. del no iquifib and departamento de química biológica, facultad de farmacia y bioquímica, uba, junín , c aad buenos aires, argentina e. coli thioredoxin (trx) is a monomeric / protein of amino acids with a fold characterized by a central beta sheet surrounded by alpha helices. two subdomains are topologically noticeable, but it is unclear whether their folding occurs in a concerted fashion. subdomain trx - has been extensively studied as a model of a partially folded, with no tertiary or persistent secondary structure. this work describes the expression and characterization -by circular dichroism (cd), uorescence emission, size exclusion chromatography, chemical cross-linking and light scattering-of a novel engineered fragment (trx - ) lacking the last stretch of amino acids. after refolding from inclusion bodies, trx - shows a strong propensity to form soluble oligomers endowed with distinctive optical properties unlike those observed for the full protein. although trx - also shows signi cant changes in secondary structure, trp residues appear to occupy rigid and apolar environments. these ndings support the existence of an alternatively folded form for trx - . in addition, the secondary structure content of chemically synthesized peptide trx - and its ability to complement fragment trx - upon refolding were also evaluated by cd. taken together, the data herein presented shed light upon issues such as the distribution of information content relevant for folding along the polypeptide chain in regard to conformational stability. with grants from anpcyt, ubacyt and conicet. thermal aggregation of two "beta-protein" models at different ph values v. vetri, f. librizzi, v. militello, m. leone physical and astronomical sciences, univ. of palermo, italy & infm the structural stability of proteins strongly depends on the environment and the lost of this stability may trigger a partial unfolding, leading in turn to the formation of aggregates. such processes have been extensively studied also in view of their biotechnological and medical implications. in fact a large number of diseases is associated with protein misfolding and aggregation. conformational changes play a keyrole in the aggregation processes and have their onset under particular external conditions. the aggregation pathways and the topology of the obtained supramolecular structures sizeably depend on the details of the involved conformational changes, which are determined by the details of the external conditions. here we present an experimental study on thermal aggregation processes of two model proteins mainly composed from structures: -lactoglobulin and concanavalin a, at different ph values. the conformational changes of the proteins (whose association state depends by ph) and the aggregation pathways were monitored by intrinsic and suitable external dyes uorescence. at the same time, the growth of supramolecular structures was followed by measuring the rayleigh scattering of the excitation light. secondary structure changes were followed by circular dichroism measurements. the results show that at different ph values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes. a. verma , t. herges , w. wenzel iwr, forschungszentrum karlsruhe, po box , , karlsruhe, germany, int, forschungszentrum karlsruhe, po box , , karlsruhe, germany ab-initio protein structure prediction and the elucidation of the mechanism of the folding process are among the most important problems of biophysical chemistry. investigations of the protein landscape may offer insights into the folding funnel and help elucidate folding mechanism and kinetics. we investigate the landscape of the internal free-energy of the amino acid villin headpiece with a modi ed basin hopping method in the all atom force eld pff , which was previously used to fold several helical proteins with atomic resolution. we identify near native conformations of the protein as the global optimum of the force eld. more than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. from , independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free energy landscape and characterizes the ensemble of metastable conformations. these emerge as similar in secondary structure content, but differ in the tertiary arrangement. exploring the free energy landscape of a folded protein by means of afm stretching experiments m. vassalli , b. tiribilli , l. casetti , a. torcini , a. pacini , a. toscano isc -cnr, florence -italy, csdc, univ. of florence -italy, anatomy dept, univ. florence -italy the aim of our work is to study the mechanically induced folding and refolding of single proteins by means of an atomic force microscope (afm). the resulting data will be analyzed with theoretical methods, both to determine the folding pathway and to gain information on the energy landscape of real systems. recently, experiments employing atomic force microscopy have shown that mechanical and thermal unfolding share several common features. we are using an afm to perform mechanical stretching experiments on single biomolecules. the experiments that can be performed are particularly well-suited to reconstruct the folding-unfolding pathways as well as the free energy landscape of the examined protein. in particular we are interested in the free energy pro le associated to titin and elastin, by considering a periodic loading of the afm cantilever, instead of the usual linear ramp, and measuring the force as a function of displacement. these experiments will be complemented by theoretical and numerical studies. molecular dynamics simulation of simple models but including the experimental geometry, will allow to examine in detail the effect of different experimental procedures (periodic loading versus linear ramp) proposed to reproduce equilibrium energy landscapes. moreover, we will investigate the limit of applicability of the jarzynski's equality which has been claimed to be able to be used to extract equilibrium results from non equilibrium measurements. association of subunits is a prerequisite for formation of the native structure of the dimeric ipmdh a. varga, É. gráczer, i. hajdú, p. závodszky, m. vas institute of enzymology, biological research center, hungarian academy of sciences, budapest, hungary to answer the question whether subunits are autonomous folding units, or their association at an early stage of folding is required for formation of the native protein structure, denaturation-renaturation experiments were carried out with the dimeric isopropylmalatedehydrogenase (ipmdh). denaturation was induced by guanidine hydrochloride, renaturation was initiated by dilution and followed by activity measurements and uorimetry. reactivation is a complex process with an initial lag phase, indicating the presence of an inactive intermediate. the kinetics of the process is independent of protein concentration, suggesting that association of the two polypeptide chains takes place much faster than the rate limiting rst order isomerisation step(s). restoration of protein uorescence during renaturation is also protein concentration independent, biphasic process, however the initial lag phase is replaced by an even faster burst increase of uorescence. the rst step leads to formation of an intermediate with a native-like uorescence spectrum. based on our experiments the following mechanism is proposed for refolding of ipmdh: d+d i i n , where d is denaturated monomer, i and i are inactive dimer intermediates, n is native dimer, that means initial association of the polypeptide chains during refolding is a prerequisite for formation of the native dimensional structure of ipmdh. we describe a new beamline for optical biophysic in construction on the synchrotron soleil. the high briliance of the synchrotron beam, associated with its tunability on a broad part of the electromagnetic spectrum make it an excitation source of choice for several biophysical optical techniques. the disco beamline we present will consist of three endstations : . the circular dichroism (cd) endstation will bene t from the inclusion of the energies accessible in the vuv wavelength range ( - nm) and from the natural polarization of the synchrotron beam. cd spectra of proteins covering a broad range of wavelenghts will enable better and ner structural analysis. moreover, new biological chromophores such as sugars which absorb in the deep uv will be accessible in cd. . the mass spectrometry endstation,will bene t from an ionisation beam with even greater energies (down to nm) comprising nebulisation at atmospheric pressure. photoionisation of macromolecular bio-structures without any solvent restriction will produce perfect analytes for mass spectrometry, . the multiparametric imaging endstation, build on a confocal microscope, will use the great tunability of the synchrotron radiation ( - nm) to excite samples at many wavelenghts simultaneously. the temporal component of the beam will allow natural lifetime imaging by phase modulation -demodulation. predictive all-atom protein folding with stochastic optimization methods the prediction of protein tertiary structure, in particular based on sequence information alone, remains one of the outstanding problems in biophysical chemistry. according to the thermodynamic hypothesis, the native conformation of a protein can be predicted as the global optimum of its free energy surface with stochastic optimization methods orders of magnitude faster than by direct simulation of the folding process. we have recently developed an all-atom free energy force eld (pff ) which implements a minimal thermodynamic model based on physical interactions and an implict solvent model. we demonstrated that pff stabilizes the native conformation of several helical proteins as the global optimum of its free energy surface. in addition we were able to reproducibly fold several helical proteins (the amino acid (aa) trp-cage protein, the villin headpiece ( aa), the conserved headpiece of the hiv accessory protein ( aa), the headpiece of protein a ( aa) and the -helix bacterial ribolsomal protein l ( aa), as well as several beta-sheet peptides. we used several stochastic optimization methods: the stochastic tunneling method, an adapted version of parallel tempering, basin hopping techniques and distributed evolutionary optimization strategies. we discuss advantages and limitations of this approach to de-novo allatom protein structure prediction. the independent thermal unfolding simulations of gb have been performed. physical property parameters of protein structure were chosen to construct a -dimension physical property space. then the -dimension property space was reduced to dimensions principle component property space. under the property space, the unfolding pathway ensemble of gb was obtained. the pathway ensemble likes a funnel that was gradually emanative from the native state ensemble to the unfolded state ensemble. the unfolding trajectories have the similar variable trend during the native state and the transition state ensemble. during the unfolded state, the unfolding trajectohries were divided into two types that one includes only one trajectory and the other include trajectories. the rst type of unfolded state was a discontinuity step distribution model, which is not random distribution. the second type of unfolded state was a near ellipsoid distribution model and a near random. there were substantial overlaps of unfolded state, indicating that thermal unfolded state consists of a con ned set of property values that makes the number of unfolded state of protein to be much smaller than that was believed before. the protein circular dichroism data bank (pcddb): a bioinformatics and spectroscopic resource b. a. wallace , l. whitmore , r. w. janes dept. of crystallography, birkbeck college, university of london, school of biological sciences, queen mary, university of london we describe the development and creation of the protein circular dichroism data bank (pcddb), a deposition data bank for validated circular dichroism spectra of biomacromolecules. its aim is to provide a resource for the biological and bioinformatics communities, by providing open access and archiving facilities for circular dichroism spectra. it is named by parallel with the protein data bank (pdb), a long-existing valuable reference data bank for protein crystal structures. it will permits spectral deposition via userfriendly web forms and will include automatic reading of a range of data formats and data mining from le headers to facilitate the process. it will be linked, in the case of proteins whose crystal structures and sequences are known, to the appropriate pdb and sequence data band les, respectively. a series of validation tools that will provide reports on data quality are included (and are accessible as stand-alone software). it is anticipated that this data bank will provide readily-accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality assurance and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies. the relation of n-terminal residues and structural stability of l-chain apoferritin k. yoshizawa , k. iwahori , y. mishima , i. yamashita crest/jst, atrl-matsushita, nara institute of science and technology the denaturation of apoferritin by acidic solution was studied. ferritin, the ubiquitous iron storage protein, represents a well known polymeric assembly that is highly resistant to chemical and physical denaturants. it is a cage-shaped protein which is composed of subunits. natural vertebrate ferritins are copolymers of two different subunits, l-and h-chains. in the recombinant h-chain apoferritin (rhf), the structural stability is decreased by deletion of the n-terminal residues. we studied the effect of n-terminal residues of recombinant l-chain apoferritin (rlf) on the acidic ph denaturation and re-assembly. we constructed rlf and mutant rlfs which are lack of (fer ) or (fer ) amino acid residues from the n-terminus and investigated their stability by cd spectra. among three, fer has the least endurance against ph decrease. in the case of fer , the re-assembly of subunits into apoferritin can be performed by increasing solution ph without causing the by-product while huge aggregations are caused in fer and fer . the structural comparison of three mutants indicates that the hydrogen bonds of inter-and intra-subunits decrease by the loss of the n-terminal residues. therefore, it is elucidated that the hydrogen bonds of inter-and intra-subunits from n-terminal residues affect the molecule stability and re-assembly of l-chain apoferritin. pressure perturbation calorimetic studies of solvation, unfolding and aggregation of proteins r. winter university of dortmund, physical chemistry, otto-hahn-str. , d- dortmund pressure perturbation calorimetry (ppc) was used to study the solvation and volumetric properties of various proteins in their native and unfolded state. in ppc, the coef cient of thermal expansion of the partial speci c volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. the effects of ph and various chaotropic and kosmotropic cosolvents (glycerol, sucrose, urea, guhcl, salts, etc.) on the solvation and unfolding behavior of the proteins was also investigated, and the observed volume and expansivity changes are correlated with further thermodynamic and spectroscopic properties of the systems. depending on the type of cosolvent and its concentration, speci c differences are found for the solvation properties of the proteins, and the volume change upon unfolding may even change sign. taken together, the data obtained lead to a deeper understanding of the solvation process of proteins in different cosolvents in their native and unfolded states. in addition, the effects of con nement and crowding on the solvational properties of the proteins were studied. finally, the use of ppc for studying intermolecular interactions and aggregation (amyloidogenesis) phenomena of proteins (e.g., insulin, prp) will be discussed. the in uence of semisynthetic derivatives of phenolic lipids on activity of yeast abc pumps phenolic lipids are the natural amphiphilic long-chain homologues of orcinol ( , -dihydroksy- -methylbenzene). they occur in numerous plants and microorganisms. resorcinolic lipids exhibit high af nity for lipid bilayer and biological membranes and are able to modify the activity of membrane enzymes (e.g. pla , ache). the in uence of semisynthetic derivatives of phenolic lipids on yeast pdr protein activity was studied by spectro uorimetric method using the potentiometric uorescence probe dis-c ( ). the probe is expelled from s. cerevisiae by abc pumps and can conveniently be used for studying their performance. two pump-competent s. cerevisiae strains and different pump-free mutant strains were used to experiment to check the effect of the semisynthetic derivatives of phenolic lipids on activity of abc transporters. two of these derivatives, named . and . , seem to affect the activity of pdr pumps. their in uence on activity of yeast plasma membrane multidrug resistance abc pumps is concentration-dependent. in uence of lipid membrane composition on pglycoprotein activity k. bucher, s. d. krämer, h. wunderli-allenspach department chemistry and applied biosciencies, eth zurich, switzerland p-glycoprotein (p-gp), a membrane atpase expelling many structurally unrelated compounds, is one of the major contributors to multidrug resistance. it is proposed that substrates bind to it within the membrane and are exported from there out of the cell. p-gp substrates are generally hydrophobic and their binding to the transporter is governed by their ability to partition into the membrane. the intimate association of both p-gp and its substrates with the membrane suggests that p-gp function may be regulated by the composition of the lipid bilayer. as detergents in uence the membrane properties and have been shown to affect p-gp atpase activity, we developed virtually detergent-free proteoliposomes to investigate the in uence of the membrane environment on the atpase activity of p-gp. the basal and substrate induced atpase activity was dependent on the cholesterol level of egg phosphatidylcholine (pc) membranes. the compound concentration at half maximal activation of p-gp (k m ) in proteoliposomes correlated with the af nity of the respective compound to liposomes consisting of the same lipids as the proteoliposomes tested. in conclusion, the basal and drug-induced atpase activity of p-gp is strongly dependent on the cholesterol content in detergent-free p-gp/egg pc/cholesterol proteoliposomes. m. berchel , j. jeftic , t. benvegnu , j.-y. thepot , d. plusquellec enscr umr cnrs , campus de beaulieu, rennes, université de rennes , umr cnrs , rennes bipolar lipids found in archaebacterial membranes, generally termed bolaamphiphiles, induce increased stability in membranes exposed to environments such as acidic conditions, high temperatures, high salt concentrations and/or absence of oxygen. we have synthesized a spin labeled unsymmetrical bolaamphiphile that selforganises in water solutions in multilamellar vesicles and shows slow ip-op phenomenon in comparison to conventional liposomes. generally, the ip-op from the exovesicular to the endovesicular membrane surface is a relatively slow process, which is due to the high energy barrier in transferring the polar amphiphilic heads through the lipophilic membrane. it can be involved in membrane transport mechanisms and in facilitating the transport, cells have evolved to use various supramolecular strategies. the half-life of the ip-op is estimated to more than twelve hours. we are now modulating the ip-op rate by incorporating chemical modi cations such as addition of cyclopentanes, double or triple bonds into the bridging chain of the molecule, in order to control the membrane transport via the ip-op mechanism. transport activity of the monocarboxylate transporter is increased by carbonic anhydrase h. m. becker, j. w. deitmer abt. allgem. zoologie, tu kaiserslautern, kaiserslautern the enzyme carbonic anhydrase (ca), which catalyses the conversion of co and h o to bicarbonate and protons, is present in nearly all animal cells, and is highly expressed in astrocytes. it is known that ca can bind to several membrane transporters, forming a transport metabolon and thereby enhancing the transport activity of the protein. in this project we have studied the functional interaction of the enzyme with the monocarboxylate transporter (mct ), which transports lactate and other monocarboxylates together with protons and is believed to play a pivotal role in the metabolite shuttling between astrocytes and neurons. therefore we expressed mct and then injected ca into xenopus oocytes. our results indicate a direct binding of ca to the mct , leading to a ca-induced increase in acid/base ux mediated by the transporter. interestingly, the effect was insensitive to the ca inhibitor ethoxyzolamide and to the nominal absence of co /hco , but disappeared when binding of ca to the mct was hindered. it seems, that ca, bound to the mct , mediates local buffer capacity by removing protons transported into the cell via the mct . this helps to stabilise the proton gradient close to the cell membrane, and thereby enhances the transport activity of the mct . these ndings suggest that ca can enhance metabolite-acid/base transport, by forming a transport metabolon with the mct . melibiose permease of e.coli (mel b) is a membrane bound ioncoupled sugar symporter that uses the favorable na , li , or h electrochemical potential gradient to drive cell accumulation ofor -galactosides. cysteine scanning mutagenesis, electrophysiological (ssm -solid supported membranes) and uorometric measurements, were used in order to better understand the role of speci c parts of the protein in the function of this symporter. the ssm technique combines a rapid solution exchange with the high sensitivity of planar lipid membranes. it employs a solid supported membrane as a capacitive electrode and allows the time resolved investigation of charge translocation during the catalytic cycle of such transporters as na /solute symporters. in order to obtain some more precise information about the function of mel b symporter, starting from the c-less melb, the mutant g c was constructed and from electrical, spectro uorimetric and fret measurements carried out on this mutant, in the absence and in the presence of speci c inhibitors, conclusions were drawn about the possible role of the helix iv in the function of the symporter. two-dimensional crystallization of co-reconstituted ca +-atpase, phospholamban and sarcolipin the sarcoplasmic reticulum ca -atpase and its regulators phospholamban (plb) and sarcolipin (sln) form a primary control mechanism in the recovery of resting state calcium levels in the myocardium. defects in the regulation of ca -atpase by plb and sln are central determinants in cardiac contractility and disease states such as cardiomyopathy. given the signi cance of these proteins, the structural details of their regulatory mechanisms remain an important future goal for the clinical improvement of heart disease. using co-reconstitution into proteoliposomes at low lipidto-protein ratios, we have examined the effects of mutation on the functional properties of plb and sln, revealing novel insights into calcium pump regulation. in addition, these same co-reconstituted proteoliposomes have been used for structural studies by electron cryo-microscopy. in an attempt to better de ne the structural interactions between plb, sln and ca -atpase, we have sought methods for the production of large two-dimensional crystals suitable for high resolution electron crystallography. we previously utilized the co-reconstituted proteoliposomes to produce long, tubular crystals suitable for helical reconstruction. our new procedure comprises three steps -co-reconstitution, membrane fusion, and crystallization -producing large two-dimensional crystals suitable for high resolution structural studies. herein, we will present our latest results characterizing the structural interaction between plb, sln and ca -atpase. t. v. demina , n. s. melik -nubarov , h. frey , e. e. pohl state university, chemistry department, moscow, russia, institute of organic chemistry, johannes-gutenberg-university, mainz, germany, humboldt university, charite, institute of celland neurobiology, berlin, germany multidrug resistance (mdr) of tumours is associated with overexpression of the p-glycoprotein responsible for an active drug ef ux from cells. block copolymers of ethylene oxide and propylene oxide (pluronics) are known to cause a pronounced chemosensitization of tumour cells. the effect may be due either to speci c polymer -protein interactions or to unspeci c lipid bilayer disturbance. we have shown recently that amphiphilic copolymers with various hydrophilic and hydrophobic blocks can disturb lipid bilayers. importantly, that block copolymers of propylene oxide and glycerol (ppo-pg) with hyperbranched "corona" induced larger effects then pluronics with linear polyethylene oxide chains. in the present work we have shown that ppo-pg copolymers increase dox cytotoxicity towards human erythroleukemia (k is , k /dox) and breast carcinoma (mcf /dox) resistant cell lines. using confocal and two-photon microscopy, we demonstrated that these copolymers accelerated dox penetration into resistant cells, inhibited efux and caused drug redistribution into nuclei. a clear correlation between the ability of the polymers to disturb lipid bilayers and favour drug accumulation in mdr cells was disclosed. this nding points to an unspeci c mode of the copolymers' chemosensitizing activity. voltage dependence of processes related to electrogenic membrane transporters electrogenic membrane transporters, such as the sodiumbicarbonate cotransporter (nbce ), may induce dependence on membrane potential upon processes which are innately voltageindependent. we tested this hypothesis by heterologously coexpressing electrogenic nbce from human kidney in oocytes of the frog xenopus laevis with the electroneutral rat monocarboxylate transporter mct . the apparent intracellular buffer capacity was increased by nbce expression and became voltage-dependent by mm/ mv membrane depolarisation. lactate transport via the mct not only became enhanced after co-expression with nbce , but also dependent upon membrane potential. injection of carbonic anhydrase caii from bovine erythrocytes into oocytes enhanced the ef cacy of nbce activity, identifying an additional, ca-sensitive, membrane current via nbce . our results show that nbce adds voltage-dependent buffer capacity to the cytosol; this is suggested to be the prime cause for enhancing acid/base-coupled transport and conferring membrane potential dependence on transporters which are stoichiometrically electroneutral. these interactions may have functional consequences for cells and tissues, where electrogenic and electroneutral processes interact, such as in brain, heart and muscle. . by using carboxy-snarf- as ph-sensitive uoroprobe and microspectro uorimetry, we now show that nhe activation is due to jun kinase (jnk) activation, resulting from reactive oxygen species (ros) produced during metabolism of b(a)p and might involve lipid raft. when analysing b(a)p-induced apoptosis, we have found that cariporide signi cantly reduces both nuclear fragmentation and caspase- like activity. we further show that nhe activation and/or alkalinization affects the mitochondrial ros production detected during the apoptotic cascade, likely via an effect on the complex iii of the electron transport chain. altogether, our results suggest that apoptotic xenobiotics, such as benzo[a]pyrene, induce an early activation of nhe that might play a signi cant role in the subsequent mitochondria-dependent apoptosis. conformational dynamics of a lactose proton symporter j. c. holyoake, m. s. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. transporter proteins are an essential class of proteins, catalysing the transfer of molecules across membranes. increasing numbers of transporter structures are becoming available, opening the way to study their dynamic properties using computational techniques. we present a study on the conformational dynamics of the lactose proton symporter lactose permease using molecular dynamics simulations. these simulations exhibit large scale conformational changes from the initial intracellularly open conformation to a more closed conformation that may be signi cant to the transport mechanism. the conformational change is analysed to identify the contributing motions. effects of nortriptyline and chlorpromazine on anthroylouabain-labeled na,k-atpase e. a. guevara , m. l. barriviera , a. hassón-voloch , s. r. louro departamento de física, puc-rio, rio de janeiro, brazil, instituto de biofísica carlos chagas filho, ufrj, rio de janeiro, brazil the effects of nortriptyline and chlorpromazine (cpz) on the uorescence properties of anthroylouabain (ao)-treated na ,k -atpase of electrocyte membranes from e. electricus are studied. na ,k -atpase oscillates between two major conformations e and e during ion transport cycle. the cardiotonic steroid ouabain speci cally inhibits this enzyme binding to the e conformation. the uorescent label ao presents increased uorescence when binding to the ouabain site of na ,k -atpase. tricyclic drugs such as the antipsychotic cpz and the antidepressant nortriptyline inhibit na ,k -atpase activity in the micromolar range. for the e enzyme, but not e , nortriptyline was found to increase the uorescence in a concentration dependent manner, suggesting a further stabilization of e . for both conformations, cpz induces negligible uorescence change up to µm. the uorescence of atpasebound ao, however, strongly increases upon ultraviolet exposure after cpz treatment at concentrations around µm. fluorescent products of cpz-photodegradation were studied in pure buffer and in the presence of membranes. the results suggest that cpz binds to na ,k -atpase and photolabels amino-acid residues near the ouabain binding site. how important is protein exibility for transport through ion channels? a. a. gray-weale university of sydney certain ion channels are selective for k+ over other ions, but the geometry of the pore does not explain selectivity because thermal uctuations are too large. i extend the usual treatment of ion channels with molecular dynamics simulation by calculating the static and dynamic pair correlations between monovalent ions and ion channels (gramicidin-a and kcsa), and also between certain small, complex cations and the gramicidin channel. this means not only the radial-distribution functions or the density pro les, but also correlations between the ion and the mass and charge densities of various regions of the protein. the advantage of this approach is that it systematically identi es the elements of the protein and modes of motion that contribute to selectivity, and illustrates the decay of correlations. recently, noskov et al. [ ] showed that thermal uctuations protect selectivity. my results on the interaction of ions with carbonyl groups agree with theirs, but take the analysis further to higher correlations. the key new element is the study of the time-correlation functions that describe the motion of the ions through the channel, borrowing methods originally developed for the study of dense or even supercooled liquids. the surface-enhanced infrared absorption spectroscopy (seiras) is used for the investigation of two membrane proteins, the cytochrome c oxidase (cco) and the bacteriorhodopsin (br). of central interest are the transport-mechanisms of electrons (cco) and ions (br). the main parts of the setup are an infrared light source, a hemispherical si-crystal, in which the beam is internal re ected and a plexiglas cell with buffer solution (see schematic scetch). the beam is totally re ected on the inner at surface of the crystal, but the evanescent wave excites surface plasmons in a chemically adsorbed gold-layer on the crystal (attenuated total re ection spectroscopy-atr). the protein to be analysed is attached at the gold surface and can absorb certain wavelengths. the gold is need for the surfaceenhancing effect. cco plays a major role in the respiratory chain, the retinal protein br is a photosynthetic protein. the cco is immobilized on the gold surface via the af nity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (nta) surface. for the br we incorporate the protein in a lipid membrane, which is attached on the gold surface by the sulfuric bindings of , -di-o-phytanyl-sn-glycerol- -tetraethylene glycol-d,l-lipoic acid ester lipid (dptl). the sensitivity of this method is further enhanced by modulation of an external parameter, like the electric potential. effects of copper ions on the escherichia coli growth and proton-potassium exchange copper ions are required for the function of many important enzymes in escherichia coli but can cause a number of toxic cellular effects also. it's interesting to reveal the in uence of copper ions on the growth of bacteria and proton-coupled membrane systems. upon transition of e. coli mc wild-type culture to stationary growth phase a decrease in redox potential (e h ) from the positive values ( + mv) to the negative ones (of - to - mv), resulting h production by formate hydrogenlyase (fhl) have been studied. copper increased a latent growth phase duration as well as delayed a logarithmic growth phase in concentration-dependent manner. during the anaerobic growth, the production of h was strongly inhibited in the presence of cucl ( mm). . mm cucl was inhibited h production under experimental conditions with glucose. the inhibitory effect of copper ions ( . mm) on n',n'dicyclohexylcarbodiimide (dcc)-sensitive h /k exchange was also observed: k uptake was decreased and the stoichiometry of dcc-inhibited ion uxes varied. interestingly, these effects on h and k uxes were absent for the mutant hd (hyc-operon for hydrogenase was deleted). we suggest that copper ions, inhibiting the activity of fhl, have an effect on h /k -exchanging mechanism which is the proton f f -atpase associated with k uptake trk system. this effect may be due to the relationship of fhl with the ion-exchanging mechanism above under fermentation at alkaline ph. lateral diffusion in tethered bilayer membranes m. jung, v. atanasov, w. knoll, i. koeper max planck institute for polymer research, mainz, germany tethered bilayer membranes (t'blm) provide a useful platform for the investigation of bilayer membranes as well as embedded membrane proteins. we have developed a modular system, which is suitable for surfaces providing gold and oxide surface coatings. these systems serve as a quasi natural environment for the study of membrane proteins, being functionally incorporated into a lipid bilayer, which is covalently bound (tethered) to the substrates. functionality could be shown using electrochemical methods. here we present a study of these systems, basically the membrane itself, using uorescence recovery after photobleaching (frap) studies in order to investigate lateral motion in the lipid bilayers. lateral mobility is essential for successful incorporation of large membrane protein complexes. we will present rst results of experiments that try to differentiate motions hindered due to the tethering from diffusion in free oating or suspended bilayers. the information gained in this study will serve for improvements in the chemical structure of the tethered molecules. we will develop the system as a basis for bio sensing applications, where embedded proteins will serve as actual sensing elements. a. d. ivetac, j. campbell, m. s. p. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. atp-binding cassette (abc) transporters form an important superfamily of membrane proteins which couple atp hydrolysis to the active transport of diverse compounds across the cell membrane. their biomedical relevance is highlighted in examples such as multidrug resistance to antibacterial and anticancer agents, and cystic brosis. the availability of crystal structures of three complete bacterial abc transporters provides an opportunity to study structurefunction relationships at the atomic level. in this work, we carry out multi-nanosecond molecular dynamics simulations of the vitamin b importer from e. coli (btucd), with both the complete multimeric transporter embedded in a phospholipid bilayer and the soluble subunits in a membrane-free environment, in an attempt to elucidate some of the conformational changes which arise during the transport event. atp-bound and atp-free structures are used to investigate the effect of nucleotide on the system. a range of analytical techniques have been applied to assess the dynamic behaviour of the protein during the simulations, which includes measurements of: conformational drift, residue exibility, transmembrane domain (tmd) movement, concerted protein motions, nucleotide-binding and translocation pathway changes. an in-vitro method was designed to measure transmembrane transport rates. liposomes were prepared by extrusion with dipalmitoyl phosphatidylcholine (dppc) and optionally cholesterol, and loaded with a peptide (zinc-insulin tagged with a uorescent group or bsa). after removal of the non-encapsulated peptide from the liposome solution by gel ltration, the release of the peptide from the liposomes was monitored by uorescence as a function of time at various temperatures. the transport was greatly accelerated by the presence of a speci c proprietary excipient molecule (cyclopentadecanolide -cpe ™), which effectively triggered the release of the peptide. a mathematical model was developed to quantify these results. a semi-empirical nonlinear equation involving four parameters ts the protein release pro les. then a neural network predictions model was used to correlate the different release condition parameters and the four semi-empirical tting parameters based on the experimental data sets. most release data t well with the mathematical model, further supporting our theory of a two step release mechanism. phenyltin the well known group of organotin compounds that exhibit toxic properties in relation to the biological systems are phenyltins. no studies have been performed as yet to establish directly whether organotins such as diphenyltin dichloride ( dpht) and triphenyltin chloride (tpht) cross the lipid bilayer. we have performed experiments that showed transfer of those compounds across the lipid bilayer using the stopped-ow technique and desorption of those compounds from a monolayer using the langmuire technique. obtained results demonstrate that dpht and tpht rst adsorb onto the lipid bilayer surface, in diffusion controlled manner, within a very short time ( . s), whereas the membrane passing was observed in a minute's time range. the long time kinetics show a complex dependence on the kind of compound, its concentration and the presence of cholesterol in the membrane. the desorption of both compounds from the monolayer to water subphase occurs in a minute's time range. these observations may explain the known fact, that the in uence of organic, amphiphilic tin (and also lead) compounds is more toxic than that of inorganic ones.the phenyltins much easier (compared with tin or lead ions ) penetrate e.g. blood -brain barrier. [ under the resting conditions ca concentration in agonistsensitive ca stores re ects a balance between active uptake mediated by a ca -atpase (serca) and passive ef ux of ca . this ca leak appears to be a common property of ca -storing organelles, but the nature of the leak in submandibular acinar cells remains unclear. we have studied the ca leak pathways in the endoplasmic reticulum (er) of acinar cells of rat submandibular salivary gland by directly measuring concentration ca in the er ([ca ] er ) in mag-fura /am preloaded cells while [ca ] i was clamped at a resting level with a egta/ca mixture. we have shown that thapsigargin (tg) or ca -free buffer treatment completely blocked ca uptake by serca after the rst minute of superfusion and caused a ca leak represented by continuous decline in [ca ] er . this ca leak from the er was not sensitive to tg, heparin and ruthenium red and therefore appears to be independent of the serca, the insp receptor and the ryanodine receptors. however, treatment with puromycin ( . - mm) to remove nascent polypeptides from er-ribosome translocon pores increased ca leak from the er by a mechanism independent of the serca, insp or ryanodine receptors. thus we conclude that basal ca leak from the er of submandibular acinar cells occurs through translocon pores in the er membrane. r. b. kishore, j. reiner, e. edgu-fry, a. jofre, k. helmerson physics laboratory, national institute of standards and technology we have developed a procedure to make lipid and polymer nanotubes of up to one cm long and nm in diameter, from the surface of giant liposomes and polymersomes, using micro uidics and optical tweezers. the liposomes and polymersomes were formed, using elcetroformation method, from phospholipids and amphiphilic diblock copolymers, respectively. the polymer tubes were made extremely robust by cross-linking them using chemical reactions. we are currently studying the transport of molecules in the crosslinked nanotubes for use in nano uidic networks. p-glycoprotein (p-gp) is an active membrane transporter capable of expelling out of the cell a large number of potentially cytotoxic amphiphilic molecules with unrelated chemical structures. as a consequence, p-gp may be responsible for multidrug resistance of tumors against chemotherapy (mdr); it also plays a key role in absorption, biodisposition and elimination of many pharmaceuticals. to understand the molecular mechanisms of the transmembrane drug transport mediated by pgp, it is highly desirable to design a convenient assay for measuring both p-gp atpase activity and p-gp transport function. to do so, we used inside-out native membrane vesicles, prepared from mdr cells and containing high amounts of p-gp. we took advantage of the speci c property of a uorescent dye, the carbocyanin jc- , known to be expelled out of mdr cells: above a critical concentration (the "cjc"), this dye forms j-aggregates which emit a uorescence at a wavelength very different from that emitted by the monomer. in the presence of mgatp, the p-gp-containing vesicles accumulated jc- , which exceeded locally the cjc and thus formed intraluminal j-aggregates; these aggregates allowed accumulated jc- both to be sequestered inside the vesicles, by dramatically slowing down its passive backdiffusion, and to be speci cally detected. kinetic characterization of this transport suggests that jc- is rst translocated to the exoplasmic lea et of the vesicle membrane before its internalization into the aqueous phase of the vesicle lumen. interaction between the energy metabolism and externally applied electric elds in yeast cells electric elds are often used for biophysical or biomedical treatment of biological cells, e.g. cell fusion or killing of cells. however, only a few data about the possible mechanisms of electrosensitivity of biological cells are available. since electrostimulation always induces depolarization of biomembranes, an impact of the energy metabolism is obvious due to the regeneration of electrochemical gradients by the expenditure of cellular energy. our aim is to investigate the interactions between externally applied electric elds and the aerobic/anaerobic energy metabolism of yeast cells. for this, we have constructed a new electrical interface for local stimulation of biological cells with variable duration and amplitude. when applying short lasting electrical pulses to yeast cells, we nd a direct response of the energy metabolism (measured by nadh-uorescence) to these pulses. a sudden and fast decrease in nadh is followed by a slower recovery of the uorescence signal. these nadh-signals are abolished in the presence of antimycin a or kcn, demonstrating the importance of mitochondrial energy production for this phenomenon. we attribute these changes to the immediate break down of atp as a consequence of the regeneration of the membrane potential (atpases) and the slower regeneration of atp by mitochondrial respiration. new insights of hypericin blood transport and its incorporation into the plasma membrane b. m. macri , g. stoian , m. l. flonta department of animal physiology and biophysics, faculty of biology, university of bucharest, department of biochemistry, faculty of biology, university of bucharest, bucharest, romania hypericin (hyp) is one of the active compounds from hypericum perforatum (an herb usually prescribed as antidepressant). the bioavailability of this hydrophobic molecule is a very important issue for medical applications. the goal of our work was the study of hyp blood transport mechanisms. techniques of absorbtion spectroscopy, electrophoresis and uorescence microscopy were used in order to de ne the properties of hyp-albumin and hyp-lipoproteins complexes and to explain the action of hyp at the plasma membrane level. hyp bind to several electrophoretic (sds-page) bands evidenced by mice plasma migration. both albumin and lipoproteins bind to hyp, forming complexes, during the blood transport process. different types of lipoproteins from males and females plasma mice were evidenced by gradient electrophoresis to bind hyp. hyp-albumin complex was also identi ed by absorbtion spectra, and the ratio a /a has a ph-dependence. hyp interaction with plasma membranes was also examined on cell culture by uorescence microscopy, and hyp plasma incorporation is a dose-and incubating time-dependent process. our results partially elucidate the plasma fractions that bind hyp, contributing to its blood transport. this study proposes a new mechanism of hyp cellular insertion, discussing its plasmatic membrane penetration due to its high hydrophobicity. the overexpression of p-glyoprotein (p-gp) is one of the major causes of multidrug resistance (mdr) in cancer chemotherapies. many p-gp inhibitors have been designed to reverse the mdr effect, but the structure-activity relationships of the p-gp inhibitors and substrates still remain largely unknown. until now, it is still very challenging to obtain the high-resolution p-gp structures and currently only low-resolution electron microscopy structure is available. this has caused the structure-based design of "p-gp-ignoring" therapeutic agents a remote goal. however, the recent determination of x-ray crystal structures of bacterial lipid a transporter, msba, has provided eligible structure templates for homology modeling of p-gp. we have therefore conducted explicit solvent molecular dynamics simulations of the fulllength ef ux pump, human p-gp, in an excessively hydrated popc bilayer to re ne the homology model. both free and atp-bound forms of p-gp have been simulated. the entire system consists of more than , atoms. our molecular dynamics simulations have shown that the overall architecture of p-gp remained very stable for tens of nanoseconds, while the observed membrane undulation was rather large. the simulation results have allowed us to investigate the conformational changes of p-gp upon atp binding in the ef ux process and to predict the possible binding site of various known substrates and inhibitors. the re ned structure models of p-gp by our simulations could be used as the basis for further drug design. electroporation (ep) is a phenomenon where increased permeability of cells exposed to an external electric eld is observed. the induced transmembrane voltage presumably leads to the formation of aqueous pores in the phospholipid bilayer, which increases permeability of the membrane for molecules and ions. ep is currently used in many biomedical applications including transfer of genes and electrochemotherapy of tumors. still, the molecular mechanisms of the process are not fully explained. recently it was proposed that ep could be monitored in real-time by measuring electric conductivity of tissue. so far the studies focused mostly on a single pulse, however in biomedical applications usually several pulses are used. in our study we used a train of electric pulses to analyse the relationship between electric conductivity and cell permeabilization. current-voltage measurements during and after pulse application were performed in dense suspension of cells. conductivity changes were analysed numerically using nite elements method and compared with the percentage of permeabilized cells. we obtained a transient increase in conductivity above a certain voltage with complete relaxation in < s. substantial changes in conductivity are also due to the diffusion of ions through membrane pores and osmotic swelling. we further show that relation between conductivity and permeabilization level is indirect. interaction of quinolones with bacterial porin ompf: uorescence quenching studies quinolones are widely used antibiotics witch develop their antibacterial action by inhibition of important bacterial enzymes. consequence of the internal location of their target of action, the translocation of this drugs trough the outer membrane is an essential step for their antibacterial action. in vivo studies have been showing that ompf is important for the entrance of some of these antibiotics, but the exact degree of involvement of this protein in the transport of the different members of this group of antibiotics, remains unknown. in this study, the quenching of the intrinsic tryptophan uorescence of ompf, in presence and in the absence of the drugs and by two distinct quenchers, was used as a rst approach, to elucidate ligandinduced structural changes and consequently prove the differential involvement of ompf in the entrance of these antibiotics in the bacterial cell. the results obtained reveal that the degree of interaction with the protein is related with the hydrophobicity of the different antibiotics. this kind of evidence suggests that the entry by the porin channel is not the only path used by these antibiotics and that it is more important for the latest generations of this group because of their increased hydrophilic characteristics. inhibition of multidrug resistance-associated protein mrp and kv channels by natural polyphenols k. michalak, a. teisseyre, b. ania-pietrzak department of biophysics, wroclaw medical university, poland resistance to cytotoxic agents remains a major obstacle to successful chemotherapy in cancer. best-characterized form of drug resistance is caused by the overexpression of genes encoding membrane drug pumps, like p-gp or mrp . in present study, activity of several plant polyphenols ( avonoids and stilbene) against mrp has been studied using functional assay based on ef ux of mrp uorescent substrate. very recently, a role of kv . potassium channels in proliferation of various cancer cells was suggested. in our study the effect of the plant polyphenols on voltage-gated potassium channels kv . was investigated by patch-clamp electrophysiological method. some of studied compounds were found to be active inhibitors of multidrug resistance-associated protein mrp and voltage-gated potassium channels, and their properties are promising for further research in the eld of anticancer activity of natural products. the transport mechanism of melibiose permease: a study using electrical measurements and uorescence techniques k. meyer-lipp , c. ganea , t. pourcher , g. leblanc , k. fendler max planck institut für biophysik, frankfurt, germany, c. davila medical university, bucharest, romania, cea, université de nice so a antipolis, nice, france the melibiose permease (melb) of escherichia coli is a membrane bound carrier that uses the favorable na+, li+, or h+ electrochemical potential gradient to drive cell accumulation of alfa-galactosides (melibiose, raf nose) or beta-galactosides (methyl- -thio-beta-dgalactopyranoside). electrophysiological techniques have proved to be extremely useful tools to investigate the mechanism of ion transfer across the membrane by ion-coupled transporters. using a solid supported membrane (ssm) as a capacitive electrode a rapid solution exchange can be combined with the high sensitivity of planar lipid membranes and allows time resolved investigation of the charge translocation during the catalytic cycle of na+/solute symporters. this technique has been combined with uorescence measurements, which report on structural changes during the substrate transport process of the carrier. we have used time resolved tryptophane uorescence, uorescence energy transfer with a uorescent sugar substrate and site speci c uorescence of a label attached to a cysteine residue on the protein. this allowed us to identify conformational transitions during the reaction cycle of the melibiose permease. we could assess their electrogenicity and determine rate constants. a kinetic model for na+ and melibiose binding and transport is presented. electrophysiological characterization of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into dened lipid bilayers. we present an automated method for reconstitution of membrane proteins into lipid bilayer membranes, that substantially reduces both the reconstitution time and the amount of protein required. we have applied this well-de ned system to the characterization of a novel mitochondrial uncoupling protein, ucp and demonstrated that ucp exhibits protonophoric function exclusively in the presence of fatty acids, similar to that previously shown for its homologue ucp . the membrane conductance was proportional to the concentration of the reconstituted ucp in presence of oleic acid or eicosatrienoic acid, and was inhibited by atp. amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. for the study of the ca -atpase from sarcoplasmic reticulum, previous experiments have revealed both advantages and disadvantages to the use of a polyacrylate-based amphipol, a - . these issues have been reinvestigated using four different amphipols. size exclusion chromatography showed that, although a - aggregates in the presence of millimolar concentrations of calcium -an effect that probably accounts for most of the aggregation of atpase/a - complexes observed in our previous work-, aggregation can be avoided by resorting to a sulfonated version of a - . we also found that all amphipols tested slowed down the rate of calcium dissociation from its binding sites and reduced atpase activity, while protecting the solubilized protein against denaturation. this suggests that association with the polymer may damp the protein's dynamics, perhaps due to the multipoint attachment of the polymer to its hydrophobic transmembrane surface. such a "gulliver" effect could contribute both to the protection of membrane proteins against denaturation and to the reversible inhibition of serca a. clc proteins are found from prokaryotes to mammals. they function as plasma membrane chloride channels or provide neutralizing anion currents for v-type h -atpases that acidify compartments of the endosomal/lysosomal pathway. vesicular clcs have been thought to be cl -channels, in particular because clc- and clc- mediate plasma membrane cl -currents upon heterologous expression. we have shown, however, that these two mainly endosomal clc proteins rather function as electrogenic cl /h exchangers, resembling the transport activity of the bacterial clc-e that has been crystallized. neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion ux to proton counter-transport. clc- and clc- may still compensate the charge accumulation by endosomal proton pumps, but are expected to tightly couple vesicular ph-gradients to cl -gradients. calorimetry and mechanics of ca transporting systems in rat myocardial bigeminies a series of new n-oxides of tertiary amines (nta) was checked for its biological activity. individual compounds differed in the length of substituted alkyl chain. the primary goal was to nd if they can be used as effective antioxidants and, to what degree they modify used model (liposomes) and biological (erythrocytes, algae and cucumber) membranes. various methods were used in order to do that. a mechanism of the interaction between nta and membranes was studied by measuring their potency to hemolyse erythrocytes, to in uence a phase transition temperature in dppc liposomes, to change a membrane potential of algae cells. the measure of the interaction of nta with cucumber cells were potassium leakage, chlorophyll content and inhibition of growth of hypocotyls. antioxidative abilities of nta were determined by measuring their ef ciency to protect erythrocytes against membrane lipid oxidation induced by uv irradiation and by comparising their antioxidative ef ciencies with that of trolox (vitamin e analogue) in chromogen experiments. the mostly widely employed mechanism of drug extrusion in bacteria is via membrane transport proteins called ef ux pumps. in gram-negative bacteria, multidrug resistance is conferred by tripartite complexes, rather than by a single transport protein. through these systems, a wide range of substrates is expelled from the cytoplasm, through the periplasmic region, to the exterior of the cell. among these complexes, the acrab/tolc system in escherichia coli is formed by an inner membrane ef ux pump, acrb, an outer membrane protein, tolc, and a periplasmic protein known as an adaptor, acra. the components of this complex are studied, in order to provide insights into drug transport in bacteria. here we present a dynamics study on mexa, homologue of acra from pseudomonas aeruginosa. the protein has been studied by molecular dynamics simulations in bulk water. a structural adjustment by the periplasmic protein is required in order to engage both the bottom part of the om protein and the top region of im protein. the dynamics on mexa reveals a exible behaviour of the protein in water. the major concerted motions observed are the hinge-bending of the two domains, and the rotation of the -barrel domain. these can be related to the adaptation of mexa (and acra) to the om and im proteins during the process of assembly in forming the complex, and during the opening of the channels. electrophysiologic study of ap in chara corallina -indication of its biochemical nature the speci c conductance's of aqueous solution of electrolytes (viz.naf, nacl, nano , na so , kf, kcl, kno , k so , mgcl , cacl , fecl , mncl ,crcl ,cucl , cocl , )have been measured across peritoneum at temperatures between( - ) c.conductance attains a maximum limiting value at higher concentrations for each electrolyte due to a progressive accumulation of ionic species within the transmembrane region. the membrane becomes more and more conductive to incoming ions and attaining a limiting value due to the fact that an electrically neutral pore, which is speci c for a particular ion, is unlikely to contain more than one type of ion. consequently, at high electrolyte concentration, the pore saturates and the conductance's approaches a limiting value. the values of speci c conductance measured follow the sequence for anions; so > cl > no > f . whereas for the cations the sequence is k > na ; ca > mn > co > cu > mg ; cr > fe . the energy of activation for the cations as well as for the anions follows the sequence (for cations): the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosysytem ii (lhcii) were investigated after freeze-thaw cycle in criotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in cryotoxic medium (nacl). in the presence of the cryoprotective compound (sucrose) freezing damage is less pronounced and is not affected by the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus in the process of freeze-thaw treatment are discussed. spectral and redox characterization of the novel heme ci in the cytochrome b f complex . this is an indication of different spin delocalization in the primary donor, for the mutant being typical of a monomeric oxidized bchl. considering the fact that the properties of both isolated and membrane-associated mutant rcs were similar, we conclude that missing bchl molecule from the mutant rc was the result of the introduced mutation but not of the protein puri cation procedure. authors acknowledge the support by the russian brf. we created two site-directed mutants, a s and l i in the d protein of photosystem ii in thermosynechococcus elongatus. both mutations are within the binding pocket of the primary quinone acceptor (q a ). we investigated the effects of the mutations in vivo and in isolated psii. while the l i mutant exhibits characteristics similar to the wild type, the a s mutation effects q a charge recombination measured by thermoluminescence and uorescencedecay. these results strongly indicate that the a s mutation induce a shift in the redox potential of q a . the a s accelerates the rate of photoinhibition, an effect consistent with the negative shift in the redox potential. epr was used to measure the temperature dependence of the electron transfer from q a to q b in the a s mutant. it was found to be indistinguishable from the wild type despite the difference in the midpoint potential of q a . this is taken as an indication as a gating mechanism on the acceptor side of psii similar to that in bacterial reaction centers. protochlorophyllide oxidoreductase takes an abnormal reaction pathway below the glass transition g. durin , d. j. heyes , c. n. hunter , d. bourgeois ibs and esrf, grenoble, france, krebs institute and r hill institute for photosynthesis, shef eld university, shef eld, uk motions through the energy landscape of proteins lead to biological function. at temperatures below a dynamical transition ( - k), the activity of some proteins cease. in this work, we describe an enzyme that, instead, engages into a non-productive pathway below k. protochlorophyllide oxidoreductase (por) catalyzes the reduction of protochlorophyllide (pchlide) into chlorophyllide (chlide), a key step in chlorophyll biosynthesis. por is one of the two enzymes known to require light for catalysis. when illuminated with gentle light at k, the complex of t. elongatus por with pchlide and nadph transforms into a nonuorescent intermediate. upon warming, several uorescent intermediates develop, and at k chlide is released. when illuminated at temperatures below k, por behaves differently. if gentle light is used, the reaction can not start. instead, if a blue laser source is used, the initial complex disappears, like at k. however, upon warming, a new intermediate develops that uoresces at nm and leads to a dead-end product. by using uorescence microspectrophotometry, we have measured the solvent glass transition temperature of the system to be k. the solvent glass transition, possibly controlling a por dynamical transition, may be the determinant that switches the enzyme reaction pathway from a non productive to a productive one. the nonproductive pathway results from a two-photons absorption mechanism, whereas the productive pathway is a one-photon mechanism. sensory rhodopsin ii from n. pharaonis (npsrii) forms a complex with its cognate transducer nphtrii in a : stoichiometry . light activation of npsrii leads to a movement of helix f which triggers a rotation of tm in nphtrii . the mechanism of signal transduction through the hamp region to the cytoplasmic domain of the transducer is still unknown. structural information exists for the transmembrane and cytoplasmic regions, however the hamp domain is not yet characterized. in order to obtain structural information on this domain, twenty-four residues in the membrane adjacent region ( - ), and six residues in the following region were spin labeled and investigated by cw and pulsed x-band epr. to analyze the overall architecture of the complex, doubly spin labeled variants between the transducer and the receptor were also engineered. depending on their function, the absorption spectra of rhodopsins can be tuned by the protein over a wide range. a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. we compute and compare the classical electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (br), halorhodopsin (hr), and sensory rhodopsin ii (srii). these proteins are an excellent test case for understanding the spectral tuning of retinal. the absorption maxima of br and hr are very similar, while the spectrum of srii is considerably blue shifted. we nd that the electrostatic potential is similar in br and hr, but differs signi cantly in srii. a quantum mechanical model of a particle in a box with a step potential can qualitatively relate the differences between the electrostatic potentials of the proteins to the relative shifts of their absorption maxima. by decomposing the electrostatic potential into contributions of individual residues, we could identify six residues that are responsible for the differences in electrostatic potential between the proteins. three of these residues are close to the retinal, while the other three residues are more then angstroem away from the retinal. the counterion of the schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials. effect of uv-a radiation on thylakoid membranes with different organization p. ivanova, a. dobrikova, t. markova, s. taneva, e. apostolova institute of biophysics, bulgarian academy of sciences, so a, bulgaria the effect of uv-a ( - nm) radiation on the energy transfer and the photosynthetic oxygen evolution of thylakoid membranes from pea mutants was investigated. the membranes have different pigment composition, stoichiometry and organization of pigment-protein complexes. the aim of our work was to nd out whether uv-a induced damage is affected by the altered content and/or oligomerization of the main light-harvesting chlorophyllprotein complex (lhcii) in thylakoid membranes. the data for the effect of uv-a radiation on the oxygen evolution demonstrate that: (i) the inhibition of photosystem ii (psii)-mediated electron transport and ash-induced oxygen yields strongly depend on the amount of lhcii; (ii) the increase of the s o populations of psii centers in darkness is more pronounced in thylakoid membranes with smaller amount of lhcii; (iii) the inhibition of the oxygen evolution is related to the reduced number of the functionally active psii centres; (iv) the degree of impairing of active psii centres depend on the amount and oligomerization of lhcii. the results also show that the altered content and organization of lhcii in uence the uv-a light-induced changes in the energy transfer between psii and psi and within the supramolecular lhcii-psii complex. the effects of uv-a radiation on leaves and isolated thylakoid membranes are compared. sudden polarisation -a large change in the electric dipole moment between the excited and the ground state -is a well-known phenomenon for retinal chromophore. some early models of the energy transduction mechanism in bacteriorhodopsin (br) even attribute a primary functional role of that. however, it was apparently unrecognized that the maxwell theory intuitively predicts the appearance of an ultrafast transient electromagnetic radiation due to this dipole moment change. here we show that the existence of this type of radiation can be derived from semiclassical quantum electrodynamics as a second order phenomenon. in optical terms it corresponds to the previously unstudied resonant case of optical recti cation. recently we experimentally observed a major component in the fs coherent infrared emission of oriented purple membranes of br corresponding well to this effect (groma et. al, proc. natl. acad. sci. , , ). our theory predicts that such a signal holds detailed information on the dynamics of excited state polarization, opening a new branch of impulsive spectroscopy on asymmetric systems. beyond optical recti cation we found a complex phase a coherent oscillation living for a few ps, i.e. much longer than the excited state of br. fitting analysis resulted in at least seven vibrating modes in the - cm region, while windowed fourier transform indicated time-dependent frequency distribution. a. ghignoli, g. cercignani, s. lucia, g. colombetti istituto di bio sica, cnr -pisa, dipartimento di fisiologia e biochimica, università di pisa, italy the life cycle of ophryoglena ava, a histophagous ciliate dwelling in fresh waters, reportedly includes several stages that feature morphology changes and different phototactic responses. previous studies on the phototactic responses in o. ava during its phase of maximal positive phototaxis led to an action spectrum with two main peaks at and nm, and a minor peak at nm. starting from those results, we analyzed the phototactic response at various cell ages, using three broad-band interferential lters (fwhm = nm) centred respectively at , and nm, and constructed dose-effect curves for each band. a higher photosensitivity at nm, and lower photosensitivies with the other two lters ( and nm) have been observed at any cell age. however, the photosensitivities in the blue and orange regions show a different time course vs. cell age with respect to the photosensitivity in the green region. measures were also carried out on cells whose feeding cycle was altered by a -day starvation (a double time with respect to the standard protocol) before being fed at t = . the maximal photoresponse values reached by starved cells are lower than the highest values reached with standard cultures; in other words, a general reduction of the phototactic response is observed. these results suggest that, while feeding optimally induces cell division, it does not generally reset all cellular functions. a. quaranta , f. lachaud , y. pellegrin , p. dorlet , m.-f. charlot , s. un , a. aukauloo , w. leibl service de bioénergétique, cea-saclay, bât. , gif-sur-yvette cedex (france), laboratoire de chimie inorganique, bât. , université paris-sud, orsay (france) coordination complexes based on a photoactive rutheniumpolypyridyl moiety linked to simple, rigid ligands with binding sites for transition metals, are developed to mimic the light induced charge separation and water oxidation processes taking place in the photosynthetic apparatus. inspired by the structure around the donor side of photosystem ii a family of phenanthroline based ligands holding an imidazole, a phenol or an indole unit simulating the amino acids histidine, tyrosine and tryptophan in the oxygen evolving complex, were developed as models for proton-coupled electron transfer. in some of the molecules investigated the hydrogen bonding interaction present in the natural system is reproduced. combined data from photophysical, spectroelectrochemical studies and dft calculations evidenced the photogeneration of a phenoxyl or a tryptophan radical upon excitation of the chromophore in presence of an external electron acceptor, therefore mimicking the electron trade between p and tyrz-hist . finite element model to predict the electric potential distribution in ps i containing vesicles c. p. a. pennisi , e. chemineau , e. greenbaum , k. yoshida center for sensory motor interaction, aalborg university, denmark, chemical sciences division, oak ridge national laboratories, usa, facultad de ingeniería, uner, argentina photosynthetic reaction centers (rc) are integral membrane proteins and molecular photovoltaic structures. recently, it was suggested their use as triggers of voltage-gated ion channels in excitable cells, where a certain voltage threshold has to be reached to evoke a response (kuritz et al., ieee trans. nanobiosci. in press ). experimental studies with rc's reconstituted in lipid vesicles have shown different values of transmembrane voltage, depending on parameters like light intensity, rc concentration and membrane passive properties. ultimately, the purpose of this work is to have a tool to estimate the proximity, number and density of rc's required near a voltage-gated channel to activate an excitable cell. as a starting point, we aim to predict the spatial distribution of the membrane potential in vesicles. a nite element model was realized using a commercial package (femlab, comsol a/s). the three-dimensional distribution of the electrical potential near a single rc in the surface of a spherical vesicle was calculated. in terms of density, in conditions of saturating light, a minimum of , e rcs/cm is needed to develop a potential of mv, capable to activate voltage-gated sodium channels. microsecond time-resolved x-ray diffraction study of purple membrane t. oka , k. inoue , m. kataoka , n. yagi faculty of science and technology, keio university, japan, japan synchrotron radiation research institute (jasri), japan, nara institute of science and technology, japan the structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of Å by time-resolved x-ray diffraction experiment utilizing synchrotron x-rays from an undulator of spring- . the xray diffraction measurement system, used in coupling with a pulsed yag laser, enabled to record diffraction pattern from purple membrane lm at a time-resolution of µsec over the time domain of µsec to msec. the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosystem ii (lhcii) were investigated after freeze-thaw cycle in cryotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in a cryotoxic medium (nacl). in the presence of a cryoprotective compound (sucrose) freezing damage is less pronounced and independent of the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus during the freeze-thaw process are discussed. we have studied the effect of a cytokinin meta-topolin (mt, m) on senescence-induced changes in the photosynthetic apparatus of detached primary leaves of wheat (triticum aestivum l. cv. hereward). the senescing leaves were kept under continuous light conditions. mt signi cantly slowed down the senescenceinduced decrease in chlorophyll content and markedly stimulated violaxanthin zeaxanthin (z) conversion. the high z content was maintained even after an hour in darkness. mt treatment caused also the appearance of an emission band f peaking at - nm. this emission band is attributed to aggregates of lightharvesting chlorophyll a/b-binding proteins (lhc), the production of which is associated with a higher z content. the presence of lhc aggregates in mt treated leaves was documented also by electron microscopy imagines. besides the lhc aggregation, mt induced also a decrease in photosystem i content which was documented by electrophoresis and k-uorescent spectra. supported by grants frvs / and msm . recently high resolution images of bacterial photosynthetic membranes have revealed the organization of membrane proteins in these native membranes. the organization revealed is remarkable, and all the more so when we realize that these specialized, protein rich, membranes differentiate from the cytoplasmic membrane which has a more complex composition and is richer in lipids. analysis of the protein organization in these specialized membranes from several different bacteria suggest that the organization results from a phase separation of several different contiguous phases. in order to better understand our observations we have undertaken an examination of the different phase behaviors that are possible for membrane proteins considered as a two dimensional colloid. monte-carlo modeling of the phase diagram of this system shows the importance of interaction distance in the determination of system behavior. transcription of our observations on the model systems to the photosynthetic membranes suggests that electrostatic and elastic forces in the membrane are of particular importance in determining the high level order of membrane proteins. the recent crystal structure of photosystem i (psi) from synechococcus elongatus shows two quasi-symmetric branches of potential electron transfer cofactors including primary donor (dimer of chlorophylls p ), monomeric chlorophylls a and a and quinone a , bound to the psaa/psab heterodimer. so far, it is not clear if both potential electron transfer pathways are active in this process or only one of them. to solve this issue, we studied a set of mutants with methionine coordinating the primary electron acceptor, a , replaced by histidine, leucine, or serine in either of two branches. our results obtained with a technique of femtosecond transient absorption spectroscopy show that both branches are equally active in electron transfer. mutation in either branch slows the forward electron transfer between a and a from ps in wilde type psi to - ns in all these mutants. this strong effect is explained by signi cant change in the redox midpoint potential and change in the position of a by the mutations. i. s. zaharieva department of biophysics and radiobiology, faculty of biology, university of so a an approach to the investigation of structural and functional properties of the photosystem ii supramolecular complex in native photosynthesizing objects based on the registration of delayed chlorophyll a uorescence is developed. using a disc phosphoroscope, we register simultaneously: i) changes of the intensity of millisecond delayed uorescence (decayed in . - . ms time range) during the transition of the photosynthetic apparatus from dark to lightadapted state; ii) changes of the intensity of delayed uorescence decaying in different subintervals of the investigated time range; iii) dark relaxation curves at different moments of the transition; iv) changes of the intensity of prompt chlorophyll a uorescence. the analysis of these data allows the correlation of the delayed uorescence characteristics to particular processes occurring in the photosystem ii complex -proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein complexes caused by different stress factors, for example temperature. three-dimensional structure of major light-harvesting antenna of photosystem ii from cucumber h. yan , z. liu , k. wang , t. kuang , j. zhang , l. gui , x. an , w. chang national lab of biomacromolecules, institute of biophysics, chinese academy of sciences(cas), datun road, chaoyang distr., beijing , china, lab of photosynthesis and environmental molecular physiology, institute of botany, cas, nanxincun, xiangshan, beijing , china the major light-harvesting antenna complex of photosystem ii (lhc-ii), the most abundant integral membrane protein, functions in light capture, energy transfer/distribution and photoprotection. lhc-ii from different species or conditions shows different spectral properties and variation in polypeptide and pigment components. this indicates some speci c function-related alterations in the organization of lhc-ii. here we report a . -Å crystal structure of cucumber homo-trimeric lhc-ii, organized in a perfect virus-like icosahedral particle. the electron-density map shows the reasonable existence of a chlorophyll (chl) a/b mixed binding site in the complex. the occurrence and locus of lactucaxanthin (lac) was seen directly for the rst time. based on the credible structure information, a mechanism of the energy transfer, regulation and excess excited energy dissipation under high light condition was proposed. coherent anti-stokes raman scattering microscopy (cars) is a new approach for chemical imaging of molecular systems within cells and tissues, with high sensitivity, high spatial resolution, and three dimensional sectioning capabilities, without using uorophores that are prone to photobleaching. this technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. the epi detected (e-cars) and forward detected (f-cars) intensities depends on the shape, the size of the sample, as well as the index of the solvent. in this presentation, after introducing the cars microscopy technique, we show the rst cars studies of the refractive effect of the sample, comparing the e-cars and f-cars signals for different diameters of polystyrene beads, in different refractive index solvents. we present several simulations, comparing forward-detected and backward-detected signals in different sized polystyrene beads, embedded in different index solvents, and we show that, the backwardre ected f-cars dominates the experimentally epi-detected signals. furthermore, we demonstrate experimentally and theoretically that the maxima of forward and epi-detected signals are generated at different positions along the z axis in the sample. we nally discuss how index mismatch in cells can alter cars images. the effects of static magnetic elds on humans have been the subject of continuous investigations. since one of the major static magnetic eld sources is nuclear magnetic resonance imaging (mri), the present study aimed to investigate the effects of . t magnetic eld that is produced by mri on humans. the study is carried out with voluntary and healthy young men from to years of age. the subjects informed about the purpose of the study at the beginning. the subjects exposed to minutes of . t static magnetic eld by means of putting the subjects into the magnetic resonance unit. ml blood was taken from each subject one minute before and one minute after exposure. t and t relaxation times and trace elements were measured in of pre and post exposure plasma of the subjects. the obtained post exposure values were compared with pre-exposure values of the subjects. pre and post exposure results were analyzed by means of student t-test. evaluation of tumor response of breast cancer patients by diffusion weighted mri k. a. danishad , v. seenu , u. sharma , p. k. julka , g. k. rath , n. r. jagannathan department of nmr, department of surgery, department of radiotherapy, all india institute of medical sciences, new delhi, india diffusion weighted mr imaging (dwi) measures the diffusion of water molecules in tissues and is quanti ed by apparent diffusion coef cient (adc). dwi can be used to differentiate tumors from normal tissue and also can be used to monitor the response of tumor to chemotherapy. thirteen healthy volunteers and twelve patients were recruited for the study. dw images were obtained prior to therapy (n= ) and after three cycles of therapy (n= ). the mean adc value of tumors ( . x . mm /s) was signi cantly less (p < . ) compared to the normal tissue ( . x . mm /s). decrease in adc in tumor is due to an increase in the cellularity which restricts the diffusion of water molecules. in patients receiving neo-adjuvant chemotherapy, the adc values were higher ( . x . mm /s) and were closer to that of the normal tissue (p < . ), indicating response of the tumor to chemotherapy. the post-therapy increase in adc is due to the cell damage caused by the therapeutic agents which increases the fractional volume of the interstitial space, causing an increase in the mobility of water. the study showed that dwi can be used non-invasively to assess the response of breast cancer patients to neo-adjuvant chemotherapy. quanti cation by optical imaging of gene electrotransfer in mouse muscle and knee optical imaging was evaluated for monitoring and quanti cation of the mouse knee joint and tibial cranial muscle electrotransfer (et) of a luciferase encoding plasmid. the substrate of luciferase (luciferin) was injected i.p or locally in the muscle or the knee joint. luminescence resulting from the luciferase-luciferin reaction was measured with a cooled ccd camera. luminescence of the knee joint and muscle were higher after local than after i.p injection of luciferin, but both measurements were highly correlated. local injection procedure was adopted. a signi cant correlation was observed between measurements in vivo and in vitro on the same muscle. reproducibility of individual luminescence measurements was also veri ed, and the luminescence levels were clearly dependant of the amount of plasmid injected. in vivo luciferase in the electrotransfered knee joint was detected for two weeks. intramuscular electrotransfer of . or µg of plasmid led to stable luciferase expression for days, whereas injecting µg plasmid resulted in a luminescence fall two weeks after electrotransfer. these decreases were, at least partly, related to the production of antibodies against luciferase. thus, optical imaging was shown to be a relevant technique to quantify variations of luciferase activity in vivo in one given tissue. furthermore, evaluating the effective amount of luciferase in tissues from in vivo luminescence levels requires calibration since it relies on conditions of the enzymatic reaction and light absorption. acute effect of corticosterone on nmda receptormediated ca + elevation in mouse hippocampal slices m. saito , s. sato , h. osanai , a. hirano , y. komatsuzaki , s. kawato department of physics and applied physics, college of humanities and sciences, nihon university, department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo corticosterone (cort) is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. we examined the rapid effects of cort on n-methyl-d-aspartate (nmda) receptor-mediated ca signals in adult mouse hippocampal slices by using ca imaging technique. application of nmda caused a transient elevation of intracellular ca concentration followed by a decay to a plateau within sec. the min preincubation of cort induced a signi cant decrease of the peak amplitude of nmda-induced ca elevation in the ca region. the rapid effect of cort was induced at a stress-induced level ( . - µm). because the membrane non-permeable bovine serum albuminconjugated cort also induced a similar rapid effect, the rapid effect of cort might be induced via putative surface cort receptors. in contrast, cort induced no signi cant effects on nmdainduced ca elevation in the dentate gyrus. in the ca region, cort effects were not evaluated, because the marked elevation of nmda-induced ca signals was not observed there. in vivo subcellular structures recognized with phase k. nagayama , r. danev , n. usuda , y. kaneko , k. nitta , a. nakazawa , k. atsuzawa , m. tanaka , m. setou okazaki institute for integrative bioscience, okazaki, japan, fujita health university, school of medicine, toyoake, aichi, japan, saitama university, saitama, japan, tokyo metropolitan institute of gerontology, itabashi-ku, tokyo, japan phase contrast transmission electron microscopy has been developed to enable a high contrast and a high resolution observation for unstained ice-embedded samples. to enhance the image contrast, two methodologies have already been developed; i) scattering contrast for stained samples with small aperture diaphrams and ii) defocus contrast for unstained or stained samples with deep defocusing. the former prevails in histochemical sciences and the latter is popular in electron crystallography. both methods, however, have a common drawback that the contrast is only improved by impairing the image quality. this drawback can be removed with use of the phase contrast method using phase plates, which has traditionally been used in visible light microscopy. due to the severe obstacle of the charging of phase plates, however, the idea has not yet been materialized. we have solved the phase-plate charging problem. an experiment kv with tem for a whole cell from cyanobacterium unstained and ice-embed ful lled the expectation. only weak and vague contrast was obtained for the conventional image of the cell even with a very deep defocus. contralily a high-contrasted image has appeared for phase contrast images, where various ne structures are clearly recognized. this may be a rst example to observe nanometer scale structures in details in the intact cell. other examples including intact state intravesicular structures will be shown. j. lichtenberger, p. fromherz max-planck-institute for biochemistry we cultured bovine chromaf n cells on an array of electrolyteoxide-silicon eld-effect transistors (eos fet) and monitored granule secretion. by stimulation with barium chloride, vesicles are released into the narrow sheet of electrolyte between the chip surface and the plasma membrane. the interaction of released protons with the silicon dioxide surface of the chip alters the threshold voltage of the transistor and gives rise to a measurable signal. simultaneously performed measurements with a carbon bre showed a correlation of the transistor signals and amperometric current traces. we conclude that the transistors are able to monitor exocytosis on a single vesicle level. to elucidate the role of protons, we destroyed the proton gradient across the vesicle membrane by nigericin and valinomycin. as a result a massive reduction of the transistor signals was induced, whereas there was only little change of the amperometric records. we conclude that released protons are responsible for the detection of vesicles with transistors. the individual transistor records of vesicle exocytosis can be explained by combining the dynamics of the exocytotic event with the diffusion in the cell-chip junction. transistor recording of exocytosis does not depend on the electrochemistry of transmitters. as many kinds of exocytotic vesicles contain a large amount of buffered protons it can be applied to numerous kinds of exocytotic events, independent on the nature of the transmitter. we tried various solvents for the solubility of the uorescent product, and found that the product was insoluble in water and most organic solvents. a quite bright uorescence emitted by the particles was observed by uorescent microscope when emitted by uv nm. sem indicated that the size of the particles was µm µm, depending on the reaction time and phospholipid concentration in hexane solution. endothelial cells from human vein grew better on the surface prepared from the particles than the culture plate, implies a possible application as a new type of biomaterial as a coating material for medical devices, and as a uorescent tracer for human bodies. confocal microscopy of the phototactic ciliate f. salina. fabrea salina is a marine heterotrich ciliate, which dwells in salt ponds. in previous works we have described the phototaxis and the uorescence properties of a hypericin like endogenous pigment in an albino strain. we have recently obtained a heavily pigmented strain from the saline of torre colimena (taranto, italy). we have used confocal microscopy to characterize the uorescent properties of this strain and to compare them with those of the albino strain. the results obtained by one and two photon confocal microscopy show that, as in the albino strain, the uorescence intensity of the pigment is higher in dead cells than in the living cells. the excitation and emission spectra are quite similar in the two strains and this is also true for the uorescence lifetime, which is about ns. all together, these measurements indicate that the pigment of the new strain belongs to the family of hypericin-like chromophores. the analysis of different confocal planes shows that the pigment is localized not only in granules under the somatic membrane in the cellular body, as currently thought, but also in the cilia. some experiments of fotobleaching "in situ" con rm this result, that might have important implications in the understanding of the mechanisms of the photomotile responses of f. salina and probably of other heterotrich ciliates. there is no doubt that modern physician should have the knowledge of basic sciences as physics, chemistry and biology. furthermore, the biophysics is incorporated into the curriculum of most european medical schools. at medical school in zagreb, the course of physics and biophysics is positioned in rst and forth year of study. the students learn basic physics phenomena of structure of matter, mechanics, thermodynamics, electromagnetism, optics and acoustics applied on the human body. additionally, the interactions of the body with the surrounding are thoroughly discussed as the basis for different diagnostic methods. the arguing at our school is still going on where to include the content of this course. should it be the autonomous course or the part of physiology and radiology courses in the problem based learning approach?! so far at zagreb medical school the biophysical courses are autonomous structured according to the biophysics programs at other european universities. the highlighting is on seminar work and lab, encouraging the students for individual learning. the seminars are made more vivid and instructive for students by inclusion of different model devices constructed in our department learning on science is also learning how scienti c knowledge is produced. in this sense, issues related to the dynamics of science should be brought into focus in science education. the idea has support in the "declaration on science and the use of scienti c knowledge", particularly in the statement that "science curricula should include science ethics, as well as training in the history and philosophy of science and its cultural impact". thinking on rst year undergraduate students, we are interested in an integrated approach of that kind of themes. this presentation describes a practical teaching module included in the basic biophysics course for biochemistry majors. organized in case studies, it deals with stories of biophysics (and biochemistry), and addresses the role of the biophysical approach in the progress of the life sciences. the module follows the whole course, and consists in small exercises on the way a given understanding has been constructed explored within the practice trend of contemporary science studies. examples of the chosen stories -anchored in the subjects covered in the lectures of the course -relate to the search for the mechanism of energy production through atp-synthase, the development of radioactive labelling techniques, and the discovery of protein water channels. beyond their value as cultural legacy and as motivating tools, the insight they might provide is vast. axiomatic theory of biophysics q. zhao china rehabilitation research centre, beijing, china. until now, all approaches to interpret biology by applying principles of physics have been announced failure. the achievement of biophysics is limited in area to provide essential tools for biological research and biophysics is far from to be the basic theory of biology. to resolve it requires the fundamental research about the logic features of biophysical processes of biology. we promoted system logic and then protein thermodynamics structure theory. based upon it, the axiomatic theory of biophysics and biology could be developed. our result shows that the real understanding of biology or biophysics must be constructed based upon new thinking methods. a successful ligand-receptor docking methodology depends strongly in the ef ciency of the global optimization algorithm used to explore the ligand conformational space. in this work we have implemented and analyzed the performance of a new exible ligand-receptor docking methodology. this methodology uses as optimization method a multisolution version of the generalized simulated annealing algorithm adapted to problems with box constraints. a grid-based methodology, considering the receptor rigid, and the gromos classical force eld are used to evaluate the ligand-receptor scoring function. the methodology was tested in redocking (ligand within it own protein conformation) and cross-docking (ligand within another protein conformation) experiments for ve hiv protease-ligand complexes with known threedimensional structures. all ligands tested are highly exible, having to conformational degrees of freedom. the implemented docking methodology was able to redock successfully all exible ligands with a success ratio % and a mean rmsd lower than . Å with respect to the corresponding experimental structures. in the cross-docking experiments we observed a strong dependence of the mean success ratio with respect to the protein structure used as reference. in situations we observed a mean success ratio % and % in cases among the possible ones. s. aida-hyugaji , h. nakagawa , j. nomura , m. sakurai , d. tokushima , t. takada , u. nagashima , t. ishikawa tokai university, japan, tokyo institute of technology, japan, nec corporation, japan, national institute of advanced industrial science and technology, japan irinotecan is a widely-used antitumor drug that inhibits mammalian dna topoisomerase i. however, overexpression of abcg can confer cancer cells resistance to sn- , that is, the active form of cpt- . in the present study to develop a platform for the molecular modeling to circumvent drug resistance associated with abcg , we have characterized a total of fourteen new sn- analogues by some typical properties, which were evaluated by molecular orbital (mo) calculations and neural network (nn) analysis. the nn was rst applied to estimate hydrophobic properties (logp) of the analogues. thereafter, the electrostatic potential (esp) and the solvation free energy ( g) were evaluated by mo calculation. these indexes were found to be well correlated with the drug resistance ratio experimentally observed in abcg -overexpressing cells. it is suggested that hydrophilic analogues carrying oh-or nh -groups are good substrates for abcg and therefore exported from cancer cells. in contrast, sn- analogues with cl or br atom at those positions have similar logp values and high af nities toward the putative active site of abcg , however they were not substrates of abcg . from these results, it is strongly suggested that hydrogen bond formation with oh-or nh -groups are critically involved in the transport mechanism of abcg . a. agopian , j. depollier , e. gros , g. aldrian-herrada , p. clayette , n. bosquet , g. divita crbm-cnrs, route de mende, montpellier, france., spi-bio-cea fontenais aux roses, france reverse transcriptase (rt) plays an essential role in the replication of hiv and constitutes the main target for the development of aids therapies. the biologically active form of hiv rt is a heterodimer of two subunits, p and p , each consisting of distinct subdomains: the ngers, the palm, the thumb, the connection and the rnase h subdomain, the latter only present in p . we have demonstrated that formation of fully active rt is a two-step process involving rapid association of the two subunits (dimerization) followed by a conformational change (maturation). thanks to the crystal structure of rt we have identi ed a new class of inhibitors based on short peptide motifs derived from the dimer interface. we rst identi ed a short mer peptide (pep- ) derived from the tryptophanrich motif of the connection subdomain that blocks dimerization of rt and ef ciently abolishes hiv- replication. pep- interacts preferentially in a pocket involving residues trp and phe on p . we then designed mer peptides derived from the thumb domain which inhibit rt maturation as well as viral replication when delivered into cells. taking into account these results we propose that dimerization of rt constitutes a potential target for the design of more speci c new antiviral drugs. . the success of gene therapy largely relies on the availability of vectors that would deliver the genetic material ef ciently to the target cells with a minimal toxicity. in this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight ( kda) chitosan derivatives grafted with dodecenoyl (ddc) groups at different percentages ( , , and %). in the absence of dna, the critical micellar concentration (cmc) of these derivatives in mm mes buffer ph . was found to be strongly dependent on the percentage of ddc but not on ph or salt concentrations. this indicates that the ddc groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with dna. next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid dna with these derivatives at different ph, salt concentrations and n/p ratios (expressed in charged units of chitosan amines to dna phosphates). we found the smallest and more positively charged complexes were obtained at ph . and n/p= in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the dna phosphate groups. biophysical and biological examination of dna/lipids complexes particles of virus-like structure designed for in vivo gene transfer d. durand , m. schmutz , b. lebleu , a. r. thierry lure, centre universitaire paris sud, orsay, france, institut henri sadron, strasbourg, france, laboratoire des défenses antivirales et antitumorale, umr , montpellier, france the structure of complexes made from dna and suitable lipids (lipoplexes lx) was examined by cryo electron microscopy. we observed a distinct concentric ring-like pattern with striated shells when using plasmid dna. these spherical multilamellar particles have a mean diameter of nm with repetitive spacing of . nm with striation of . nm width. small angle x-ray scattering (saxs) con rmed cryoem data and revealed repetitive ordering of . nm, suggesting a lamellar structure containing at least a dozen layers. this concentric and lamellar structure with different packing regimes was also observed by cryoem with linear dsdna, ssdna and oligodeoxynucleotides. for the rst time, dna chains could be visualized in dna/lipid complexes. such speci c supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of dna in a liquid crystalline phase. cryoem of t phage dna packed either in t capsides or in lipidic particles showed similar patterns. saxs suggested an hexagonal phase in lx-t dna. thus, both lamellar and hexagonal phases may coexist in the same lx preparation or particle and transition between both phases may depend upon equilibrium in uenced by type and length of the dna used. organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry. engineering self-assembly peptides for targeted delivery of therapeutics and imaging agents s. s. dhadwar, m. sung, k. kawamura, j. gariépy department of medical biophysics, university of toronto, canada peptide-mediated delivery systems have recently emerged as a means to substitute or augment conventional drug and gene delivery technologies. these approaches are versatile and easily designed to incorporate a number of speci c attributes required for ef cient delivery of therapeutic and imaging agents. in particular, self-associating peptide domains can be utilized to construct stable and structurally well-de ned protein-like assemblies displaying a series of cell-routing functions. more speci cally, a peptide-based self-assembling intercellular delivery vehicle was designed by incorporating the -residue long tetramerization domain of the human tumor suppressor protein p (hp tet). the resulting peptide tetramer displays termini within its structure that allows for the simultaneous presentation of distinct cell targeting signal or functional domains. the fusion of polycationic sequences to the hp tet domain promotes the cellular import of the resulting constructs into eukaryotic cells. this internalization event was dramatically enhanced for such multivalent peptides in relation to their monomeric counterparts. peptides containing a nuclear localization sequence along with a polycationic sequence were found to shuttle reporter plasmids ef ciently to the nucleus of cells. these results have important implications in the design and construction of novel targeted delivery vehicles. mechanisms of non-covalent peptide mediated cellular delivery of therapeutics: a biophysical study s. deshayes , m. c. morris , a. heitz , p. charnet , g. divita , f. heitz crbm -cnrs fre montpellier france, cbs -cnrs umr -inserm u montpellier france two different cell-penetrating peptides mpg and pep- were shown to promote non-endosomal intracellular delivery of non-covalent bound cargos, namely nucleic acids and proteins; respectively. in order to identify the peptide mediated internalization pathway, we undertook conformational investigations of both peptides with and without associated cargos and checked the conformational consequences of the presence of phospholipids. from the conformational point of view, pep- behaves differently from mpg. cd analysis revealed a transition from a non-structured to a helical conformation upon increase of the concentration while mpg remained nonstructured. determination of the structure by nmr showed that in water, it's a-helical domain extends from residue to . cd and ftir indicated that pep- adopts a helical conformation in the presence of phospholipids while mpg is in a -sheet form. adsorption measurements performed at the air-water interface were consistent with the helical form. pep- did not undergo conformational changes upon formation of a particle with a cargo peptide. in contrast, we observed a partial conformational transition when the complex encountered phospholipids. for mpg, interactions with nucleic acids generated a partial folding into -sheet which was more pronounced in the presence of lipids. electrophysiological measurements showed that both peptides, whether associated or not with their cargo, can induce transmembrane pore-like structures. self-assembly of hydrolysed alpha-lactalbumin into nanotubes j. f. graveland-bikker , k. g. de kruif nizo food reseach, ede, netherlands, van´t hoff laboratory, netherlands nanotubes are formed by self-assembly of partially hydrolysedlactalbumin, a kda milk protein. there are several promising applications of these -lactalbumin tubes, in food, pharmacy and nanotechnology. we studied the mechanism of self-assembly, the structure and the properties of the nanotubes. limited proteolysis of the -lactalbumin (by a serine protease) makes the molecule prone to self-assembly. in the presence of ca tubular structures are formed. other divalent ions like mn and zn can also induce tubular self-assembly, while mg leads to random aggregation. light scattering showed that the self-assembly is reversible, which is of relevance for controlled release applications. on the other hand, we could also make stable tubes by cross linking, which would be a requisite for several other applications. from afm and saxs measurements, we obtained values for the outer diameter: nm; and the inner diameter: nm. afm and cryo-em revealed the helical structure of the tube wall; it is a right-handed helix. by performing nano indentations with afm we determined mechanical properties of the tubes. the tubes were shown to be relatively resilient upon small deformations; the elastic modulus is of the order of . gpa. targeted delivery of photosensitizers into the cancer cell nuclei enhances their cytotoxic ef cacy the search for new pharmaceuticals has raised interest in locallyacting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities, e.g. photosensitizers used in anticancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. earlier we have produced a number of modular recombinant transporters for locally-acting drugs comprising several functional modules for cell-speci c targeting, internalization, escape from intracellular acidic vesicle, and targeting to the nuclei of melanoma cells overexpressing melanocortin receptors. here we describe new transporters on the basis of epidermal growth factor which are speci c for a wide variety of cancers. these transporters possess all necessary functional activities and deliver photosensitizers into the nuclei of human carcinoma cells to result in photocytotoxic effects almost orders of magnitude greater than those of nonmodi ed photosensitizers. characterization of mixtures of dna and nonionic polymeric agents for gene delivery in muscle j. m. gau , j. lal , l. auvray laboratoire mpi -lrp, umr cnrs , université d´Évry val d´essonne, Évry cedex, france, argonne national laboratory, ipns, south cass avenue, illinois , usa a strategy to cure muscle disease is to introduce genes (dna) into the muscle cell to correct or to add genes. nonionic polymeric agents have emerged as an ef cient vector to deliver dna in the muscle. these polymers protect dna from extracellular nuclease degradation by allowing the dna diffusion throughout the muscle tissue. there is at present no understanding about how nonionic polymers enhance transfection in the muscle. the kind of interactions between these nonionic agents and dna, dna-nonionic polymeric agent mixtures and cell membrane are currently unknown. also the structure of dna-nonionic polymeric agent mixtures is not yet well de ned. more information is needed to improve this delivery system. neutron scattering (contrast variation) and light scattering were used to investigate the interaction between: dna and nonionic polymers (pvp, di-and triblock copolymers). furthermore, electrical measurements with the same polymer complexes and black lipid membrane were also performed. depending on the polymer type there is either direct interaction with dna or in other cases polymers exhibit strong interaction with the lipid membrane. an explanation for transfection ef ciency of these nonionic agents in gene delivery to muscle will be given. high throughput in-silico screening against exible protein receptors b virtual screening of chemical databases to targets of known threedimensional structure is developing into an increasingly reliable method for nding new lead candidates in drug development. based on the stochastic tunneling method (stun) we have developed flexscreen, a novel strategy for high-throughput in-silico screening of large ligand databases. each ligand of the database is docked against the receptor using an all-atom representation of both ligand and receptor. in the docking process both ligand and receptor can change their conformation. the ligands with the best evaluated af nity are selected as lead candidates for drug development. using the thymidine kinase inhibitors as a prototypical example we documented the shortcomings of rigid receptor screens in a realistic system. we demonstrate a gain in both overall binding energy and overall rank of the known substrates when two screens with a rigid and exible (up to sidechain dihedral angles) receptor are compared. we note that the stun suffers only a comparatively small loss of ef ciency when an increasing number of receptor degrees of freedom is considered. flexscreen thus offers a viable compromise between docking exibility and computational ef ciency to perform fully automated database screens on hundreds of thousands of ligands. maturation and inhibitor design of sars-cov cl protease based on a product-bound crystal structure severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. here we report that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the c-terminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the crystal structure of this product-bound form shows that the active site has a p pocket that binds the gln side chain speci cally. in addition, the p and p sites are clustered together to accommodate large hydrophobic side chains. the tagged c a mutant protein served as a substrate for the wildtype protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of t analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme (k d = . nm) than for the mutant (c a) containing the n-terminal (k d = . nm) or the c-terminal extra amino acids (k d = . nm). taken together, the study here provides insights to the design of our new structure-based inhibitors. nevertheless, a signi cant proportion of patients do not respond to this therapy, and adverse effects are common. here we report the delivery and expression of recombinant mycobacterial dna vaccines in vivo and demonstrate the ability of multicomponent dna vaccines to enhance th -polarized immune responses. splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against bladder tumour cells. we used four combined recombinant bcg dna vaccines (multi-rbcg) for electroporative immunotherapy in vivo, and found that tumour growth was signi cantly inhibited and mouse survival was prolonged. increased immune cell in ltration and induction of apoptosis were noted after treatment with multi-rbcg alone, with the interleukin- (il- ) vaccine alone, and-most signi cantly-with their combinations. thus, electroporation immunogene therapy using multi-rbcg plus il- may be an attractive regimen for the treatment of bladder cancer. this approach presents new possibilities for the treatment of bladder cancer using recombinant bcg dna vaccines and il- dna vaccine. the cell-penetrating peptide (cpp) pep- is capable of introducing large proteins into different cell lines, maintaining their biological activity. two mechanisms have been proposed to explain the entrance of other cpps in cells, endosomal-dependent and independent. we evaluated the molecular mechanisms of pep- mediated cellular uptake of -galactosidase ( -gal) from e. coli, in large unilamellar vesicles (luv) and hela cells. fluorescence spectroscopy and immuno uorescence microscopy were used to study the translocation. internalization of -gal into luv and protein functionality in hela cells were detected by enzymatic activity. -gal translocated into luv in a transmembrane potentialdependent manner. likewise, -gal incorporation was extensively decreased in depolarized cells. furthermore, -gal uptake efciency and kinetics were temperature-independent and -gal did not co-localize with endosomes, lysosomes or caveosomes. therefore, -gal translocation was not associated with the endosomal pathway moreover transmembrane pores were not detected. these results indicated that the protein uptake in vitro and in vivo was mainly, if not solely, dependent on a physical mechanism governed by electrostatic interactions between pep- (positively-charged) and membranes (negatively-charged). peptide the dramatic acceleration in the identi cation of new nucleic acidbased therapeutic molecules has provided new perspectives in pharmaceutical research. however, the development of nucleic acidand peptide-based therapeutics is limited by their poor cellular uptake and traf cking. with the aim of addressing these issues, we have designed a family of short amphipatic peptides for delivery of nucleic acids (mpg) and of peptide/pna (pep). these carriers consist of a hydrophobic moiety and a nls-derived hydrophilic domain. they form stable non-covalent complexes with peptides, proteins, sirna or pna without any requirement for prior covalent cross-linking. both mpg and pep carriers enter cells rapidly, in a process involving membrane disorganization, independently of the endosomal pathway. mpg ef ciently delivers short odns and sirna into a wide variety of mammalian cell lines, without interfering with their biological function. pep signi cantly improves delivery of pna and peptides. both carriers were used for the delivery of sirna or antisense pna targeting the cell cycle regulatory protein cyclin b in an animal model and were found to block tumor growth upon intravenous injection. we believe that mpg and pepbased technologies will contribute signi cantly to the development of basic and therapeutic applications. probing the bound conformation of acetylcholinesterase (ache) inhibitor at the binding site c. g. kim, x. zhao, s. goodall, a. watts department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk acetylcholinesterases are the enzymes which preferentially hydrolyze acetyl esters (such as ach or acetyl-â-methylcholine), containing amino acid residues in the eeache form and arranged as a -stranded â-sheet surrounded by á-helices. the protein is ellipsoidal in shape, with approximate dimensions of Å by Å by Å. inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of alzheimer's disease. an understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and speci city. interaction between the ligand, amino- -methyl- -( tri uoroacetylbenzyl-oxymethyl)quinoline (r ), and ache inhibitor has been studied by advanced solid-state nmr through double-quantum chemical shift and distance measurements. combining solid-state nmr data and docking simulations, conformation of the ache inhibitor at the active site has been predicted. in vivo, heat shock proteins (hsps) being stress-inducible chaperones can attenuate detrimental consequences of ischemic insults, inammation, neurodegenerative diseases, etc. also, intracellular accumulation and chaperone activities of some hsps may contribute to improved cell survival following uv or ionizing radiation. in models of pathological states and their treatment, we used special virusbased vectors for overexpression of hsp or hsp in cell cultures to confer cytoprotection under simulated ischemia/reperfusion. in parallel, similar cytoprotection was achieved after pretreatments of the cells with a pharmacological hsp inducer, geranylgeranylacetone. the cytoprotective effects were manifested in the lesser extent of oxidative modi cation and aggregation of cellular proteins, better preservation of the cytoskeleton, faster restoration of energy metabolism and the improved post-stress cell survival. in the other model, we treated normal and tumor cells with an inhibitor of the chaperone activity of hsp , geldanamycin. only the drug-treated tumor cells became more sensitive to gamma-irradiation; such results characterize this drug as a potentially selective radiosensitizer of tumors. taken together our data demonstrate promising approaches to clinically bene cial manipulating the levels of expression and/or chaperone activity of hsp(s) by means of gene therapy or pharmacotherapy. characterizaton of proteins from human pleural uid r. jain, s. kumar, n. singh, s. sharma, t. p. singh all india institute of medical sciences, new delhi ,india the samples of human pleural uid were obtained from both healthy subjects and patients infected by tuberculosis. after the preliminary processing these samples were run in independent lanes of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the two lanes indicated variations in the intensities of a few bands and some new bands were also observed in the infected samples. these were characterized by determining their nterminal sequences. the new bands which had low density were carefully identi ed and cloned. some of the common bands that showed intensity variations were characterized. these were matrix metalloproteins, secretory phospholipasea , transferrin and ceruloplasmin. they were also studied with maldi-tof and their molecular weights have been determined. some of these proteins have been crystallized and their detailed crystal structure determinations are in progress. biophysical study of non-lethal stress response of cultured dc f cells stress factors may induce two kinds of responses in living cells: either cell death or adapting mechanisms. our aim was to search for non-lethal effects of various stress conditions on cultured hamster lung broblasts (dc f cells) as well as to assess the recovery time after stress removal. dc f cells were cultured in standard conditions and were submitted to stress, either by incubation with chemical substances (sodium arsenate, sodium nitroprusiate) and drugs (bleomicine and statins) either by irradiation (uv, he-ne laser). the doses and exposure times were chosen as to avoid cell death. after stress removal, cells were allowed to recover and the recovery time period was measured. structural and functional parameters were evaluated before and after stress, as well as during recovery. by now, experimental models for the in vitro study of non-lethal stress inducing factors have been set up. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. the viral maturation requires a main protease ( cl pro ) to cleave the virus-encoded polyproteins. we report here that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the cterminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the tagged c a mutant protein served as a substrate for the wild-type protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of the quaternary structures for the tagged and mature proteases by analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme than for the mutant (c a) containing the n-terminal or the c-terminal extra amino acids. taken together, the study here provides insights to the design of our new structure-based inhibitors. characterisation of macromolecular transport in physiologically relevant mixed ecm based gels s. lelu, a. pluen school of pharmacy and pharmaceutical sciences, university of manchester (uk) the extracellular matrix (ecm) a complex gel made of hyaluronic acid, collagen and proteoglycans (pg) impedes the penetration of macromolecules especially in tumours, and may compromise the success of novel therapies. though recent in vivo investigations pointed out that, not only ha but brillar collagen its content and organisation and its interactions with pg were involved in macromolecular transport hindrance, transport mechanisms relating the macromolecular drug and these ecm components are unknown. in this study we seek to evaluate the determinants of passive transport mechanisms of biomacromolecules in complex gels made of ha, collagen using uorescence techniques (frap and confocal re ection microscopy (crm)) and rheology. focus was on conditions relevant to tumours and, initially, on low collagen and relatively high ha content. rheology experiments showed that mixed systems containing less than mg/ml of ha present higher elastic modulus ge than pure ha network or pure collagen gels. interestingly crm and frap studies revealed similarities for collagen and mixed gels: the organisation and spacing of the collagen bres did not change and the ratio of the diffusivities (d/d ) of dextran m and igg were not different but higher than those in ha networks. systems with higher collagen content are under investigation to complete the characterisation of transport. encapsulation of clone vector dna by cationic diblock copolymer vesicles for gene delivery a. v. korobko , j. r. van der maarel leiden university, the netherlands, national university of singapore, singapore we will discuss the design, control, and structural characterization of cationic copolymer vesicles loaded with dna. these vesicles serve as a model system for diverse applications such as gene delivery, micro-arraying techniques and packaging of dna in congested states. encapsulation of dna was achieved with a single emulsion technique. for this purpose, an aqueous puc or pegfp-n plasmid solution is emulsi ed in an organic solvent and stabilized by an amphiphilic diblock copolymer. the neutral block of the copolymer forms an interfacial brush, whereas the cationic block complexes with dna. a subsequent change of the quality of the organic solvent results in a collapse of the brush and the formation of a capsule. the capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. inside the vesicles, the dna is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarisers and the increase in uorescence of labeled dna. the compaction ef ciency and the size distribution of the vesicles were determined by light and electron microscopy, respectively, and the integrity of the dna after encapsulation and subsequent release was con rmed by gel electrophoresis. we demonstrate the gene transfer ability of this new carrier system by the transfection of encapsulated pegfp plasmid into hela cancer cells. cellular transduction of nucleotide kinases to improve the activation of nucleoside analog prodrugs m. konrad , c. monnerjahn , s. ort , a. lavie max-planck-institute for biophysical chemistry, goettingen, germany, university of illinois at chicago, chicago, usa the objective of our study is to improve therapeutic enzymeprodrug systems by generating catalytically superior nucleoside and nucleotide kinases that are essential for activation of nucleoside analogs. compounds, such as azt for the treatment of hiv infections, acv and gcv used against herpes virus, or the anticancer compounds arac and gemcitabine, can enter cells only in the unphosphorylated state (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with dna replication. we have rst designed mutants of the human tmp kinase (htmpk) that phosphorylate aztmp up to -fold faster than wildtype. expression of this enzyme in human cells leads to -fold higher intracellular concentrations of azttp and to enhanced hiv inhibition. second, the prodrugs acv and gcv are not phosphorylated by human kinases, but are converted to their monophosphate forms by hsv -tk which is used in enzyme/prodrug-dependent cancer suicide gene therapy. we generated enzyme variants which show selective and ef cient phosphorylation of gcv. third, an engineered human dck variant catalyzes more ef ciently the activation of the prodrugs arac and gemcitabine. thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intracellular transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. docking-molecular dynamics studies on the peroxidase site of prostaglandin endoperoxide h synthase prostaglandin endoperoxide h synthases- and (pghs- and ) catalyze the rst step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. arachidonic acid is transformed into prostaglandin g (pgg ) at the cyclooxygenase site of the enzyme and the -hydroperoxide oxygen-oxygen bond of pgg is subsequently cleaved by reaction with haem at the distinct peroxidase site (pox) to produce prostaglandin h (pgh ). herein we present a plausible productive conformation obtained by docking calculations for the binding of pgg to the pox site of pghs- . the enzyme-substrate complex stability was veri ed by a -ps molecular dynamics simulation. structural analysis unveils the requirements for enzyme-substrate recognition and binding: the pgg -hydroperoxide group is in the proximity of the haem iron and participates in a hydrogen bond network with the invariant his and gln and a water molecule, whereas the carboxylate group establishes salt bridges with the remote lysines and . the interaction of the peptide lah with anionic lipids during dna/rna delivery to eukaryotic cells a. j. mason , a. martinez , c. leborgne , a. kichler , b. bechinger faculté de chimie, université louis pasteur, strasbourg, france, généthon, evry, france the histidine rich amphipathic peptide lah has antibiotic and dna delivery capabilities. the peptide has a strong af nity for anionic lipids found in the outer membrane of bacterial membranes and has shown evidence of higher transfection activity against transformed over healthy tissue in culture. it has been proposed that anionic lipids can ip-op to reach the cytoplasmic monolayer. here they neutralise the cationic transfection complexes thereby causing release of oligonucleotides into the cytoplasm. we were, therefore, particularly interested to test for the role of the acidic lipid phosphatidylserine (ps) in mediating lah -mediated delivery of dna ef ciency. to understand the potential peptide-lipid interactions in more detail, solid-state nmr experiments on model membranes have been performed. p mas nmr on mixed phosphatidylcholine (pc)/ps and pc/phosphatidylglycerol (pg) membranes has been used to investigate speci c lah interactions with anionic lipids. by using deuterated lipids and wide-line h nmr when probing lipid chain order, it is demonstrated that lah preferentially interacts with ps over pc. lah thereby effectively disorders the anionic ps lipid fatty acyl chains. the lipid chain destabilising effect of lah and also lah analogues can then be compared with their transfection ef ciency for dna or sirna in cell culture to aid in rational peptide vector design. virtual screening is now widely accepted as a basis for drug discovery thanks to signi cant improvement and good hit rates [ ]. however, it is still highly cpu-consuming. at the same time, the number of protein-ligand complexes described at the atomic level is rising and the sequence similarity is used for structure and function predictions. new approaches are being developed to take advantage of the available structural data and the huge number of protein sequences in order to allow better tuned virtual screening. new web servers are being built to ease and to speed up the whole process (http://abcis.cbs.cnrs.fr/kindock/). integrating these servers into a pipeline dedicated to molecular modelling (http://abcis.cbs.cnrs.fr/atome/) shall allow both the re ned validation of modelled active sites as well as the oriented screening for the primary caracterization of potential ligands. an ideal drug delivery system should own the following characteristics, the rst is the targeting of therapeutic agent to the speci c site of their action. the second is the controlled delivery of a therapeutic molecule or protein in a pulsatile or staggered fashion. the third is the achieving sustained zero-order release of a therapeutic agent over a prolonged period of time. in this study, a new drug delivery system combined these characteristics was provided, which contains azobenzene derivatives (ab lipid) as an on-off switch incorporated into liposomes. the drastic release of calcein was observed on the rst uv irradiation of ab lipid to the cis isomer, while a suppressed release was observed when irradiated with the rst visible light. after that, the slope of release pro le became coincident. furthermore, calcein release was greatly increased after uv irradiation of ab lipid to the cis isomer and the drug release was greatly suppressed after vis irradiation of ab lipid to the trans isomer. we can control the release rate of calcein from ab lipid/egg pc mixed liposomes by uv or vis light irradiation. tryptophanase (trpase), a bacterial enzyme with no counterpart in eukaryotic cells, produces l-trp pyruvate ammonia and indole. it was suggested that indole is essential for bacteria multiplication and bio lm formation. bio lms destroy equipment and food and cause many illnesses. most synthesized quasi-substrates inhibit trpase at mm range. an optimal and speci c inhibitor of trpase may eliminate indole production and prevent bio lm formation. x-ray crystallography of the holo-wt e. coli trpase soaked with l-trp and the known mechanism of trpase activity should provide the information for the design and synthesis of active-sitespeci c quasi-analogs. utilizing the chromogenic substrate s-(onitrophenyl)-l-cysteine, the following michaelis-menten kinetics analyses determined the mode of trpase inhibition by trp and quinone based quasi-analogues and the corresponding ki values (in µm): dl- -alanyl- , anthraquinone, noncompetitively, ; trypthophan ethylester, competitively, ; acetyltryptophan, uncompetitively, . ; s-phenylbenzoquinone-l-tryptophan, uncompetitively, . plp-l-trp, inhibited irreversibly only the apo form of trpase and may serve for structure-determination purposes. further attempts are being made to synthesize improved trpase inhibitors, i.e., in the nm range. polyelectrolyte multilayer lms (pem) adsorbed on biomaterial surfaces are a new way to create a controlled release system. using biodegradable polymers, the lms can be degraded in vivo and release active molecules. in this work, we demonstrate the possibility of tuning the degradability of polysaccharide pem in vitro and in vivo. chitosan and hyaluronan pem (chi/ha) were either native or cross-linked (cl) using a water soluble carbodiimide (edc) at various concentrations in combination with nhydroxysulfosuccinimide. the in vitro degradation of the lms in contact with enzymes was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy after lm labeling with chi fitc . whereas the native lms were subjected to degradation, the cl lms were more resistant to enzymatic degradation. films made of chitosan of medium molecular weight were indeed more resistant than lms made of chitosan-oligosaccharides. in addition, macrophages could degrade all types of lms and internalize the chitosan in vitro. the native lms implanted in vivo in mouse peritoneal cavity for a week showed an almost complete degradation whereas the cl lms were only partially degraded. these results suggests that the polysaccharides pem are of potential interest for in vivo applications as biodegradable coatings and that degradation can be tuned by controlling lm cross-linking. membrane electroporation -tool for therapeutic electrotransfer of drugs and gene dna e. neumann, s. kakorin physical and biophysical chemistry, faculty of chemistry, university of bielefeld, germany membrane electroporation (mep) is a new electrical high voltage scalpel, transiently opening the cell membranes of tissue for the penetration of foreign substances. due to the enormous complexity of cellular membranes, many fundamental problems of mep have to be studied at rst on model systems, such as the curved bilayer membranes of unilamellar lipid vesicles. electrooptical and conductometrical data of unilamellar liposomes indicate that electric eld pulses cause not only the formation of membrane electropores but also shape deformation of the liposomes, both processes mutually affecting each other. the primary eld effects of mep and cell deformation can trigger a cascade of numerous secondary phenomena, such as pore percolation and transport of small and large molecules across the electroporated membrane. the chemical mep theory represents a molecular physico-chemical approach to electrochemomechanical pore formation, yielding transport parameters, such as permeation coef cients, pore fractions and pore sizes. the pore concept is successfully applied to rationalize optimization strategies for biotechnological and medical applications of mep. in silico elucidation of xenobiotic processing loops k. nakata , y. tanaka , t. nakano , t. ishikawa , h. tanaka , t. kaminuma national institute of health sciences, tokyo, japan, tokyo medical and dental university, tokyo, japan, tokyo institute of technology, yokohama, japan, hiroshima university, hiroshima, japan one of the important challenges for drug designers is to predict and analyze how drugs are absorbed, distributed, metabolized and excreted (adme) in the body. these processes highly correlated with toxicity of drugs and are actively studied in pharmacology. two classes of proteins, the drug metabolizing enzymes such as cytochrome p s (cyps) and transporters, are the target of such adme/tox research. it was relatively recent that these two classes of proteins are synthesized by the genes that are the target genes of the nuclear receptors. nuclear receptors are ligand-activated transcription factors that form a superfamily. in case of humans there are nuclear receptors almost half of whose ligands are identied, leaving some as true orphans. thus it was now recognized that these nuclear receptors play the role of sensors of drugs and other xenobiotic substances including environmental chemical pollutants and nutritional ingredients, while the drug metabolizing enzymes and the transporters are the processors which carry the actual cleaning jobs. we have started to elucidate the feedback loops that are formed by the xenobiotic ligands, nuclear receptors, their target genes, their product proteins, and their feedback actions on the ligands. the work is being carried out on our background database on the ligands and their receptors called kibank, and search programs for target genes of nuclear receptors algorithmically. the most recent results will be presented at the presentation. the major route for drug entry into cells is permeation across lipid bilayers. due to methodological limitations there are only few studies on permeation of drug-like molecules across lipid bilayers. an assay developed in our lab allows the direct measurement of lipid bilayer permeation of aromatic carboxylic acids (acas). tb , which forms a uorescent complex with acas, is entrapped in liposomes and aca entry is determined from luminescence increase. lipid bilayer permeation was ph-dependent, following a henderson-hasselbalch function with a plateau for the neutral and the anionic species, respectively. in contrast to the expectations of the ph-partition hypothesis, permeation of the anionic species was only to magnitudes lower than that of the neutral species, leading to anion-controlled permeation at ph . , independently of bilayer state and lipid composition. permeation across bilayers with a biologically relevant lipid composition was signi cantly slower than across egg-phosphatidylcholine membranes. the in uence of single lipids, such as cholesterol, was dependent on the structure and ionization state of the permeant. permeation coef cients of the neutral species correlated better with the polar surface area (psa) than logp oct , therefore psa is a better predictor for bilayer permeation of the neutral species of small acas than logp oct . interplay between polymerized liposomes physicochemical properties and composition and citotoxicity this study was aimed at investigating whether there is an interplay between diacetylenic polymerized liposomes physicochemical properties and lipid composition affecting citotoxicity in vitro. unsaturated , -bis( , -tricosadiynoyl)-sn-glycero- -phosphocholine with saturated , -dimiristoyl-sn-glycero- phosphocholine in molar ratio : , were combined to give a chemically modi ed membrane by uv-polymerization. biophysical characterization was carried out determining the hydrophobic factor and hydrodynamic radius. citotoxicity was evaluated through haemolytic capacity on bovine red blood cells and indirectly by capacity of induction of lipid peroxidation on microsomes or mitochondrial membranes. the haemolysis percentage in presence of dc , pc/dmpc is less than that induce by polymers used in dentistry. the data obtain suggests that the polymerized lipids can not induce lipid peroxidation on natural membranes. the polymerized diacetylenic liposomes showed less interaction with serum proteins than non polymerized and lower citotoxicity as compared with natural lipids. also cell viability was determined in cell line nih t after exposure to lipids systems under study. the hydrophobic factor showed further augmentation for polymerized liposomes and is discussed in relation to in vitro stability. the above results suggest that polymerized and non-polymerized liposomes would serve as an effective delivery vehicle. s. sonar, s. d´souza, k. p. mishra radiation biology and health sciences division,bhabha atomic research centre,mumbai- , india liposomes offer new approaches for drug delivery through their encapsulation to alter pharmacodynamic properties of loaded drug leading to reduction in toxicities and/ or improved ef cacy. for prolonged systemic circulation, the liposomes size has been shown to be limited to nm or less. the ethanol injection method is an excellent technique for the formation of liposomes of < nm without the need of sonication or extrusion. the present study was aimed to produce liposomes encapsulating doxorubicin in minimum procedural steps. liposomes were prepared using distearoyl phosphatidylcholine and cholesterol, distearoyl phosphatidylcholine, cholesterol and oleic acid. the effects of different operational conditions for vesicle production and drug encapsulation were evaluated, with a view to achieve process cost to a minimum, suitable size and high encapsulation ef ciency. although high ef ciency of doxorubicin encapsulation was obtained by 'active' or 'remote' loading process in dspc/chol system, it was poor in one-step injection method. oleic acid was included to cut down the active loading by ph-gradient. dspc/chol/oa systems spontaneously loaded doxorubicin with encapsulation ef ciency of % and nal drug to lipid molar ratio upto . . the mean diameter of the vesicles was + nm. the method offers liposomes of small size with high loading. design of peptides with consecutive dehydro phenylalanine residues r in order to develop general rules for the design of peptide conformations with consecutive alpha, beta-dehydro phenylalanine-residues, peptides were synthesized, crystallized and crystal structures and molecular conformations were determined. following conclusions were drawn based on the structural data: -peptide unit sequences with two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions adopt an unfolded s -shaped structure with dihedral angles phi-psi centred at , . -the peptides containing two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions • form two overlapping type iii beta -turns (incipient -helix). • with branched beta -carbon residue only at (i+ ) position adopt a conformation with two overlapping types ii and iii beta -turns. • with branched beta-carbon residues such as val and ile at both (i+ ) and (i+ ) positions form two overlapping types ii and i beta -turns. the consistency in the formations of these conformations makes the design of peptides with alpha,beta ?-dehydro -residues a useful and highly predictable method for developing speci c ligands for various biological applications including drug design. binding of cationic porphyrin to isolated double-stranded dna and nucleoprotein complex k. zupán , l. herényi , k. tóth , z. majer , g. csík institute of biophysics and radiation biology, semmelweis univ., budapest, hungary" biophysics of macromolecules, german cancer research center, heidelberg, germany, department of organic chemistry, eötvös loránd univ., budapest hungary the complexation of tetrakis( -n-methylpyridyl)porphyrin (tmpyp) with free and encapsidated dna of t bacteriophage was investigated. to identify binding modes and relative concentrations of bound tmpyp forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. spectral decomposition, uorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of tmpyp, e. g., external binding and intercalation both in free and in encapsidated dna. tmpyp binding does not in uence the protein structure and/or the protein -dna interaction. concentrations of tmpyp species were determined by comprehensive spectroscopic methods. our results facilitate a qualitative analysis of tmpyp binding process at various experimental conditions. we analyzed the effect of base pair composition of dna, the presence of protein capsid and the composition of buffer solution on the binding process. protein crystallization and structural study of upa protease domain with active site serine mutation the urokinase (upa) system is composed of upa, its receptor (upar), and inhibitor (pai). it plays important role in various physiologic processes, including brinolysis, cell adhesion, and signal transduction, and has been recognized as a target for intervention in tumor growth and tumor metastasis. we constructed an active site mutant of upa protease domain ( - ) with three mutations (c a, n q, and s a) and expressed it as secreted protein in pichia pastoria with ppicza vector. the secreted mutant was captured from culture medium by a cation exchange column and then further puri ed on a gel ltration column. the puri ed mutant was then crystallized by sitting drop vapor diffusion method with several precipitant conditions: ( ) . - . m ammonium sulphate, - % peg , mm sodium citrate ph . or mm sodium phosphate ph . , . % sodium azide. ( ) . m ammonium sulphate, . m lithium sulphate, mm sodium acetate at ph . , ( ) . - . m sodium formate, mm sodium acetate at ph . . ( ) . - . m sodium chloride, mm sodium acetate at ph . . the crystals were of varying quality but generally diffracted from . Å- . Å with inhouse x-ray source. the structure of this upa mutant and its complex with various inhibitors will provide a platform for rational upa inhibitor design. abstract in this work, the linear interaction energy (lie) method was used to calculate the binding free energies of hiv- integrase (in) and a series of dicaffeoyl -or digalloyl pyrroliding and furan derivatives inhibitors. the model of binding free energy prediction for homogeneous inhibitors of hiv- in has been obtained with a root-mean-square deviation (rmsd) of . kj/mol and estimated to be a precise model with good prediction capability. in addition, the probable binding mode of this series of inhibitors with hiv- in was proposed by using molecular docking and molecular dynamics (md) simulation methods. our results indicate that caffeoyl -or galloyl group of inhibitors have close interaction with a hiv- in conservative dde motif. a speci c non-competitive inhibitor of a small g protein/gef complex on the protective role of selenium and catechin in cadmium toxicity s. Özdemir , s. dursun , s. toplan , n. dariyerli , m. c. akyolcu istanbul university, cerrahpasa medical faculty, department of biophysics, turkey, istanbul university cerrahpasa medical faculty, department of physiology, turkey cadmium as heavy metal is toxic and carcinogenic for organisms. cadmium perform their effects on living organisms by accumulation in blood and various tissues. due to their accumulation in various tissues and in blood, tissue antioxidant enzyme systems are affected. the present study was planned to determine the possible protective roles of selenium and catechin against the toxic effects of considered heavy metals. the study has been performed in wistar albino type rats which divided into four groups as control and cadmium, cadmium+selenium, cadmium+catechin received groups. besides cadmium as heavy metal, selenium concentration determinations were performed in blood, liver and kidney tissues of each group of rats. in the same tissue samples besides lipid peroxidation measurements, glutathione, glutathione peroxidase and superokside dismutase enzyme activity determinations were also performed. the accumulation of heavy metals was determined in blood, liver and kidneys after cadmium administration during experimental period. in the tissue of experimental group animals there was an increased lipid peroxidation but decreased antioxidant enzyme activities were observed. while effects of selenium in decreased toxicity of cadmium have been detected, there was no statistically signicant effect of catechin observed. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. we demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. we determine the motor density along a tube by using uorescence intensity measurements. we also perform a theoretical analysis describing the dynamics of motors and tube growth. the only adjustable parameter is the motor binding rate onto microtubules, which we measure to be . +/- . s . in addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. we nd that the number of motors pulling a tube can range from four at threshold to a few tens away from it. the threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traf c regulation in cells. kinesin and dynein move a peroxisome in vivo: a tug-ofwar or coordinated movement? c. kural, p. r. selvin, k. hwajin, g. goshima, v. i. gelfand university of illinois at urbana-champaign, usa we have used fluorescence imaging with one nanometer accuracy (fiona) for analysis of organelle movement by conventional kinesin and cytoplasmic dynein in a cell. we can locate a green uorescence protein (gfp)-tagged peroxisome in cultured drosophila s cells to within . nanometer in . milliseconds, a -fold improvement in temporal resolution, suf cient to determine the average step size to be nanometers for both dynein and kinesin. furthermore, we nd that dynein and kinesin do not work against each other in vivo during peroxisome transport. rather, we nd that multiple kinesins or multiple dyneins work together, producing up to times the in vitro speed. engineering a bio-molecular walker h. jankevics, j. e. molloy division of physical biochemistry, mrc national institute of medical research, the ridgeway, mill hill nw aa, london, uk in this work we describe the design and development of a biomolecular walker based on the motile system found in certain ciliated protists. the motile system is driven by the binding of ca ions and in contrast to other commonly studied motor proteins is independent of atpase (amos et al., ) . the motor protein is a kda ca-binding protein called spasmin (maciejewski et al., ; itabashi et al., ) which belongs to the ef-hand family of calcium binding proteins called calmodulins. upon calcium binding, spasmin is thought to undergo a large conformational change as it binds its own target peptide and we wish to exploit this in order to create our own novel molecular walker.we have created a recombinant spasmin with sequence tags to enable speci c immobilization and various conjugate chemistries. cystein mutants have been introduced at speci c points in the protein to enable attachment of other small molecules, for example uorophores. we are now optimising protein expression and puri cation to maximise the yield of active protein on which we can perform the conjugate chemistries. we will characterize the structural changes using biophysical methods such as circular dichroism, analytical ultracentrifugation, electron microscopy, afm and total internal re ection uorescence spectroscopy on single molecules. the force generation in muscle arises from direct interaction of the two main protein components of the muscle, myosin and actin. the process is driven by the energy liberated from the hydrolysis of atp by myosin. the interaction is performed by cyclic interaction of myosin with atp and actin, and at least six intermediates are proposed for actomyosin atpase in solution. the powerful dsc technique allows the derivation of heat capacity of proteins as a function of temperature. from the deconvolution of the thermal unfolding patterns it is possible to characterize the structural domains of the motor protein. in this work we tried to approach the temperature-induced unfolding processes in different intermediate state of atp hydrolysis in striated muscle bres. we have extended the experiments to study the ber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin where the inorganic phosphate (p i ) was substituted by the phosphate analogue orthovanadate. the dsc transitions were analyzed in different buffer solutions (tris and mops) to get information about the temperature dependence of ph on the conformational changes. single kinesin motor proteins walking through the searchlight s. verbrugge, l. c. kapitein, e. j. peterman vrije universiteit, de boelelaan , hv, amsterdam, the netherlands the dimeric motor protein kinesin steps by a hand-over-hand mechanism. this means that the centre of mass moves with nm steps, while the two motor domains, the one after the other, move nm to the next binding site on the microtubule. the molecular details of what happens during a step are not fully understood, partly because of lack of time resolution in wide-eld, single-molecule uorescence experiments. we set out to develop an approach to study the motility of kinesin with a time resolution below a millisecond (a single step takes on the order of milliseconds). this approach allows us to look into the mechanochemistry and coupling of the two kinesin motor domains while they are stepping. our method is based on confocal microscopy and we study the uorescent properties of single labeled motors while they walk through the confocal laser spot. we present the experimental details of our approach and show our results on human kinesin constructs that are speci cally labeled in the tail. we show that our approach enables us to study the mechanism of kinesin with a much higher time resolution than what was achieved before with single-molecule uorescence experiments. movement of coupled single-headed kinesins analysed by a brownian-ratchet model . the analysis of this system is expected to provide insights into the mechanism underlying the motility of conventional double-headed kinesin, espetially the roles played by individual heads. we would like to clarify whether the experimentally observed behaviors that are supposed to be caused by a pair of single-headed kinesins can be explained by a simple brownianratchet model, which is successful in describing the motion of an unconventional single-headed kinesin kif a. our model consists of two brownian motors (ratchets) separated by a xed distance r. the velocity and other quantities of the coupled motors are calculated by solving the fokker-planck equation with various choices of r and other parameters. then, assuming a certain probability distribution of r associated with random attachment of kinesin heads on a bead in the experiment, the statistical properties of the motion of the coupled brownian motors are analysed. the force-velocity relation observed experimentally is found to be consistent with the present model with appropriate choices of the model parameters. the adequacy of the parameter choice needs to be con rmed by other experiments. optical trap with fast programmable feedback loop to study rotary molecular motors t. pilizota, f. bai, r. m. berry clarendon laboratory, univeristy of oxford an optical trap with back-focal plane detection and fast programmable feedback has been developed for the study of rotary molecular motors. a helium-neon laser ( nm) is used for position detection and a solid state bre laser ( nm, w cw) forms the trap. acousto-optic de ectors (aods) controlled by a digital signalling processing board are used to achive programmable feedback loops with exible control options and speeds up to khz. several modes of feedback are demonstrated, controlling both bead position (x,y) and angle (r, ). polystyrene beads or bead pairs can be held at set (x,y) or , and the set-point can be changed while the program is running. for example, feedback can be used to move a bead or a bead pair in a circle. results of using the system to study the bacterial agellar motor are presented. a dimeric -d lattice gas as model for molecular motors collective dynamics p. pierobon , t. franosch , e. frey ludwing-maximilian universitaet, münchen, germany, hahn meitner institut, berlin, germany, arnold sommerfeld center for theoretical physics, münchen, germany the transport of molecular motors along microtubules closely resemble the dynamics of a driven lattice gas of dimers without conservation of particles. the unidirectionality, asymmetry and stochasticity of the motion are encoded in the well studied totally asymmetric simple exclusion process (tasep). we extend the model to a more realistic one, including attachment and detachment kinetics and extended (dimeric) particles. we study the stationary phase diagram by means of monte carlo simulations combined with a continuum description (based on an extended mean eld theory). we also evaluate the domain wall theory nding out the effective potential con ning the phase interface into the bulk. a. e. wallin , j. lisal , r. tuma department of physical sciences, pobox , fin- , university of helsinki, finland, institute of biotechnology, university of helsinki, finland molecular motors often consist of two or more subunits that cooperate to convert chemical energy into mechanical motion. hexameric helicases and viral packaging atpases constitute a special class of molecular motors that translocate along nucleic acids. recent structural and spectroscopic characterization of these motors revealed that their enzymatic cooperativity does not result from cooperative binding [ - ]. in order to understand this new type of cooperativity we simulated the kinetics of a single hexameric motor by multiple coupled stochastic reactions using the gillespie algorithm [ ] . in contrast to analytical methods, our direct simulation allowed us to investigate the kinetics with an arbitrary model for cooperativity between the subunits. simulations on the kinetics of the hexameric rna packaging motor p from dsrna bacteriophage [ ] with different cooperativity mechanisms provided insight into the rnamediated cooperativity and yielded a sound theoretical basis for the interpretation of experimental results [ ]. the viral infectivity factor (vif) encoded by hiv- is a small basic protein that strongly modulates the viral replication and is required for pathogenicity. vif is packaged into hiv- particles through a strong interaction with genomic rna and is associated with viral nucleoprotein complexes. moreover vif acts during the early stages of the viral infection (capsid disassembly, reverse transcription) as well as during the late stages of virus replication (virus assembly and maturation of the virion). however the effect on early stages is probably a consequence of a defective assembly and / or virion maturation. understanding the rna-binding properties of vif would contribute to elucidate the role played by vif in the regulation of the genomic rna traf cking in the cytoplasm to unable ef cient packaging, and the prevention of cellular inhibitors from altering hiv- rna. in this context, we have characterised the interactions of recombinant vif with hiv- genomic rna by uorescence spectroscopy, and determined the af nity of the protein for synthetic rnas corresponding to various regions of hiv- genome. taken together our results demonstrate cooperative and speci c binding. in particular, we showed that vif has a high af nity for the 'untranslated region of hiv- genomic rna. s. bernacchi , e. ennifar , k. toth , p. walter , j. langowski , p. dumas cnrs upr -strasbourg (france), dkfz -heidelberg (germany) we have used the dimerization initiation site (dis) of hiv- genomic rna as a model to investigate hairpin-duplex interconversion by using a combination of uorescence, uv-melting, gel electrophoresis and x-ray crystallographic techniques. fluorescence studies with molecular beacons and crystallization experiments with -nucleotide dis fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on rna concentration, and not on cooling kinetics. with natural sequences able to form a loop-loop complex (or 'kissing complex'), concentrations as low as µm in strands are necessary to obtain a majority of the hairpin form. on the contrary, when kissing-complex formation was made impossible by mutation in the loop, a majority of hairpins was obtained even at µm in strands. this mutated sequence also showed that kissing-complex formation is not a prerequisite for an ef cient conversion to duplex in presence of salts. we proved that this happens through hairpins engaged in a cruciform intermediate, but not from free strands after hairpin melting. supporting this view, the very rst step of formation of such a cruciform intermediate could be trapped in a crystal structure. such a mechanism might be biologically signi cant beyond the strict eld of hiv- rna dynamics. generation of rna dimeric form of the human immunode ciency virus type (hiv- ) genome is important for the viral replication. the dimerization initiation site (dis) has been identi ed as a short sequence that can form a stem-loop structure with a selfcomplementary sequence in the loop and a bulge in the stem. a mer dis rna fragment, dis , spontaneously formed "loosedimer" and was converted into "tight-dimer" by supplement of nucleocapsid protein ncp . nmr chemical shift analysis for dis in the kissing-loop and extended-duplex dimers revealed that three dimensional structures of the stem-bulge-stem region were similar between the two types of dimers. therefore, we determined the solution structures of two shorter rna molecules corresponding to the loop-stem region and the stem-bulge-stem region of dis , and the solution structures of dis in the kissing-loop and extendedduplex dimers were determined by combining the parts of structures. the mechanism of conformational conversion will be discussed based on the solution structures and the molecular dynamics analysis. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. aminoglycoside binding to hiv- dis kissing-loop complex: from crystals to cells e. ennifar , j.-c. paillart , a. bodlenner , p. pale , r. marquet , p. dumas cnrs upr , strasbourg -france, cnrs/université louis pasteur strasbourg umr , strasbourg -france all retroviral genomes consist in two homologous single stranded rnas. dimerization is an essential step for viral replication. hiv- dimerization initiation site (dis) is a strongly conserved stem-loop in the ' leader region of the genomic rna. it was shown in vivo that alteration of the dis dramatically reduces viral infectivity. we have previously solved crystal structures of the dis kissing-loop complex. analysis of these structures revealed an unexpected resemblance between the dis kissing-loop and the s ribosomal aminoacyl-trna site (a-site), which is the target of aminoglycoside antibiotics. we have shown that some aminoglycosides specifically bind to the dis kissing-loop complex with an af nity and geometry similar to that observed in the a-site. in agreement with these previous results, we have now solved highresolution crystal structures of the dis kissing-loop complex bound to four aminoglycosides. these structures show that, as expected, two aminoglycosides are bound per kissing-loop complex. importantly, the binding is observed not only in vitro on large hiv- genomic rna fragments, but also on infected cells. moreover, we showed that some of these aminoglycosides stabilize the kissingloop rna dimer, which is consistent with the observation in crystal structures of numerous direct and water-mediated drug-rna contacts. these structures are currently used as starting points for designing potential new drugs targeted against the viral rna. modeling the long range entropy of rna: w. k. dawson , k. fujiwara , k. yamamoto , g. kawai chiba institute of technology, - - tsudanuma, narashino, chiba, japan, international medical center of japan, - - toyama, shinjuku-ku, tokyo, japan non-coding rna appears to make up a large part of the human genome. a reliable rna structure prediction program is needed to understand the structure of this non-coding rna. we recently developed a new way to model the long range entropy in rna and applied it to rna secondary structure prediction. in some of instances, the new approach is able to achieve far better predictions than the state of the art secondary structure programs even given exactly the same parameters. predictions using this method tend to show distributions that are funnel shaped. a new and important parameter in these calculations is the persistence length (a measure of the correlation and exibility of the rna). (url: http://www.rna.it-chiba.ac.jp/ vsfold/vsfold /) this new approach has now been extended to prediction of pseudoknots. the method is a heuristic wherein the hierarchical folding hypothesis is used to nd the pseudoknots as the rna secondary structure is folding, and corrections to that secondary structure are made to accommodate the pseudoknot. it is able to do these searches in roughly n^ time. the model is consistent with the hierarchical hypothesis and it is possible to estimate rna folding times that are of the correct order of magnitude using this model. with further adaptations to account for the size, shape and variablity of amino acid residues (hydrophobicity etc.), the model also appears to be transferable to protein folding problems. a. v. melkikh ural state technical university, ekaterinburg, russia a model of the genome as a gene network capable of receiving information about the environment and performing some operations on genes has been considered. the evolution rate of replicators for the mechanism of random mutations has been estimated. it was shown that the evolution rate under random actions is negligibly small for real dimensions of genomes of replicators [ ]. it was inferred that only a deterministic mechanism of the evolution can explain the known evolution rate of replicators. a deterministic model of the evolution has been proposed. the basic principles of this model include: ) information about the replicators evolution is encoded in the conformational states of proteins; ) the conformational language of proteins is translated into the language of nucleotide sequences during the evolution; ) the structure of genes is controlled such that the transition to a nearest free ecological niche takes a minimum time (at a preset restriction on the control). transfer rnas are synthesized as part of longer primary transcripts that require processing of both their ' and ' extremities in every living organism known. the ' side is matured by the quasiuniversally conserved endonucleolytic ribozyme, rnase p, while removal of the ' tails can be either exonucleolytic or endonucleolytic. the endonucleolytic pathway is catalysed by an enzyme known as rnase z. rnase z cleaves precursor trnas immediately after the discriminator base in most cases, yielding a trna primed for addition of the cca motif by nucleotidyl transferase. rnase z is found in the vast majority eukaryotes and archaea and in about half of the sequenced bacteria. it is often essential for growth and mutations in one of the two genes encoding rnase z (elac ) have been linked with prostate cancer in man. in this poster we present the crystal structure of bacillus subtilis rnase z at . Å resolution ( ) resolved by mad method and propose a model for trna recognition and cleavage. the structure explains the allosteric properties of the enzyme and also sheds light on the mechanisms of inhibition.it also highlights the extraordinary adaptability of the metallohydrolase domain of the b-lactamase family for the hydrolysis of covalent bonds. ( )i. most rnas undergo several steps of post-transcriptional modi cation before carrying out their assigned functions. one of the major modi cations is the splicing process, by which non-coding introns are removed from the coding exons. splicing can be performed by autocatalytic, self-splicing introns (e.g. group ii introns), i.e. catalytic rna or ribozymes. group ii introns, which occur in bacterial genomes and in organellar genes of plants, funghi and lower eukaryotes, consist of a conserved set of six domains. domain recognizes the '-exon through a - base pairing interaction formed by two regions within the intron (exon binding sites, ebs and ebs ) and the last - nucleotides of the '-exon (intron binding sites, ibs and ibs ). as the correct recognition of ibs by ebs is crucial for a successful splicing event we are investigating the structural and metal ion requirements of this part by various spectroscopic techniques, e.g. nmr. our data shows that the hairpin including ebs consists of a helical region followed by an unstructured single stranded part, which is ready for splice site recognition. the results of the structure analysis will be presented. financial support by boehringer ingelheim fonds (fellowship to d. k.) and the swiss national science foundation (snf-förderungsprofessur to r. k. o. s.) is gratefully acknowledged. structural basis for the antigene and antisense properties of modi ed dna:dna and rna:dna duplexes e. c. m. juan , t. kurihara , j. kondo , t. ito , y. ueno , a. matsuda , a. takenaka graduate school of bioscience and biotechnology, tokyo institute of technology, graduate school of pharmaceutical sciences, hokkaido university, faculty of engineering, gifu university oligonucleotides containing polyamines are currently being evaluated as potential antigene and antisense compounds. those with -(n-aminohexyl)carbamoyl- '-deoxyuridine ( n u) and its '-omethyl derivative ( n u m ) exhibit improved nuclease resistance and form stable duplexes with their dna and rna targets. x-ray structures of these duplexes have shown good correlation between the conformational changes and the observed chemotherapeutic properties. the amide groups of the modi ed uracil bases form six-membered rings through the intra-molecular nh-o hydrogen bonds, so that the aminohexyl chains protrude into the major grooves. some of the terminal ammonium groups are involved in intra-duplex interactions with phosphate oxygen anions, whereas the others interact with those of the adjacent duplex. such interactions contribute to the stability of duplex formation. the '-o-methyl modi cation in n u m shifts the ribose ring toward the c '-endo conformation and in uences duplex stability. observed changes in the dimensions of the minor grooves and in the hydration structures are also well correlated to nuclease resistance and duplex stability. group ii intron ribozymes catalyze selfsplicing in bacterial genomes as well as in organellar genes of lower eucaryotes. for correct structure and function these ribozymes need speci c concentrations of monovalent and divalent cations such as k and mg . most of these ions are used for charge screening, but some are also bound to distinct sites ful lling various speci c tasks. the conserved secondary structure of group ii intron ribozymes consists of six domains grouped around a central wheel. here the focus is set on domain (d ) of the yeast mitochondrial intron ai , a hairpin of nucleotides, which is crucial for catalytic activity. the -nucleotide bulge in d is known to be exible and acts as a metal ion binding platform. we have investigated the binding of different metal ions (mg , ca , mn , cd ) to this platform by uorescence spectroscopy. for this the bulge site adenosine in d was replaced by the uorescent nucleotide base analogue aminopurine ( ap). the binding data ts to an equation describing a binding to a single class of sites. the titration experiments not only reveal different dissociation constants for the tested metal ions but also indicate different effects on the bulge structure. financial support by the swiss national science foundation (snf-förderungsprofessur to r.k.o.s) is gratefully acknowledged. modi ed nucleosides and across the anticodon loop interactions in trna u. b. sonavane, k. d. sonawane, r. p. tiwari national chemical laboratory, pune , india in several interesting trna molecules, the ( th ) as well as the ( th ) nucleoside are hyper modi ed. as an example, unique hypermodi ed nucleosides mcm s u and ms t a are crucial in human trna lys , which acts as a primer in hiv replication. modi ed nucleosides may facilitate or hinder across the loop interactions. large substituents in th and th modi ed nucleosides if oriented suitably may also interact with each other. across the loop interactions may lead to unconventional anticodon loop structures also affecting exibility of the anticodon loop. this may restrict or enlarge synonymous codon choice and decoding during protein biosynthesis. except for trna asn (with interacting q and t a ), our studies show conventional 'open' loop structure -free of across the loop interactions, for a number of interesting trna anticodon loops with diverse hyper modi ed nucleosides at both of these locations. molecular dynamics simulations of hydrated anticodon arm of trna asn show persisting interaction involving the diol group of q and carbonyl group of ureido linkage in t a . additionally, the hoogsteen edge of th adenine base participates in hydrogen bonding with watson -crick edge of rd base and thus contributes to unique loop structure of trna asn . resulting suboptimal q:c base pairing leads to unbiased reading of u or c as the third codon letter. absence of queuosine modi cation, q happens to be also associated with uncontrolled rapid proliferation of cells and malignant growth. structural properties of ctg/cag repeats, and preliminary x-ray analysis of cug repeats y. sato, k. kimura, a. takénaka graduate school of bioscience and biotechnology, tokyo institute of technology, yokohama, japan. the human genome contains so many different types of repetitive sequences. some of them are tandem repeats of trinucleotides. their unusual expansions cause genetic diseases such as type myotonic dystrophy (dm ) and huntington's disease (hd), the unit sequences being ctg and cag, respectively. the numbers of repeats of the two complementary sequences change independently during dna replication or repair. the direct origin of dm is, however, the transcribed rna fragments with cug repeats, which forms a speci c structure and inhibits other protein syntheses. in the present study, structural versatilities of such dna and rna fragments has been examined. native pages of (cug) n show that the hairpin structure with even number is more stable than that with odd number. this difference might be ascribed to the structural difference at the hairpin head. the pages also show that duplex formation is dependent on coexisting cationic species and their concentration. crystal data of (cug) (a=b= . , c= . Å, and the space group r ) suggest that the asymmetric unit contains the rna fragment. an approximate crystal structure solved by molecular replacement techniques at . Å resolution shows that the rna fragments form a duplex similar to an a-form rna. context-dependent selection of promoter in a natural selection-type evolution reactor h. nagayasu, y. ageno, x. t. ma, y. husimi saitama university, saitama, japan using an isothermal ampli cation of hairpin dna/rna (developed by g.joyce), we drove a natural selection-type evolution reactor taking the speci c growth rate as the tness. we used hiv- rt and thermot rnap at c. starting from the random promoter pool, we selected the strongest promoter at c, which was separated by hamming distance from the strongest promoter at c. the latter was found to be identical to the natural t promoter. when we used a simple random pool, the selection process showed one-step convergence. when we used a random pool with a speci c short sequence at the upstream anking region of the random region, we observed an evolution process as convergencedivergence-convergence of the promoter sequence, driven by deletion of the speci c short sequence. this context-dependent selection was found to come through a neutral path, judged from the tness measurement. intronic sirna and mirna, and dna methylation gif sur yvette, france. abnormal activation of small g proteins is involved in several human diseases. small g proteins are activated by gdp/gtp exchange, which is stimulated by their guanine nucleotide exchange factors (gefs). thus, small g protein/gef complexes appear as emerging targets for interrupting signalling networks regulated by small g proteins in pathological contexts. the activation of small g proteins of the arf family is initiated by exchange factors which carry a sec catalytic domain stimulating the dissociation of the bound gdp nucleotide. the structure of the arf -gdp-arno reaction intermediate, trapped by a mutation of the catalytic glutamate, was recently solved by x-ray crystallography (pdb code: r s) acknowledgements: the work was supported by contract k- j. q. yin, f. chen, y. tan, t. gu institute of biophysics, chinese academic sciences, beijing, china sirna/mirna can ef ciently induce mrna cleavage or translational repression at the posttranscriptional level in a sequencespeci c manner. recently, it has been shown that these small rnas guide genome modi cation in mammalian cells. however, their ability to direct cognate dna methylation has been con rmed so far only in plants, and their biogenesis, functions, and modes of silencing genes are yet elusive. here, we report that small rnas derived from intron regions of some genes can target homologous dna sequence in promoter, 'utr or 'utr regions of genes in different human tumor cells. surprisingly, we also discovered that endogenous sirnas from introns of genes possessed a large number of target mrnas by using bioinformatics, and con rmed their existence in human cells with northern blot analysis. intronic small rnas generated by sliceosomes can form mature mirnas or sirnas through the processing of drosha and /or dicer. rt-pcr analysis indicated that vector-based small rna repressed expression of homologous genes at the transcriptional and/or translational levels. western blotting demonstrated that the expression of some proteins was greatly reduced or completely inhibited owing to promoter methylation. these ndings reveal that the expression of some genes can incredibly control cell activities at both protein and rna levels. our results also suggest that these small rnas may regulate gene expression in different modes of action. role of stacking in speci c recognition of capped rna by the cbc protein the stacking interaction involving -methylguanine moiety of mrna ' terminal cap (m g) and aromatic amino acid side chains is a common feature of all known cap-binding proteins. the crystal structures of the human cap binding complex (cbc) showed its induced folding upon m gpppg cap analogue binding. stabilization of the cbc-m gpppg complex by sandwich stacking of m g in between y and y is additionally enhanced by stacking of the second base of capped mrna with y . gibbs free energy of the association of various cbc mutants with the synthetic cap analogues, m gpppg, m gpppu, and m gtp, has been determined by uorimetric titration. preference of the wild type (wt) cbc for the dinucleotide analogues is also observed for the y a mutant, with the energy loss of . - . kcal/mol. however, all proteins with the mutated second stacking partner y prefer to bind m gtp compared with m gpppg. the binding of m gtp to the y a mutant is only . kcal/mol less favourable compared with the wt cbc. these divergences may be ascribed to smaller entropic costs of conformational rearrangement of cbc in the case of a smaller ligand, which can nd more favourable contacts when its second part is not ef ciently 'constrained' by stacking with y . supported by kbn p a annealing of the tar dna hairpin to a complementary tar rna hairpin, resulting in the formation of an extended duplex, is an essential step in the minus-strand transfer process of hiv- reverse transcription. in this work, we use gel-mobility-shift analysis to follow the kinetics of this reaction in the absence or presence of hiv- nc prepared by solid-phase peptide synthesis. to elucidate the reaction pathway, we use either the complete -nt tar hairpins or truncated -to -nt minihelices (mini-tar) derived from the top part (i.e. hairpin loop) of tar. assays were also carried out with mutant tar constructs. the annealing kinetics were studied systematically as a function of dna concentration and temperature. we show that the annealing initiates through a weak loop-loop kissing interaction, followed by a much slower conversion step, which results in formation of the extended duplex. nc facilitates both reaction steps, resulting in the overall -fold and -fold rate enhancement for mini-tar and tar annealing, respectively. we show that the kissing step is facilitated by the nc-induced nucleic acid aggregation, which is more pronounced for the longer tar hairpins. at the same time, the conversion steps in tar and mini-tar appear to be very similar and are similarly facilitated by nc - -fold. the later effect relays on the ability of nc to destabilize nucleic acid duplexes, and is equivalent to destabilization of a few base pairs required for the conversion initiation. linking of the n-terminus of a peptide to its encoding mrna s. ueno, h. arai, y. husimi department of functional materials science, saitama university, saitama, japanin evolutionary protein engineering, in vitro selection using a cellfree translation system has advantages of large library size and also of applicability to cytotoxic protein. although many in vitro protein selection techniques such as in vitro virus, ribosome display are developed, most of these are the techniques that link the genotype molecule to the peptide at its cterminus. we developed a method to link the genotype molecules to the nterminus of its encoding peptide. the mrna has dna-linker hybridize region, translation enhancer, initiation codon, single codon, amber stop codon, n-terminus sequence in gfp gene, his-tag and ochre stop codon. the mrna is also linked at '-terminus to sup trna via spacer and the speci c amino acid. the mrna is translated in the cell-free translation system. thus, c-terminus of the protein becomes free. moreover, in this system, the free protein of the full length is never generated. for this reason, the problem in the conventional technique, that is, the competition between the protein displayed on the genotype molecule and the free protein is eliminated. we succeeded to turn one round of the " life cycle " of the in vitro virus, judged by his-tag selection. key: cord- - cdf qpk authors: nishimoto, y.; inoue, k. title: curve-fitting approach for covid- data and its physical background date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: cdf qpk forecast of the peak-out and settling timing of covid- at an early stage should help the people how to cope with the situation. curve-fitting method with an asymmetric log-normal function has been applied to daily confirmed cases data in various countries. most of the curve-fitting could show good forecasts, while the reason has not been clearly shown. the k value has recently been proposed which can provide good reasoning of curve-fitting mechanism by corresponding a long and steep slope on the k curve with fitting stability. since k can be expressed by a time differential of logarithmic total cases, the physical background of the above correspondence was discussed in terms of the growth rate in epidemic entropy. covid- outbreak was first reported in wuhan, china, then the disease soon spread to europe, us and worldwide in the first half of . japanese government declared a state of emergency in april and people were requested to stay at home. the number of daily cases was the biggest concern and people were anxious to know the possible timing of the peak-out and settling because they have been threatened not just by the deadly disease but also by its critical impact on the economic activities. our trial approach is to keep people informed of the macroscopic trend of cases which should help them to think of their future. among epidemic analysis methods the sir theory has provided a reliable mathematical model ) , however, it requires not only infected (i) data but also suspicious (s) and recovered/died (r) data to evaluate an actual 'reproduction number ' ) . those data may include time lag and it is not regarded as a real time method. although pcr test data include a weekly periodicity and strong arbitrariness, macroscopic trends could be extracted if the arbitrariness remain consistent. therefore curve-fitting approaches could be a simple forecast tool even though it is kind of phenomenological one depending also upon people's activities. t. tomie proposed a curve-fitting with a normal distribution to answer the real time request and applied it to covid- in wuhan ) since it was known that flu epidemic followed a normal distribution ) . it offered an informative forecast, however, the model required 'ad hoc' ideas of different parameters for increase/decrease phases because the covid- profile has a noticeable feature to leave a trail in an asymmetric manner. we applied a curve-fitting with a log-normal distribution to meet the asymmetric features. this paper describes the curve-fitting approach and its physical background as specific as possible in order for people to follow it as a simulation tool for their own regions and to relieve people's concern on covid- . the fitting was carried out in the following manner. ( ) database: daily cases by country provided by csse at johns hopkins university ) ( ) model : log-normal function for daily cases a log-normal function expressed by formula ( ) has an asymmetrical distribution in fig. . a log-normal appears in the distribution of fig. log-normal function incubation periods of viruses ) . ( ) fitting : using solver in excel add-on (grg nonlinear search for least mean square) an example of spread sheet templates for a curve-fitting computation is shown in fig. . if you input daily cases data, forecasting date and initial values for undetermined coefficients a, b, c of the log-normal function in the yellow cells, excel's solver searches for an optimal coefficients' combination to minimize the variance Σ(n(t)-f(t)) less than the threshold level. the curve-fitting result is depicted in the right-hand graphs. the upper shows the fitting with daily cases and the lower shows the cumulative distribution to fit the total cases. thus curve-fitting can be carried out in the straight forward manner without any ad hoc techniques. log-normal function fittings with daily cases in japan were carried out since early april. although excel solver could not find optimal coefficients until apr. th , it suddenly became successful on the next day. since then we repeated fitting periodically, among which typical examples are shown in fig. for april th , th , th , th , may th and th , respectively. the horizontal axis is the date number originated by jan. st . 'settling' in the graphs shows a date expected that daily cases go below the / of peak cases along the fitted curve (in red). the forecast on apr. th looks a little bit pessimistic, however it is getting reliable as days go by. fig. forecast using curve-fitting with daily cases in japan at various dates these series of graphs in fig. show that significant curve-fittings are possible a few days after apr. th in the case of japan and forecast of peak-out and settling date is getting firmer day by day as depicted in fig. . the forecast peak-out date is so consistent that there is no question that daily cases reached a peak on apr. th in japan. on the other hand, a state of emergency was declared on apr. th at seven selected areas and on apr. th across japan, and the government strongly requested the public to stay at home to avoid % human contact. the effect of the government request would have come out two weeks later around apr. nd at earliest. consequently the daily confirmed cases in japan had already peaked out one week prior to the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . timing when the effect of such emergency action were expected. was the declaration of emergency state given too late or even not necessary? it was understandable that japanese had too much self-restraint because of the very first experience. however, they have to verify the cost/merit in case of upcoming waves of covid- because self-restraint in the whole society will cause huge economic damages and losses. by the way why was the curve-fitting successful after apr. th ? we had no reasoning for it and could not answer the question, just saying 'please ask the data'. why and when does the curve-fitting get successful in the course of epidemic? are there any reasons for it? we had been thinking about the question and finally came across the k value ) , which was proposed by t. nakano and y. ikeda and is defined by where t is the number of days from the reference date, and n(t) and n(t− ) are the total cases on days t and (t− ), respectively. the relationship between curve-fitting on the daily cases and the k value in japan is described in fig. . the k value shows a long downward slope since the k's summit dated apr. th while the curve-fitting was getting stable since the same date. next we looked into the same relationship in other countries. since usa total included multiple waves one after another, we chose new york as a representative. the k value graphs show long and steep slopes for italy, germany, new york and taiwan in fig. and curve-fitting was getting stable along the slopes initiated from the k's summits similarly with the case of japan. however, most profiles of the daily cases left trails as they were getting close to settling stages and the forecast of settling date got shifted later. in sweden and russia daily cases profiles show lingering patterns depicted in fig. , at first the k curves show steep slopes down to landing floors but gentle slopes are followed from the landing floors. log-normal functions still fitted the russian and swedish data but the settling period might be quite volatile and delayed to late summer. on the other hand, india, brazil and south american countries are still suffering from exponential growth and even a sign of peak-out has not appeared yet. the k curves show gentle downward slopes with small ups and downs, which leads to an unstable curve-fitting and consequently to uncertain forecast of peak-out or settling periods in fig. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint let us summarize the daily cases profile, k value at the initial, peak-out, settling and k's slope and curve-fitting stability in table . it was confirmed that the curve-fitting method had a considerably good stability even before the peak-out stage once the k curve entered to a steep slope in spite of noisy data. however, if we apply it to a forecasting tool to inform people of the epidemic outline from an outbreak through a peak-out up to a settling stage, we have to investigate the reliability of the tool carefully. there are several factors which could affect the reliability of the curve-fitting forecast although the background of them is still in a fog; ( ) the accuracy estimated by the k curve ( ) the variation caused by an asymmetric lingering effect ( ) the effect of k value at initial in japan and taiwan all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . now let us discuss on the factors which affect the forecasting reliability of the curvefitting not only from the k curve geometry but from the physical meaning of the k value. ( ) the accuracy estimated by the k curve in the previous chapter we confirmed that the fitting gets much firmer as the k curve has a steeper slope. this correlation is understandable from the k curve geometry. for example, the epidemic settling date can be estimated by an extension line along the k slope as shown in fig. ~ and the date estimation accuracy Δt is approximately given by where k' is a time differential of k which means the gradient of the k slope. it is quite reasonable that a long (small Δk') and steep (large｜k'｜) slope on the k curve makes a small Δt, that leads to a reliable forecast. ( ) the variation caused by an asymmetric lingering effect as shown in table , most of the countries' covid- data have peak-to-settling time much longer than start-to-peak one, that is, an asymmetric profile. this is the main reason why we have applied a log-normal distribution as the model function. correspondingly the k's slope has a trend of getting gentle and gentle as epidemic is getting close to settling. this spoils the reliability of forecasting of the settling period although the reliability of forecasting of the peak-out is not so much damaged by the lingering effect. what is the cause behind the asymmetric profile and the corresponding gentle slope in the k curve? asymmetries frequently appeared in the covid- daily cases data have generally been explained with political interventions such as the lock-downs and quarantines in order to urgently suppress upcoming spread peaks and to avoid the medical care breakdown. however, the asymmetries more or less occurs in most countries independent from the scale of the political intervention. for example, the lingering effect in sweden data remarkably lasted more than two months although they have a unique policy of an early acquiring of 'herd immunity' with a less intervention. ( ) the effect of k value at initial in japan and taiwan generally speaking, if the k value at initial is sufficiently high, the slope in the k curve gets steeper and the peak-to-settling time gets shorter. why does japan have a short peakto-settling in spite of its low k value at initial? we have no idea about ( ) where n(t)-n(t- ) in the numerator is the total cases during seven days from day (t- ) to day t, that is, all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . where n(t) is the daily cases of on day t and <n> is a moving average of n(t) during the previous week. although n(t) generally changes volatilely, <n> shows a smoother profile. here, note that the total cases n(t) and the daily cases n(t) have the following relationship; ( ( )) = ( ) ・・・ ( ) using formula ( ) and ( ), the k value can be expressed in a different way as shown in the following formula as long as n(t) is not zero. ｄｔ ・・・ ( ) thus the k value can be written with two factors <n>/n(t), a time-variant ratio, and d(logn(t))/dt , a time differential of logarithm of the total cases n(t). the profiles of the two factors are shown for italy, germany and india in fig. . the upper graphs show the actual data and the lower show the ones free of deviations based upon fitted curves. a time-variant <n>/n(t) is a small positive number informing a peak-out when it gets across value one. in this context the peak-out in india does not seem to be so far away. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . on the other hand what is the meaning of the factor d(logn(t))/dt ? we have been looking at graphs named 'logarithmic (of n(t))' on the dashboard shown in fig. in the covid- database of csse at johns hopkins university to find a first sign of spread settling. it reminds us that d(logn(t))/dt has a certain relationship with an epidemic settling. fig. example of logarithmic graph in database at johns hopkins univ. (source: russian data on june st , ) let us consider on the meaning of logn(t) in the formula ( ) . as described in the supplementary materials, a total cases n(t) is well fitted with a logistic function. incidentally the fermi-dirac distribution in the quantum statistics has exactly the same functional form with a logistic function although the variables, 'time' and 'energy' are different with each other. fermi-dirac statistics describe a distribution of many identical particles that obey the pauli exclusion principle when the systems reach a thermal equilibrium, that is, the maximum entropy. it is reasoned by analogy that a total cases n(t) expressed with the similar logistic function has a certain relationship with a process of entropy growth under a certain physical constraints. if we assume that logarithm of total cases n(t) is an entropy s(t) in an epidemic process, the k value expressed in ( ) can be written by the following formula ; since the time variant ratio <n>/n(t) is a positive number around one, the k value can be recognized as an entropy growth rate in the epidemic. therefore it is concluded that epidemic settling is an approach to a maximum entropy after spread. in the statistical physics a virtual 'maxwell's damon' is trying to microscopically discriminate and sort different kinds of particles in order to keep the local entropy lower. correspondingly, world countries are now trying to macroscopically reduce their economic activities through cities' lock-down and isolation of positives as if they were keeping the local epidemic entropy lower. now that we understand the k value is a good estimate of the epidemic entropy growth rate, let us be back again to the questions ( ) and ( ) discussed above. ( ) what is the cause behind the asymmetric profile? in other words why does a k curve change direction to escape zero when it is getting close to zero? from the entropy-based discussion above, k= means that the epidemic entropy s stops increasing and the local system has already reached a thermal equilibrium. however if any human interventions are given, the equilibrium will be surely disturbed and another entropy growth will get restarted. the lingering pattern of daily cases and an asymmetric distribution is worldly common and more or less an unavoidable matter because of human intervention. forecasting with curve-fitting unavoidably depends upon human activities. ( ) why is k value at initial strangely low in japan and taiwan? since the k value can be regarded as the epidemic entropy growth rate, this means that the epidemic entropy in japan and also in taiwan started growing quite slowly along the rate of doubled/ days as schematically shown in fig. ) . according to the statistical physics, the entropy growth rate generally depends upon physical conditions such as the density, pressure, strength of interaction and, of course, temperature of the local system. it can be inferred by analogy that japanese weak interaction derived from their life style, always keeping clean with an appropriate physical/social distance, contributes to the initially low entropy growth rate even though the population densities in japan and especially in tokyo are considerably high. or are there any reasons for the low entropy growth rate in japan, owing to another 'factor x' ) ? further discussions are highly appreciated. daily given covid- related news continued to depress many people and we believed that keeping the people informed of the macroscopic trend of confirmed cases should help them to set up their future plan. we applied a curve-fitting approach using an asymmetric log-normal as a model function. daily cases data in many countries were successfully fitted and timings of peak-out and possible settling could be forecasted at rather earlier stages. the reason why and when curvefitting was possible was supported by the k value as a guideline. the physical meaning behind the relationship between the curve-fitting approach and the k value was discussed in terms of epidemic entropy. the lingering effect inherent to covid- and the difference in the initial k value was also discussed. under huge social and economic sacrifices, each government is now struggling on the containment of spread by using its brake and accelerator and will be studying on 'how each country's policy contributed or disturbed' and 'what was the cost/merit'. we hope that the review with the curve-fitting method and the viewpoint of epidemic entropy will play a role in the research review and, as well, in detecting the second wave which is very likely to flare up anywhere in the world. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint let us consider on the meaning of logn(t) in the formula ( ) . in the previous chapter a lognormal function was adopted to fit data of daily cases n(t). however in order to fit data of total cases n(t), a logistic function seems suitable. a logistic function defined by formula (s ) shows step-like curves depicted in fig.s . this function has been used for analyses in the world population increase, growing process of species and also epidemic spread under the environmental constraint. actually the total cases of covid- in japan can be accurately fitted using a logistic function as shown in fig.s . fig.s depicts the time differential of the logistic function in fig.s . it can be seen that the corresponding daily cases in japan is followed as long as a symmetric distribution. therefore it can be concluded that the total cases n(t) is well-fitted with logistic functions. the expression (s ) reminds us of fermi-dirac distribution in the quantum statistics which. is given by a logistic function depicted in the formula (s ) and fig. , where β is an inverse temperature, ε is an energy of the single-particle, and μ is the total chemical potential. let us compare the formula (s ) with (s ). although the variables, 'time t' in (s ) and 'energy ε' in (s ), are different with each other, fig.s fermi-dirac distribution functional forms are exactly the same and distributions are actually very similar. fermi-dirac statistics describe a distribution of many identical particles that obey the pauli exclusion principle when the systems reach a thermal equilibrium, that is, the maximum entropy. it is naturally suggested that logistic all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint function has a certain relationship with a process of increasing entropy under some physical constraints. if we assume that logarithm of total cases n(t) is an entropy s(t) in an epidemic process, the k value expressed in ( ) can be written by the following formula ; (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint idwr bulletin data th week covid- dashboard csse johns hopkins univ epidemiology of covid- among children in china the authors thank dr. t. tomie for his original curve-fitting idea, dr. y.ymaguchi for his timely suggestion on log-normal function, prof. t. nakano for his useful advices on the k value and dr. kawata and mr. y.yokota for constructive discussions. key: cord- -vli nnk authors: takayama, naohide title: rabies: a preventable but incurable disease date: - - journal: journal of infection and chemotherapy doi: . /s - - - sha: doc_id: cord_uid: vli nnk abstract rabies is a typical zoonotic disease which has been known for more than years. to date, no effective medical therapy has been established for overt rabies. the rabies post-exposure prophylaxis (pep), which is a serial vaccination against rabies starting as soon as possible after the patient was bitten by a suspected rabid animal, is the only way to prevent death. in japan, no rabies case has been reported for about years. however, rabies is epizootic in many asian countries, where more than % of the rabies deaths in the world occur. the japanese travelers who visit these countries every year may not be aware of this fact since no rabies occurs in their own country. therefore, the risk of being bitten by a rabid animal abroad and developing rabies after returning to japan seems to be high. all medical staff should keep in mind that imported rabies cases can occur at any time. in addition, pre-exposure vaccination against rabies should be recommended to international travelers in order to ensure the preventative effect of pep. rabies is mainly a disease occurring in animals, and it is regarded as one of the most typical zoonoses. rabies is known to have the following features. ( ) the incubation period of rabies is generally very long, ranging from to months (about % of cases) to more than year ( %- % of cases) (pathogenetic features). ( ) almost % of patients who develop clinical rabies die, because the rabies virus causes fatal encephalomyelitis and no effective treatment has yet been developed. there are no laboratory tests to determine whether a person is infected with the rabies virus during the incubation period (clinical features). ( ) the host animals of the rabies virus differ among regions, even though almost every mammal is capable of contracting rabies. the main vectors are foxes in europe and canada, raccoons, skunks, and fruit-eating and insectivorous bats in the united states, dogs in asia, mongooses, jackals, and dogs in africa, and dogs and vampire bats in latin america. there are two types of epizootic rabies, namely the urban type and the sylvatic type. the former type is where the rabies virus is principally transmitted among dogs, and the latter type is where the vectors are the wildlife, such as foxes, raccoons, and mongooses (epidemiological features). rabies is caused by the rabies virus, which is an enveloped, bullet-shaped, size nm in diameter and - nm in length, single-stranded, minus-sense rna virus. , it belongs to the genus lyssavirus of the family rhabdoviridae. genus lyssavirus includes some antigenically rabiesrelated viruses (rabies-related lyssaviruses). most of these introduction half a century has passed since rabies was eradicated in japan. however, in november , two cases of imported rabies occurred, , reminding us that rabies is not a disease which only existed in the past. from a world-wide point of view, countries that are free from rabies are the exception, and there are still many areas in the world where rabies continues to occur. however, in regions where rabies is endemic, there are some areas where many patients die from rabies, and other areas where it rarely occurs. in asian are transmitted among bats, and some are reported to cause clinical rabies in humans. genus lyssavirus containing rabies virus is phylogenetically divided into two groups, phylogroups i and ii. the former includes the rabies virus, duvenhage virus, european bat virus types and , and australian bat virus; the latter includes lagos bat virus and mokola virus. , history of rabies in japan it is not known for sure when rabies appeared in human society. it is presumed that man began living with dogs about years ago, so there is a possibility that human rabies has occurred since then. the oldest document mentioning rabies as a zoonosis is the law enacted around bc in mesopotamia. the law, the eshunna code, imposed a penalty on the owner of the dog when a bitten victim died from rabies. from this description, it is understood that the causal relationship had been clearly recognized; the person bitten by a rabid dog would have overt rabies and eventually die. humans and dogs were increasing in number, and people started moving to new regions with their dogs. this movement seems to be the reason why rabies spread to various regions around the world. in japan, a large epizootic of rabies was documented in nagasaki prefecture in , and had spread to oita prefecture the following year. expanding along the main roads to sanyodo and tokaido, it eventually reached edo in , when yoshimune tokugawa was governing as the th shogun. during this epizootic, many dogs, horses, foxes, raccoon dogs, etc. were killed. in , genjo noro ( - ), one of the medical offi cials of the tokugawa shogunate, published kyoken-kosho-chiho, which is the fi rst textbook on the therapy for rabies. in this book, he reported that the sickness would become serious after a certain period of time, and eventually or out of every patients would die, even if the wound did not initially appear to be severe. furthermore, he also wrote that the best fi rst-aid treatment was to suck out the blood as quickly as possible and to apply moxa cautery to the wound. this textbook was republished in , probably because the epizootic of rabies had not ceased. little information is available on epizootics of rabies in the latter part of the edo era. we can only assume that outbreaks of rabies occurred sporadically in those days based on the statistics of the number of rabid dogs during the meiji era (from to ), when - were recorded annually. in , an epizootic of rabies occurred in nagasaki prefecture. during this outbreak, tomei kurimoto, chief physician of internal medicine at the national nagasaki hospital, gave a rabies vaccination to people who were bitten by rabid dogs for the fi rst time in japan. he made an attenuated rabies vaccine by himself, following the method of the french scientist louis pasteur. he injected this vaccine into patients who had been bitten by dogs, and as a result none of them died. in the latter part of the meiji era, outbreaks of rabies gradually increased both in number and in scale. more and more outbreaks were reported in the taisho era (from to ), mainly in large cities (table ). umeno and doi, of the kitasato institute for infectious diseases, performed a mass rabies vaccination of dogs in kanagawa prefecture and tokyo prefecture in and , respectively. as a result, the numbers of both rabid dogs and people bitten by such dogs decreased signifi cantly in both regions. however, both these numbers kept increasing outside of these regions. owing to the widespread confusion after the great kanto earthquake in , reports of rabid dogs and human rabies cases exceeded and per year, respectively, in the following years. from that time, standard rabies control methods, such as compulsory vaccination of all family dogs and the elimination of stray dogs, were enforced all over japan. consequently, the number of rabid animals steadily decreased, reaching or fewer during - . [ ] [ ] [ ] however, the number began to increase again due to the social disorder after world war ii. seventy-six cases of human rabies and over animal rabies cases had been reported in and , respectively, , which led to the rabies prevention law, enacted in (fig. ). this law requires owners to register, confi ne their dogs, and make sure that a rabies vaccination is administered. it was strictly enforced, in conjunction with the elimination of stray dogs. the number of rabid animals decreased rapidly, and no rabies cases in either animals or humans have been reported since , except for three cases of imported human rabies in and . in japan, epizootics of rabies have historically been of the urban type, where the rabies virus was transmitted among dogs, and occasionally from dogs to humans, cats, or other domestic and wild animals. during such epizootics, foxes and raccoon dogs were also infected with the rabies virus. no transmitting circle of the virus has formed among the wildlife in japan. japan was successful in eliminating rabies, although countries free from rabies are rather an exception worldwide and there are still many endemic areas. the numbers of human rabies cases differ within these areas, ranging from areas where hundreds or thousands of people die every year to regions where human rabies is very rare. the animals trans- table . numbers of rabid animals reported, regions where rabid animals occurred, and people bitten by rabid animals from to in japan (from [ ] , with permission) year no. of rabid animals no. of regions no. of people bitten % of rabies cases break out in rural and poor regions. however, the numbers of rabies deaths do not correlate with the risk of contracting rabies, because it is possible to prevent a patient from dying of rabies by giving them postexposure prophylaxis (pep), even after they have been bitten by a rabid animal. without pep, the total number of human rabies deaths in asia and africa combined is estimated to be about . in asia, % of rabies is transmitted by dogs, whereas % is from cats. in the united states, foxes, skunks, and racoons are the host animals. in latin america, dogs are the host animals of rabies. in addition to terrestrial animals, some species of bat transmit rabies in north and south america. in the united states, out of patients who contracted rabies domestically during - were infected by bats. in cases of bat bites, it is assumed that patients often miss out on the opportunity to receive pep because bite wounds from bats are too small to be noticed. humans usually contract rabies through bite wounds from rabid animals (bite exposure) because the rabies virus is highly concentrated in the saliva of infected animals. it can also be transmitted through nonbite exposure, although this rarely occurs. airborne infections, such as inhaling an aerosol of infected animal brain tissue in virus laboratories, or of contaminated air in bat-inhabited caves, have been reported. [ ] [ ] [ ] [ ] there is also one known case where a butcher became infected by skinning a cow that had died of an undiagnosed neurological disease. iatrogenic rabies cases have occurred in patients who received cornea, kidney, liver, or blood vessel graft transplantation from donors who had undiagnosed rabies (table ). to date, the only medically verifi ed cases of human-to-human rabies transmission are the cases infected through organ transplantation from undiagnosed rabies patients. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] fig. . number of rabies cases reported in humans and animals in japan after world war ii. just before world war ii, the annual number of rabid animals reported was or fewer because of the compulsory vaccination of family dogs and the elimination of stray dogs. however, rabies cases began to increase due to the social disorder after world war ii, leading to cases of human rabies in and more than cases of animal rabies in . in , the rabies prevention act was enacted. this requires dog owners to register and confi ne their dogs, and to vaccinate their dogs against rabies. this act also strictly enforces the elimination of stray dogs. as a result, the number of rabid animals decreased rapidly, and no rabies case has been reported since in either humans or animals except for three imported human rabies cases. (the last human case was reported in , and six rabid dogs were reported in .) gbs, guillain-barré syndrome; sah, subarachnoid hemorrhage mitting rabies also differ among regions, so in order to diagnose and prevent rabies it is important that we understand the epidemiology of the disease. there are no accurate statistical data of rabies deaths in every country. the number of human rabies cases is estimated to be per year worldwide, with % and % occurring in asia and africa, respectively. it is believed that in , a -year-old american woman received a right corneal transplant from a -year-old lumberman who was presumed to have died from guillain-barre syndrome. thirty days after the operation, she complained of right retro-orbital headache. in a few days, she noticed hypoesthesia on the right side of her face and diffi culty in walking, and symptoms of dysphagia and dysarthria developed. after admission to hospital, she developed fl accid paralysis, became progressively obtunded, and eventually died on day of hospitalization. a postmortem examination revealed rabies virus in the cornea, optic nerve, temporal lobe, and brain stem. in , a -year-old man received a left corneal transplant from a -year-old woman who had died from encephalitis and myocarditis in france. thirty-three days after the operation, he complained of left retro-orbital headache. over the next days he developed hypersalivation, pain and weakness in the legs, and pain on swallowing, and was hospitalized days after the operation. he became comatose on day of hospitalization and died on day . rabies virus was isolated from the patient's brain tissue, and on histopathological examination numerous negri bodies were found in the donor's brain. in , a -year-old thai woman received a corneal transplant from a boy who had died from an undiagnosed disease with mental confusion. a -year-old man also had a cornea transplant from the same donor. these recipients died and days after the operation, respectively. rabies virus was isolated from the woman's brain tissue, and negri bodies were found in the donor's brain tissue. in , two indian men received corneal grafts from a single donor. nine days later, one of these recipients, a year-old physician, reported redness, swelling, and intense pain in the operated eye. he died days after the operation. the other recipient, a -year-old man, was advised to receive pep. he received the fi rst and second doses of rabies vaccine, but refused to take the remaining doses. he experienced dysphagia along with pain, redness, and swelling in the operated eye days after the operation. two days later, he developed hydrophobia. he died days after the onset of the disease. the incubation period of the second man was more than days, probably because he had received two doses of rabies vaccine. in , a -year-old man received a corneal transplant from a -year-old man who had died from food poisoning in iran. on the same day, another -year-old man received a cornea transplant from the same donor. the fi rst patient reported nausea and paresthesia on his lips, and developed hydrophobia days after the operation. he died within the next h. the second patient was admitted to hospital with vomiting and poor general condition days after the operation. he died the following day. rabies virus was isolated from the brain tissue. in the united states, kidneys, liver, and an arterial segment were transplanted into four recipients from a common donor in . all four recipients developed encephalitis within days after transplantation, and died from rabies - days after the onset of neurological symptoms. [ ] [ ] [ ] in germany, there was an announcement on february , , that three out of six patients who had received organ transplantations from a common donor might have clinical rabies. the donor died after cardiac arrest and brain death in late . rabies was diagnosed in the donor and two of the recipients on the same day as the announcement and the next day, respectively. these cases indicate that organ transplants should not be carried out from donors who had died from encephalitis of unknown cause. at the same time, they also show that rabies is very diffi cult to diagnose intravitam. the clinical course of human rabies is divided into four phases: the incubation period, the prodromal phase, the acute neurological phase, and the coma phase. the incubation period for rabies varies from around days to year or even longer. in about % of all rabies patients, the incubation period is - months, but %- % of patients exhibited an incubation period longer than year. the longest incubation period reported was years, and was documented for a girl who migrated from laos to the united states. she had been bitten by a stray dog in laos years before the onset of clinical rabies. in general, the incubation period is shorter when the bite is to the head rather than the extremities, and is also shorter in children than in adults. during the incubation period, the rabies virus propagates in the muscle cells around the port of entry and invades the peripheral nervous system. it then migrates centrally to the central nervous system, following the fl ow within the axoplasm of peripheral nerves at a velocity of - mm per day. the symptoms of rabies fi rst appear after the virus enters the central nervous system (prodromal phase). in the prodromal phase, which lasts for - days, the patients complain of nonspecifi c symptoms such as malaise, fever, and anorexia. they may also complain of more specifi c local symptoms such as itchiness, pain, and paresthesia around the healed bite wound. the acute neurological phase continues for - days. during this phase, patients will intermittently suffer from intense anxiety, emotional agitation, and confusion. at other times they may be calm, lucid, and cooperative toward the medical staff. about % of patients will develop severe pharyngeal and laryngeal muscle spasms when they attempt to drink, or even see, water (hydrophobia). similar symptoms may also be induced when cool air blows on the face or chest (aerophobia). as a result, patients avoid drinking water, washing their hands, or feeling wind. the patient's condition gradually deteriorates. high fever, confusion, disorientation, paralysis, and general convulsions may occur, and the patient eventually falls into a coma. in the coma phase, autonomic instability becomes extremely predominant, and hypotension, arrhythmia, and hypoventilation may develop. most patients die shortly after the onset of coma if no intensive supportive care is given. no effective therapies for overt rabies have been established, so almost % of patients are destined to die. as of august , there have been only six reports of patients recovering from overt rabies. clinical and laboratory diagnosis of human rabies a clinical diagnosis might be possible if the patient could describe the animal which had bitten them and in which rabies endemic area, and also if they showed typical symptoms such as hydrophobia or aerophobia. however, it is rarely possible to diagnose rabies clinically in japan because the patient's history of animal bites is uncertain in most cases, and very few japanese physicians have experienced clinical rabies. an intravitam diagnosis of rabies could be made either by isolating the virus from saliva or cerebrospinal fl uid, demonstrating a viral antigen in skin biopsy samples or corneal impression samples using the fl uorescent antibody method, or detecting viral genes by reverse polymerase chain reaction (rt-pcr). , however, these laboratory methods are only useful after the virus has propagated into the brain tissue or disseminated to other parts of the body. they are not useful in the early stage of the disease, so it is practically impossible to diagnose rabies shortly after the onset. no medical treatment for clinical rabies has been established. treatment is mainly aimed at minimizing the clinical signs and symptoms, and especially at reducing physical and psychological pain. patients should be cared for in a private room with suffi cient sedation. the intravenous administration of morphine is effective to relieve anxiety, agitation, hydrophobia, and aerophobia. life-support measures should be avoided after rabies has been confi rmed. one case was reported of a patient who survived rabies after the use of heavy sedation in addition to antiviral medication, but other clinicians were unsuccessful using the same method. before a patient is treated with these new therapies, the patient and their family should be informed of the possibility of severe neurological sequelae even if the patient did recover. animal and human rabies still occurs in many asian countries, although neither animal nor human rabies has been reported in japan since . travelers in the endemic regions who are bitten by a possibly rabid animal need to receive pep as early as possible. the world health organisation (who) recommends the following post-exposure treatment. the bite wound should be thoroughly washed with soap and water. next, as much human rabies immunoglobulin (hrig, iu/kg) or equine rabies immunoglobulin (erig, iu/kg) as possible should be injected around the wound, and the remainder should be given intramuscularly. in addition, a tissue-culture-inactivated rabies vaccine should be administered on days , , , , and , and also on day if necessary. pep should be given to patients who request treatment even months after the bite, since an incubation period of longer than months has been reported in %- % of rabies cases. the risk of contracting rabies will be higher when the patient is bitten on bare skin as opposed to through clothing, because the rabies virus is highly concentrated in the saliva. moreover, when the face or the fi ngers are bitten, the incidence of rabies tends to be higher and the incubation period tends to be shorter than when the lower limbs are bitten. inactivated rabies vaccines marketed throughout the world are effective against lyssaviruses belonging to phylogroup i, but are not effective against phylogroup ii. in japan, a tissue-culture-inactivated rabies vaccine for human use (pcec-k) is produced by a private manufacturer, the chemo-sero-therapeutic research institute (kaketsuken). however, neither hrig nor erig is produced or imported in japan, and furthermore, the japanese government has no stock of rig. as rig is not available in japan, following the who recommendation for rabies pep is not feasible in practice. the pcec-k vaccine is prepared from an attenuated rabies strain, hep-flury, which is grown in primary cultures of chick embryo cells. it is then inactivated with betapropiolactone, followed by concentration and purifi cation. its antigen titer has not been offi cially announced. researchers in thailand reported that pcec-k is less potent than rabies vaccines produced in france and germany. during - , the number of patients visiting our vaccine clinic to receive pep after being bitten by a supposed rabid animal abroad was - per year except in , when the severe acute respiratory syndrome (sars) suddenly occurred (fig. ) . among these patients, about % and % were between the ages of - years and - years, respectively. eighty percent of the patients were bitten by animals in asian countries, and in particular thailand ( %). in detail, % of subjects were bitten by dogs, % by monkeys, and % by cats. about % of the patients attacked by animals overseas visited a local medical institute and received a rabies vaccine. the remaining % returned to japan without receiving proper treatment abroad, and visited our clinic after being warned by their family members or friends of the possibility of rabies and the need for pep. in france, cases of imported rabies have been reported since . in england, imported rabies cases were reported from to . since , cases have been reported in germany, and one case each in france, england, and taiwan. in japan, three cases of imported rabies have been reported as of july . the fi rst case was a young adult who was bitten by a stray dog in katmandu, nepal, during a personal trip. about month later, he complained of respiratory distress and died on the day of admission to hospital. he did not receive rabies pep in either nepal or japan. rabies was diagnosed based on the fi ndings of a postmortem histological examination. the second and third cases were both men in their sixties. , during their long stay in the philippines they were bitten by privately owned dogs, and they returned to japan in november without having received rabies pep. in both patients, the rabies virus was isolated from the saliva, and they were both diagnosed antemortem. by analyzing the gene, the rabies virus strains isolated were identifi ed as the strain transmitted in the philippines. in the third case, it was possible for medical staff to take the preventive measures recommended by the center for diseases control and prevention, as the diagnosis was made shortly after the clinical symptoms appeared. this case was given similar treatment to the -year-old survival case, but the treatment was unsuccessful. pre-exposure vaccination against rabies is a useful measure to prevent imported rabies. pre-exposure immunization is recommended to people who are living in, or traveling to, high-risk regions. the who recommends a dose of tissueculture rabies vaccine, with a potency of at least . iu per dose, to be given intramuscularly on days , , and . in japan, pre-exposure immunization consists of two doses of pcec-k given days apart, and an additional dose given months after the second dose. japanese travelers rarely plan their trip months or more in advance, with the exception of some public employees. in many cases, the period available before leaving japan is months at the most. when they do not have enough time to complete the three doses, we recommend taking at least doses. it is reasonably thought that patients who are bitten by a supposed rabid animal in an endemic area could effi ciently produce antirabies antibody after receiving another two or three doses of the vaccine, and that they would then be protected against rabies without using rig. it is safer for people who are scheduled to be engaged in outdoor investigations or to handle animals to receive three doses of rabies vaccine, as recommended by the who, before leaving for rabies-endemic countries. half a century has passed since rabies was eradicated in japan. however, countries free from rabies are exceptionally rare. travel to asian countries where many rabies victims still occur is easy, and only takes a few hours by airplane from japan. since japanese travelers are rarely aware of rabies, they are at great risk of being bitten by a potentially rabid animal. even if domestic human rabies cases no longer occur in japan, imported rabies cases are always possible. therefore, it is clearly important that no traveler ever carelessly puts out a hand to an animal in a rabies-endemic country. if a traveler is bitten by a suspected rabid animal in a rabies-endemic region, it is important that they receive rabies pep in a local medical institution immediately. at the same time, pre-exposure vaccination against rabies should be recommended to international travelers in order to ensure the preventive effect of pep. we should never forget that rabies is a preventable, but incurable, disease. the annual number of patients visiting our vaccine clinic to receive rabies post-exposure prophylaxis (pep). the fi rst overseas traveler bitten by a stray dog and requesting rabies pep came to our vaccine clinic in . the number of individuals receiving pep annually in our clinic remained below until . however, it exceeded in , and continued to increase from to . during this period, the annual number of japanese people going abroad was increasing. however, we cannot explain the increase in the number of patients requesting rabies pep simply by the rise in the number of japanese international travelers. one speculation is that the need for rabies pep has gradually been recognized among young japanese adults attacked by suspected rabid animals abroad because nowadays they can easily obtain information through the internet. on the other hand, not so many japanese medical institutions have rabies vaccine in stock, which leads to a concentration of patients at the limited number of hospitals capable of providing rabies pep. the decrease in the number of such patients in seems to have been caused by the outbreak of severe acute respiratory syndrome (sars) imported rabies case occurred after an interval of years -kyoto clinical course of the imported rabies case occurred after an interval of years -yokohama geneva: world health organization molecular epidemiology rabies: a historical perspective rabies: a new look at an old disease history of epidemics and prevention of rabies in the dogs in japan kyoken-kosho-chiho a study on the anti-rabic inoculation of dogs and the results of its practical application transition in epidemic and prevention of rabies on the epidemic of rabies in tokyo after the world war ii rabies control in japan rabies: update on a global disease prophylaxis against rabies a review of non-bite transmission of rabies virus infection airborne rabies transmission in a laboratory worker airborne rabies encephalitis: demonstration of rabies virus in the human central nervous system rabies transmission by a non-bite route rabies in man handling infected calf human-to-human transmission of rabies virus by corneal transplant human-to-human transmission via a corneal transplant: france human-to-human transmission via corneal transplant: thailand two rabies deaths after corneal grafts from one donor transmission of rabies by corneal graft cdc. investigation of rabies infections in organ donor and transplant recipients transmission of rabies virus from an organ donor to four transplant recipients rabies encephalitis: clinical, neuroradiological, and pathological fi ndings in transplant recipients e-alert february: cases of rabies in germany following organ transplantation the disease in man unexplained rabies in three immigrants in the united states. a virologic investigation diagnostic evaluation survival after treatment of rabies with induction of coma failure of therapeutic coma and ketamine for therapy of human rabies new anti-rabies vaccine of tissue culture origin an immunogenicity and effi cacy study of purifi ed chick embryo cell culture rabies vaccine manufactured in japan occurrence of rabies in the world and risk of rabies invasion into japan rabies questions and answers. rabies prevention and control: healthcare settings -updated the author thanks dr. naoki yanagisawa, department of infectious diseases, tokyo metropolitan komagome hospital, for his helpful suggestions in preparing this article. key: cord- -qjtifwhd authors: van diep, nguyen; sueyoshi, masuo; norimine, junzo; hirai, takuya; myint, ohnmar; teh, angeline ping ping; izzati, uda zahli; fuke, naoyuki; yamaguchi, ryoji title: molecular characterization of us-like and asian non-s indel strains of porcine epidemic diarrhea virus (pedv) that circulated in japan during – and pedvs collected from recurrent outbreaks date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: qjtifwhd background: since late , porcine epidemic diarrhea virus (pedv) has reemerged in japan and caused severe economic losses to the swine industry. although pedv vaccines have been used widely, the disease has swept rapidly across the county, and is commonly observed in ped-vaccinated farms, and has recurred in domestic herds. to better understand pedvs responsible for the reemerging outbreaks in japan, full-length spike (s), membrane (m), and nucleocapsid (n) genes of pedvs collected in japan during – , were sequenced and analyzed. results: phylogenetic analysis based on s gene sequences revealed that all the recent field pedvs were genetically distinct from the classical japanese strains, and were classified into three genotypes: north american (na), s indel, and asian non-s indel. our data suggested a possibility that multiple parental pedv strains were introduced into japan from abroad at the same time or similar times. the newly identified japanese strains showed the closest relationship to the us strains. two sublineages of japanese strains circulating in japan were similar to two sublineages identified in the us, suggesting common ancestors for these strains. in comparison with two vaccine strains used in japan, the field strains had various changes in epitope regions, glycosylation sites, and phosphorylation sites. these substitutions, particularly observed in epitope regions of the s ( , , , and ), m ( ), and n ( , , and ) proteins, may have affected antigenicity and vaccine efficacy, resulting in an unsuccessful pedv control. sequence comparisons between pedvs collected from primary and secondary outbreaks in three herds revealed that the disease has developed to an endemic stage in which pedv could persist for nearly two years in the herds or local regions, causing subsequent epidemics. conclusions: these results elucidate the genetic characteristics, origin, and molecular epidemiology of pedvs circulating in japan, as well as the pedv strains causing recurrent outbreaks. this study provides a better insight into the pedvs responsible for recent outbreaks in japan, and could potentially help to develop measures for controlling and preventing the disease. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. porcine epidemic diarrhea virus (pedv) is the etiologic agent of porcine epidemic diarrhea (ped), an acute enteritis disease that is characterized by vomiting and watery diarrhea and usually leads to high morbidity and mortality, especially in piglets [ ] . the disease was first recognized in england in [ ] , and the prototype virus, which was designated as pedv cv , was identified in belgium [ ] . subsequently, pedv has been reported in several other european countries, causing only sporadic outbreaks. since the s, it has been widespread in asia and has become an economic concern for the swine industry in many asian countries such as japan, china, south korea, thailand, and taiwan [ , ] . in late , new chinese strains of pedv were detected. these new strains were clinically more severe than the classical strains, resulting in %- % illness among infected swine herds and a %- % mortality rate among infected suckling piglets [ ] . in april , a pedv outbreak was confirmed in the us for the first time. it swept across more than states in the country, causing deaths of more than million newborn piglets during the one-year-epidemic period, and subsequently spread throughout north america, including canada and mexico [ ] . afterward, us-like ped epidemics have been reported in south korea, taiwan, japan, germany, france, and belgium [ , ] . belonging to the coronaviridae family in the nidovirale order, pedv has a single-stranded positive-sense rna genome of approximately kb in size that encodes four structural proteins (s, e, m, and n) and nonstructural proteins ( a, ab, and orf ) [ ] . among the viral proteins, the s glycoprotein is the most diverse [ ] , and plays a critical role in the process of inducing neutralizing antibodies and binding specific receptors [ ] . at least two b cell epitopes (ss and ss ) and two regions containing neutralizing epitopes (coe and c ) have been identified on this protein [ ] [ ] [ ] . the s gene is also associated with growth adaptation in vitro and attenuation of virulence in vivo [ ] , and was used as an important component in studies for understanding genetic relatedness among pedv isolates, the epidemiological status, and vaccine development [ , ] . the m protein is the most abundant component of viral protein in the envelope, and is responsible for morphogenesis, assembly, and budding [ ] . the n protein of coronavirus interacts with viral genomic rna, serving as the critical basis for the helical nucleocapsid during the viral assembly [ ] . two antigenic epitopes (nep-d and nep-d ) have been identified in the n protein of pedv [ ] . in japan, the ped-like disease was observed from late to early , but was not reported again until the outbreaks that occurred between september and june [ ] . in an epidemic of diarrhea caused by a pedv infection in , more than , suckling pigs died [ ] . afterward, there were no ped cases reported from until the first half of [ , ] . ped outbreaks first reemerged in october from a southern location (okinawa island) and spread rapidly throughout the country. until may , pedv infections were reported from more than farms in of prefectures; the infections affected over . million pigs and led to the death of half a million pigs, according to the ministry of agriculture, forestry, and fisheries of japan (http://www.maff.go.jp). currently, pedv infections and piglet mortality has decreased significantly. however, ped still occurred and frequently recurred in pig farms in japan. therefore, better insight into the causes for the reemergence of ped outbreaks is required for more efficient control and prevention of the disease. toward this goal, we sequenced and analyzed the full-length s, m, and n genes of the japanese field pedvs in comparison with those of vaccine strains used in japan, classical japanese pedv strains, and isolates from other countries. sample collection, rna extraction, and pedv detection small intestine or stool specimens were obtained from suckling piglets, post-weaning piglets, and sows presenting acute watery diarrhea at pig farms in japan, between december and february . intestinal samples were collected from dead piglets, and fecal samples were non-invasively collected immediately after excretion. thus, no aggressive actions toward the pigs were carried out for sampling purpose. samples of two major vaccine strains that have been employed in japan, p -v (nisseiken co., ltd) and -p c (kaketsuken co., ltd., japan), were collected from commercial vaccine bottles used in the swine farms. sample preparation, rna extraction, and pedv detection were performed as previously described [ ] . japanese field samples may contain both pedv variants with large deletions in the ′terminus of the s gene and pedv strains with an intact s gene [ ] . due to the complexity in discriminating, isolating, and individually sequencing pedv strains, only specimens containing pedvs with an intact s gene were used in our study. finally, pedv-positive field samples were collected from farms, located in five different prefectures in japan. these samples, along with the two vaccine samples described above, were sequenced to determine the full-length sequence of the s, m, and n genes ( table ) . all these field pedvs were confirmed to possess an intact ′-terminus of the s gene, showing only the expected prominent single dna band from pcr products on a . % agarose gel [ ] . after producing cdna, as previously described [ ] , the full-length s gene of pedv was amplified using the primer pair fs-f/fs-r ( table ) and kod fx kit (toyobo co., japan). the pcr products were used as templates for nested pcr reactions that amplified five dna fragments spanning the entire s gene using primer sets (cs -cs ), as previously reported [ ] . the m and n genes of the pedv were also amplified from the cdna using two newly designed primer sets, fmf/fmr and fnf/fnr ( table ). emeraldamp max pcr master mix kit (takara bio, japan) was used for pcr amplifications of the cs -cs fragments, as well as the m and n genes. the amplified pcr products were purified using a fastgene gel/pcr extraction kit (nippon genetics co., ltd., japan) according to the manufacturer's protocol. all sequencing reactions were carried out in duplicate, and sequences were determined in both directions using bigdye® terminator v . cycle sequencing kits (applied biosystems, ca, usa). products were analyzed using abi prism xl genetic analyzers (applied biosystems). nucleotide (nt) and deduced amino acid (aa) sequences were edited, assembled, and aligned using geneious v . . software (http://www.geneious.com), and percentage sequence divergences at the nt and aa levels were further calculated by using the same software. the obtained nt sequences were deposited in genbank under the following accession numbers: ky -ky as shown in table . unrooted phylogenetic trees were constructed using molecular evolutionary genetics analysis (mega) software, version . [ ] , with the maximum likelihood method and bootstrap tests of replicates. the best-fit nt substitution models for analysis were assessed. phylogenetic trees based on the nt sequences of the full-length s and n genes were generated using the tamura-nei substitution model with a discrete gamma distribution (tn + g). a phylogenetic tree based on the nt sequences of the m gene was created using the kimura -parameter substitution method with a discrete gamma distribution (k + g). n-glycosylation sites were predicted using a service available on http://www.cbs.dtu.dk/services/ netnglyc. phosphorylation sites were predicted using the netphos . server (http://www.cbs.dtu.dk/services/netphos) and netphosbac . server (http:// www.cbs.dtu.dk/services/netphosbac- . ). prediction of antigenic regions was performed using the emboss protein analysis tool [ ] integrated into the geneious software. identical nt sequences of the s gene were distinguished and excluded, resulting in the identification of individual sequences from the total collected field strains. phylogenetic analysis based on the s gene sequences from the japanese strains and reference strains identified from various countries revealed two major clusters: genogroup g divided into subgroups g a and g b, and genogroup g divided into subgroups g a and g b (fig. ). all japanese field pedvs in the present study fell into subgroups g a, g b, and g b. the most dominant strains belonged to g b, which comprised of current japanese strains, along with the highly virulent (north american type) pedvs isolated in the us, canada, mexico, south korea, and taiwan. two pedv of the field pedvs, jm- from miyazaki and other strains from aomori, aichi, kagoshima, and miyazaki were grouped into a monophyletic branch designated as ped-j , and they shared high nt and aa sequence identities ( . %- % and . %- %, respectively) with each other. another subclade, designated as ped-j , including japanese strains collected in miyazaki were also clustered into a segregated branch as shown in fig. the sequence data revealed that s genes from the japanese field pedvs are of - nt long, and encode proteins with - aa residues. this consequence was due to the presence of several deletions or insertions that accumulated primarily in the n-terminus of the s protein. multiple alignments of the s gene demonstrated that jm- and jm- exhibited insertions and deletions ( -nt, -nt, and -nt deletions at positions , , and , respectively, and a -nt insertion between positions and ), which are typical for s indel strains [ , ] . compared to the two vaccine strains, all japanese field pedvs were highly conserved in epitopes c and ss , except for strain jm- that had an aa substitution (l i) within c (fig. ) . additionally, compared to the vaccine strains, all the japanese field pedvs had two aa changes (l s and d s) in the epitope ss , and three serine substitutions (a s, t s, and g s) in the neutralizing domain coe (excluding jm and jm- ). notably, the aa substitutions at positions and resulted in predictable phosphorylation sites in the s protein of the japanese field strains. furthermore, different amino acids were found at the sites within the coe domain (fig. ) . regarding the prediction of high-specificity n-glycosylation sites in the s protein, most japanese strains belonging to g b exhibited eight n-glycosylation sites over the entire s protein (additional file : table s ). jm- had one additional n-glycosylation site at residue , and jm- possessed an additionally unique n-glycosylation site at position of the aa chain. two japanese s indel strains ( jm- and jm- ) had seven n-glycosylation sites, while jm- had eight n-glycosylation sites. compared with the vaccine strain, -p c , most of the japanese non-s indel strains in g b lost three n-glycosylation sites (at , , and ) and gained another nglycosylation site (at ). compared with p- v, these g b japanese strains lost three n-glycosylation sites (at , , and ) and gained four other nglycosylation sites (at , , , and ). the m gene sequences from all the japanese field pedvs and the two vaccine strains consisted of nucleotides, encoding a protein of aa. identical nt sequences were distinguished and excluded, resulting in the identification of seven unique sequences from the total collected pedvs (table ) . a phylogenetic tree based on the m gene of the japanese strains and reference strains revealed that all the sequences were divided into groups (g and g ); g was further divided into two subgroups g a and g b (fig. a) . all the current japanese pedvs fell into g , and showed the closest relatedness to the na strains and emerging strains identified in europe, south korea, and china. these field pedvs were phylogenetically distant from the vaccine strains and a classical japanese strain (jme ) of g . the m genes of field pedvs had . %- % nt identity to each other and . %- . % nt identity with the vaccine strains. the b cell epitope, wafyvr [ ] , located at position - of the m protein, was conserved in all field and vaccine strains. compared with the vaccine strains, all japanese field strains had an aa substitution (a s) belonging to an antigenic region (aa - ), as predicted by the emboss protein analysis tool (additional file : figure s a ). strain jm- had a different aa (s n) leading to the loss of a predicted serine phosphorylation site. in another predicted epitope region (aa - ), the field pedvs had one different aa (v i) compared to p- v, and two aa substitutions (f s and q e) compared to -p c . these substitutions (f s) resulted in gaining a predicted high-specificity n-glycosylation site. the n gene of all the field pedvs and vaccine strains was nt in length encoding a protein of aa. identical nt sequences of the n gene were distinguished and excluded, resulting in the identification of unique sequences. phylogenetic analysis based on the n genes from the japanese strains and reference strains revealed the classification of pedv strains into three groups: g , g , and g (fig. b) . all japanese field strains were closely clustered together into subgroup g with other us and us-like strains. the japanese field pedvs shared . %- % n gene nt sequence identity to each other and . %- . % to the vaccine strains. strain -jm from the first outbreak in miyazaki had % nt identity to eight other field strains collected in miyazaki, aichi, and kagoshima, and this is likely to be the most dominant sequence of n gene among the field strains. compared to the vaccine strains, all the field strains in the present study had aa substitutions (additional file : figure s b ); the substitution k n resulted in forming a predicted n-glycosylation site on the n protein from the field strains, while the aa substitutions a t and n s observed in field strains formed two predicted phosphorylation sites. of note, the substitutions k n and n s belong to the reported antigenic epitopes nep-d (aa - ) and nep-d (aa - ), respectively [ ] . there were three swine farms where ped outbreaks first occurred in , and then recurred in , as shown in table . farm is located in aomori prefecture, while farms and are located in the same town in miyazaki prefecture. phylogenetic analysis based on the s gene sequences revealed that three pedv strains ( jm- , jm- , and jm- ) collected from two different outbreaks on farm were closest to each other and segregated into a distinct minor branch. pedvs from the primary ped outbreaks on farm ( jm- , jm- on farm , the s, m, and n genes of pedvs identified from the recurrent outbreak ( jm- and jm- ) shared the highest ( . %, %, and . % respectively) nt sequence identities to a strain ( jm- ) collected table information of primary and recurrent ped outbreaks occurred in the three pig farms in this study farm mortality rate of piglets less than two weeks-old; b mortality rate of the piglets less than one-week-old all the information for mortality and morbidity was kindly supplied by the veterinarians responsible for these farms from the primary outbreak. the s genes of pedvs from the primary outbreak on farms and had high ( . %- . %) nt identity to each other. these strains also had identical sequences for the m and n genes. in the secondary outbreaks, pedvs from farms and also shared highly homologous genes. the s and m genes of two strains ( jm- and jm- ) from farm shared % nt sequence identities with those of a strain ( jm- ) from farm . compared with the strain collected from the primary outbreak on farm , two strains from the recurrent outbreak had aa substitutions in their s proteins (additional file : figure s ) . notably, the substitution s n lead to the loss of a predicted serine phosphorylation site. these recurrent strains also had two aa substitutions (r s and t n) in the n protein; the r s substitution formed a predicted serine phosphorylation site. comparison to pedvs from the primary outbreak on farms and , pedvs from the recurrent outbreaks had aa substitutions in the s proteins, in addition to a single aa change (a v) in the m protein and two aa changes (l i and s f) in the n protein. notably, the substitutions l i and s f occurred in the reported epitope nep-d of the n protein. since late , massive ped outbreaks have recurred in japan causing tremendous financial losses in the swine industry. despite the nationwide use of available attenuated vaccines developed decades ago [ ] , pedv has rapidly swept across the county. our sequencing data revealed that the prevailing japanese pedvs are genetically heterogeneous and can be classified into genotypes: na, s indel, and asian non-s indel. both the na and s indel types, which have been identified in recent studies in japan [ ] [ ] [ ] ] were also responsible for recent outbreaks in the us and south korea. in our study, besides the two previously reported pedv types, we identified another type of japanese pedv (designated as asian non-s indel) that is closely related to the vietnamese, thai, and chinese strains. the asian non-s indel type was collected from an early ped epidemic that occurred in southern japan where the recent ped pandemic began spreading. moreover, the asian non-s indel type appeared at a similar time to the appearance of the na and s indel types in japan [ ] . together with the absence of ped cases from to in japan [ , ] , we speculated a possibility that the three types of japanese pedv originated from one source and emerged at the same or similar time in the southern region of japan before spreading across the country. the result of this study revealed that all the field pedv strains (except jm- ) were closely related to emerging strains identified in the us, south korea, taiwan, and in european countries. of note, the japanese pedvs were genetically most closely related to the us strains; some even had identical s gene. since the recent ped pandemics in japan started to reemerge six months after those in the us, the close genetic relationship indicated that the current japanese pedv might have been introduced directly from the us, or both the us and japanese pedvs were derived from one source of origin. since april , the na type of pedv has emerged in the us. the na strains were identified subsequently in south korea, taiwan, and japan from late . another pedv type possessing typical insertions and deletions in the n-terminus of the s gene when compared to the prototype na type was first discovered in ohio in the us [ ] . this pedv type, therefore, was designated as s indel. the s indel pedvs were also identified later in recent outbreaks in japan, south korea, canada, belgium, france, germany, portugal, slovenia, and the netherlands [ ] . both highly virulent and s indel pedvs prevailing in the us are supposed to be derived from chinese pedvs as a result of recombinant events [ , , ] . in accordance with these findings, our sequence analysis showed that the s indel strains, such as ch/hbqx/ and js , had already appeared in chinese ped outbreaks during - [ , ] . with the prevalence of both na and s indel strains in the us, south korea, and japan since , we suggested that there are two major possibilities of origin for the reemerging japanese pedv strains. first, us-like strains have been directly transmitted to japan from the us or south korea. second, the same source of the us strains could have introduced the pedv into japan, from china, shortly after the us outbreaks. however, our study revealed the prevalence of the asian none-s idel pedv strain in japan, which has not been reported in the us or south korea. thus, we incline toward the possibility that multiple parent pedv strains have been introduced into japan, from china or china's neighboring countries (such as the southeast asian region), causing the recent ped pandemic. further investigation and surveillance are required to specifically identify the source of origin for the reemerging japanese pedvs. sixteen pedv strains from four different prefectures including miyazaki, kagoshima, aomori, and aichi formed a well-supported subclade (ped-j ). fourteen pedv strains from various farms in miyazaki prefecture were also grouped into a distinct subclade (ped-j ). these results suggested that strains of those japanese sublineages (ped-j and ped-j ) may derive from two common ancestral pedvs whose progeny have spread in the regions. two genetic sublineages of us strains, namely ia -co/ and mn-ia [ ] , were identified from the us outbreaks in . notably, the japanese pedv of the japanese sublineage ped-j in this study showed a close relationship with the us sublineage ia -co/ , and pedvs of the japanese sublineage ped-j showed close relatedness with the us sublineage mn-ia . this data revealed the common pattern of genetic diversity as well as the close relationship between the japanese strains and the us strains, suggesting a common ancestor for these strains. in our previous study [ ] , by sequencing a partial s gene containing a neutralizing epitope region (coe domain), we suggested that jm- may belong to the s indel type that has been circulating in japan together with the highly virulent us-like strains. in this study, by sequencing the full-length s gene, we confirmed jm- to be an s indel strain. we also identified another strain that belonged to the s indel type, jm- , although it was not predicted to be an s indel pedv in the previous study. this discrepancy arose due to the region of the partial s gene utilized in the previous report that lack the typical insertions and deletions of the s indel type. our data revealed the circulation of s indel strains in ped outbreaks occurring in japan, but these strains compose only a minor population of the reemerging japanese pedvs. these results are consistent with two recent reports of japanese pedv [ , ] . phosphorylation of viral proteins is a reversible posttranslational modification that can have major impacts on viral infection, replication, and cytotoxicity in a host cell [ ] . for infectious bronchitis virus, belonging to the coronaviruses, the phosphorylation of the nucleocapsid protein has a dramatic influence on rna binding capability and the recognition of viral rna from nonviral rna in the process of viral replication [ ] . along with phosphorylation, glycosylation is another posttranslational modification that often strongly affects the protein functions of viruses. n-glycosylation sites in the spike gene of the severe acute respiratory syndrome coronavirus were demonstrated to be crucial for viral infection [ ] . on the other hand, mutations observed in the neutralizing regions have been proposed as presenting a viral evolution, allowing escape from antibodies developed against vaccine or classical pedv strains [ ] . in this study, we characterized differences in epitope regions, high-specificity n-glycosylation and phosphorylation sites, between the vaccine and recent field strains. various substitutions observed in the s ( , , , and ), m ( ) , and n ( , , and ) proteins may have affected the antigenicity, conferring the capacity for immune evasion in the pedv field strains, and consequently, influencing the efficacy of the vaccines. in fact, despite vaccination, pedv infections still spread rapidly and are commonly observed on ped-vaccinated farms in japan [ ] . vaccination showed very low efficacy and could not mitigate the severe losses caused by ped [ ] . based on the antigenicity analysis of the vaccine and field strains, as well as the recent performance of the ped vaccines on the japanese pig farms, we suggest that the development of novel vaccines based on reemerging pedvs is necessary to control current ped outbreaks. the first ped epidemic on farm was a severe occurrence in may (table ) . afterward, obvious clinical symptoms and death caused by ped were not observed in pigs of the herd until february when the subsequent outbreak recurred with less severity than the first. although the pigs did not show any obvious symptoms of ped, the presence of pedv on this farm was detected in piglets several times by rt-pcr during the period from june to january , according to the information supplied by veterinarians responsible for this farm. phylogenetic analysis based on the sequences of the s and n genes showed that pedv strains from the primary and secondary outbreaks formed monophyletic branches. they also shared with each other the highest sequence identity of the s, m, and n genes compared to other strains used in this study. additionally, ped outbreaks were not observed on other farms within a km radius of farm , and many strict measures for establishing a high level of security were implemented on this farm. taken together, we speculated that pedv strains causing the primary outbreak induced partial protective immunity but persisted in the herd for nearly two years. subsequently, the persisted pedvs caused the secondary epidemic. this is the first report of a two-year-long persistence of pedv in a pig herd. it has been proposed that the popular circulation of pedv variants with large deletions in the s gene is associated with the persistence of the pedv on the infected farms in japan [ ] . however, we did not detect the presence of the large s deletion variants in the fecal and intestinal samples collected from this farm. other factors, including the genetic variation of causative pedv strains, may play a more important role in the disease recurrence. on farms and , ped first occurred in may , despite all the sows receiving antepartum pedv vaccinations in . afterward, the disease disappeared until early when the subsequent outbreaks recurred. there was no detection of the pedv during the interval between these two outbreaks. phylogenetic analysis based on the s and n genes revealed that the strains collected from the first outbreaks on farms and formed a monophyletic branch. moreover, these two farms were located close to each other with a distance of . km (separated by a small mountain), and the outbreaks occurred at the same time. this suggests that the pedv strains introduced onto these farms that induced the first outbreaks were derived from a common source of pedv. likewise, in the secondary outbreaks, the strains collected from these two farms also have the highest genetic identity to each other and formed minor monophyletic branches, as shown in the phylogenetic trees for the s and n genes. all the strains collected from the primary and recurrent outbreaks on these farms were closely clustered into the subclade ped-j , together with other strains collected from other farms located in miyazaki prefecture (fig. ) . this data suggested that the pedv strains causing the subsequent outbreaks on farms and evolved from pedvs that had been circulating in the miyazaki region since . there are two possibilities for the origin of the strains that caused the subsequent outbreaks on farms and . first, pedvs may have persisted silently on these farms since , evolving, and then causing the subsequent outbreaks when suitable conditions arrived in early . another possibility is that pedvs may have persisted on other pig farms, circulating in the miyazaki region since the ped occurrence, after which the pedvs were introduced again onto farms and , causing the secondary epidemics. more data is needed to elucidate exactly the origin of these pedvs strains. the occurrence of ped in these herds has partially indicated the failure of using the current pedv vaccines, which are based on old seed stock of classical japanese strains. besides, the repeated outbreaks in these herds indicate that the disease has developed to an endemic stage. these results could be used to elucidate the prevalence of pedvs and to contribute to the prevention of ped in the region. in conclusion, our study revealed that japanese field pedvs causing ped outbreaks during - could be classified into three types, and these pedvs might have been introduced from overseas at the same time or during similar periods. the disease has developed to an endemic stage in which the pedv can persist for a long time in these herds or these local regions causing subsequent epidemics. the distant genetic relationships and various aa substitutions between the japanese field pedvs and vaccine strains may be responsible for the unsuccessful ped control in japan. our findings will be useful in understanding the origin and molecular epidemiology of the pedv, and helping to develop measures for the control of the disease. additional file : table s . highly-specific n-glycosylation sites in the spike protein of the vaccine and field strains. (docx kb) additional file : figure s . amino acid substitutions in the m and n proteins between the field and vaccine strains. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines letter to the editor a new coronavirus-like particle associated with diarrhea in swine evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains new variants of porcine epidemic diarrhea virus, china cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea genetic diversity of orf and spike genes of porcine epidemic diarrhea virus in thailand incorporation of spike and membrane glycoproteins into coronavirus virions the coronavirus nucleocapsid is a multifunctional protein the identification and characterization of two novel epitopes on the nucleocapsid protein of the porcine epidemic diarrhea virus an immunohistochemical investigation of porcine epidemic diarrhoea porcine epidemic diarrhea: its diagnosis and control molecular characterization of pig epidemic diarrhoea viruses isolated in japan from isolation and molecular characterization of porcine epidemic diarrhea viruses collected in japan in us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation mega : molecular evolutionary genetics analysis version . emboss: the european molecular biology open software suite emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains new variant of porcine epidemic diarrhea virus identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus isolation and experimental inoculation of an s indel strain of porcine epidemic diarrhea virus in japan origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern china from to molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central china during - outbreaks phosphorylation events during viral infections provide potential therapeutic targets role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein specific asparagine-linked glycosylation sites are critical for dc-sign-and l-sign-mediated severe acute respiratory syndrome coronavirus entry bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during - in guangdong, china measure to mitigate loss caused by ped (porcine epidemic diarrhea) using maternal immunity the work was conducted with funding from the university of miyazaki (grant number - ) and japan society for the promotion of science (kakenhi program, grant number h ). the funders had no role in the design of the study and collection, analysis, and interpretation of data and in preparation of the manuscript. data supporting the conclusions of this article are included within the article and its additional files table s , figure s , and figure s . sequence data supporting the conclusions of this article are available in the genbank repository under the accession numbers ky -ky .authors' contributions ry and nd designed the study. ry, ms, zn, and th provided the viral samples and other materials. nd, nf, at, ui, and om conducted experiments. nd designed the primers, interpreted the sequencing data and wrote the manuscript draft. ry, ms, zn, th, at, ui, nf, and om analysis the data and revise the manuscript. all authors prepared and approved the final manuscript. in this study, the intestinal samples were collected after the death of pigs due to the virus infection in the pig farms. no pig was culled for sampling purpose in this study the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -ad i wgj authors: nan title: th international congress on amino acids and proteins : vienna, austria, august – , date: journal: amino acids doi: . /s sha: doc_id: cord_uid: ad i wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from hcu patients were compared to controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control . ( . - . ) and for hcu . ( . - . ); p ϭ . ]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ . , p Ͻ . ). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. ( nmol/g; . %); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ ; - nmol/g; . - . %); rabbit ( nmol/ g; . %); pig (sus scrofa f. domestica, artiodactyla) ( nmol/ g; . %); bovine (bos primigenius f. taurus, artiodactyla) ( nmol/g; . %); seal (phoca vitulina, carnivora) ( nmol/g; . %), and rob (halichoerus grypus, carnivora) ( nmol/g; . %). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (Ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl- -thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere rp- e, ϫ mm, µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than , . the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by fer- d/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of adults ( males and females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: individuals ( %) belonged to the adult age group, persons ( %) to the mature and ( %) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over . . quantitative determination of free and bound -nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since -nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine -nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify -nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free -nitrotyrosine in rat plasma as well as bound -nitrotyrosine in tissue samples are reported. plasma samples were spiked with , , -d - -nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z ae for the analyte and m/z ae for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl n. -nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. -fold) by comparison with -nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r Ͼ . ) within concentration range . to . nm (equivalent to . - , fmol on column) and recoveries above %. inter-and intra-assay precision was determined below % over the concentration range . to . nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range . - . nm. this curve strongly correlated with a linear model (r Ͼ . ) and slope was . Ϯ . (mean Ϯ sd). basal level of -nitrotyrosine in rat plasma was measured to be within concentration range Ͻ lod to . nm. -nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl n at °c). resulting hydrolysates were analysed by lc-esi-ms/ms and nitrotyrosine was monitored in srm mode (m/z ae for the analyte and m/z ae for the internal standard). t. guszczynski , r. b. kapust , d. s. waugh , and t. d. copeland basic research laboratory, and macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv- . with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi ; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, g; soy peptone, g; yeast extract, g; dglucose, . g; nacl, g; dipotassium hydrogen phosphate, . g in l demineralized water adjusted to ph . . fermentation was conducted in nineteen -l shake flasks, each containing ml medium, for d at °c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water : : : ) yielding . g and . g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. ) and semipreparative hplc (nucleosil c- ; ϫ mm id; µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. . the -residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. h and c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of . the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful h and c signal assignment. a complete assignment of h and c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of h and c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe ϩ are converted to coloured products, with maximum wavelength at nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by ‰ δ n. in rats fed diets containing intrinsically labeled c casein or the corresponding amino acid mixture labeled with c leucine and n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta , m. yoshitake , h. yoshida , t. yoshitake , and m. yamaguchi faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, -hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of fluorogenic reagents, o-phthalaldehyde (with -mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality . to . mol·kg Ϫ using the "isoperibol" type calorimeter at . k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re ϩ . substances (glyh) . h o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re cl ] Ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re cl ] Ϫ in the substance (glyh) [re cl ]cl together with existence of quadruple re -re bond that is described first. in the substance [re (gaba) cl (h o)]cl . h o axial position of re ϩ fragment are substituted by ligands of different kind: h o and cl Ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p- -rp column allowed separation of caseins in min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of ml/min and a temperature of °c, using a mixture of two solvents. solvent a . % trifluoroacetic acid in water and solvent b was % acetonitrile- % water- . % trifluoroacetic acid. the effluent was monitored by a uv detector at nm. the determinations were performed in the linear range of . - . mg/ml for k-casein, . - . mg/ml for α-casein and . - . mg/ml for -casein. the detection limits were . , . and . mg/ml, respectively. the validity of the method was verified. the recoveries ranged from to % for cow's milk. the precision of the method was also evaluated, the % cv being less then . %. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine ( - µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to % seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by . -and . fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from % in freshwater to % in % seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for h in freshwater, and % seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec . . . ] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was , -fold with % yield. the molecular weight of the enzyme was estimated to be kda on sds-page and kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below % with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ , were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg ]-thf-γ exhibited higher restoring activity than that of our synthetic thf-γ . phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa ) was engineered to attain a pre-designed d structure and later modified by grafting short peptide fragments from human α interferon (aag ), and insulin molecules (aag ). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa ) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet- (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α -interferon fragments was studied in vitro using human fibroblasts cell line l- and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag ) acquired ability to stimulate glucose uptake by l- cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x m -/double knockout mice, transgenic for a single-chain hla-a - m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc and ba - . analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a binding peptides resulted in candidate peptides for immunogenicity screening. we have identified antigenic peptides of which peptides were found to be immunogenic in hhd mice. interestingly of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage- were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes , e. giralt , and d. andreu centro de investigação em química da universidade do porto, portugal department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of bases including the active site. the homology of the active site and the surrounding bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was . % and . %, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of amino acid residues. there was a high degree of homology in the sequence of amino acid residues (met ϳhis ) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments - : ksglkykk (g - ) and - khvvpnev vvqrlfqvkgrr (g - ), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol , -bisphosphate (pip ). the binding of peptides g - and g - to a cluster of four pip molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of , ps. the binding of the pip molecules to the peptides g - , g - showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip . during the binding some of the pip molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by -hydroxyphthalic anhydride ( hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus (hsv- ), bovine parainfluenza virus (pi- ) and porcine respiratory corona virus (prcv), the hp proteins were shown to be active against human herpes simplex virus only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with hp-albumin, hp--lactoglobulin and hplysozyme resulted in protection against hsv- infection whereas pre-incubation with hp-α-lactalbumin had no antiviral effect. however, all hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv- infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv- glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. ) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. , syrkin a. , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c. . . . , extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt- cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration - ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt- cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration mkm ( , mg/ml) not exerting toxical effect on mt- cells. the same effect is attained having used lo in doses - ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt- cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with - % ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y b) were cultured in eagle's minimum essential medium (mem) containing % serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. ) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k- as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was mm l-glutamine, mm taurine and . unit/ml ggt, ph , and -hr incubation of °c. forty-three mm gamma-glutamyltaurine was obtained and the yield was .%. gamma-glutamyltaurine was purified by dowex ϫ column and c column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex- t . mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and Ј, Ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with amino acid residues is first translated and then processed to a mature peptide with amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex- t , and expressed efficiently in e. coli bl as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: -deoxy-d-arabino-heptulonate- -phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by . , . , . and . folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional ( -d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant protein phosphatase. the two aliquots were then subjected to -d electrophoresis. the phosphoproteins could be mapped on the -d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately % of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately - % of the total proteins resolved by a large format d gel. to reduce the number of proteins to be identified the d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a -d gel, where about mg of protein amount has been resolved, , - , protein spots can be detected, using coomassie blue staining. the spots represent the products of only - different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis , j.-f. juranville , and l. suter genomics technologies, and drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, different gene products were detected. approximately % of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. ( ) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. ( ) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution d electrophoresis and detection of phosphorylated proteins labeled with p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. ( ) differential gene expression has been followed by using s met pulse chase labelling concurrently with endothelin stimulation. at least proteins showed significant changes in expression of d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by d gel electrophoresis and mass spectrometry d. gostick , s. cohen , p. young , b. karol , j. langridge , j. randell , t. slyker , and a. jacobson micromass, manchester, u.k. waters corporation, milford, massachusetts, and bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are possible sequences for a peptide containing any of the naturally occurring amino acids and having residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms ) is set to transmit only the fragment ion of interest. the ms quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms is not mass selective, operating in the rf only mode. the first data set at low energy ( ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of da (h po ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h po moiety is not observed, however a low mass immonium ion at m/z can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ p] inorganic phosphate. d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer , , m. berger , g. lubec , and m. fountoulakis f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland institute of cancer research, and department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha- chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately proteins were identified in the mitochondrial fraction, which were the products of different genes. about % of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about % were found in the microsomal fraction as well. of the proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec and m. fountoulakis department of neonatology, university of vienna, austria gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor -alpha , elongation factor , elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli , m. g. giuffrida , e. pessione , g. dellavalle , c. barello , e. griva , and c. giunta dipartimento di biologia animale e dell'uomo, università di torino, and csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller , r. wait , l. sironi , i. eberini , m. gemeiner , e. tremoli , and e. gianazza veterinärmedizinische universität, wien, austria imperial college school of medicine, hammersmith, london, u.k. universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional ( d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra , , s. fontana , p. cancemi , g. alaimo , and s. minafra , centre of experimental oncobiology, department of cell biology and development, and institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about % of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, -bc (minafra et al., br. j. cancer, , - , ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci , l. trabalzini , d. soldateschi , e. ferro , a. paffetti , and p. martelli dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses ( kbp): ad strain genome was completely sequenced, containing about open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: ) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of ) beta, early genes, needed for dna replication, and, finally ) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis ( d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad . the phenotypic d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the - th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe , k. landry , s. taurin , s. n. orlov , and a. v. pshezhetsky sainte-justine hospital research centre, and research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp family, a molecular chaperone grp , and so-called mortalin (grp ) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis ( d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c trap cartridge it is possible to pre-fractionate the peptides before separation on a c column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c cartridge whilst they are desalted. finally the peptides are eluted from the c pre-column, at nl/min, onto a um id ϫ cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of serum samples, immunofixation identified igg , igg , igg , and igg in , , , and cases, respectively. their light-chains were k in cases and λ in cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis ( -de) with various isoelectric focusing conditions as well as by -de ( -de of the proteins extracted from agarose after serum protein agarose electrophoresis). after -de or -de, the monoclonal γ-chains were not visualized in out cases, whatever the isoelectric focusing conditions that were tested. in cases, γ-chains were only detected using alkaline ph - gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p Ͻ . ) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ . , multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis ( -de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein (hsp ), hsp ry, heat shock cognate (hsc) , alpha crystallin b chain, glucose regulated protein (grp) and grp showed aberrant expressional patterns depending on brain region. hsp . , grp and t-complex (tcp- ) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin- -gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(Ϫ)epigallocatechin- -gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec . . . ), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately hivϩ patients; not yet on anti-viral treatment (cd count to ). methods: parameters measured included: cd count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within months, significant trends are noted. the cd count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; . /cmm and % confidence interval (c ), this was confirmed by the total lymphocyte court values. after months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla , i. giovannini , j. h. siegel , g. boldrini , and m. castagneto centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r ϭ . , p Ͻ . ). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ . ϩ . (pea) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (asp) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (logglu) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in patients with pd. vitamin e concentrations were also assessed in age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups ( . Ϯ . mmoli/l for pd patients and . Ϯ . mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli , institute of biological chemistry, university of urbino, italy department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers ( tocopherols and tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and out of main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than iu ( mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite , , -trimethyl- -(b-carboxyethyl)- hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari , s. mensi , g. mercurio , c. callà , l. colacicco , e. sacco , and s. lippa department of anaesthesiology and intensive care medicine, and department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients ( females, male; aged from to years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq ). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq and vitamin e showed a positive correlation with the plasma meth levels (p Ͻ . ; r ϭ . and p Ͻ . ; r ϭ . , respectively). since vitamin e and coq are mutually dependent-molecules, the administration of meth, essential substance for coq synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: ( ϩ ) ϩ ) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content - %), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing µg of the substance in ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in % and increased visual functions in % of the cases. in % of the patients visual acuity raised by . - . . electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich- (caspase- )/ ced- (caenorhabditis elegans death protease- )-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl- interacting mediator of cell death/bcl- related ovarian death gene) and p were significantly increased only in ad frontal cortex (p Ͻ . , in all cases). cerebellar bcl- levels were significantly increased in ad (p Ͻ . ) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar , m. brózik , r. tóbi , t. szabó , j. szakonyi , b. rojkovich , p. gergely , and f. hudecz research group of peptide chemistry hungarian academy of science, budapest, central laboratory of immunology, semmelweis university, budapest, and national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting - % of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( shqestrgrsrgrsgrsgs ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)- alpha and beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il -receptor antagonist (il- ra) is a protein structurally related to il- beta that effectively inhibits the proinflammatory effects of il- . a polymorphism in the Ј-flanking regulatory region at Ϫ of the il- alpha gene, which may cause an overexpression of il- alpha and a variable number tandem repeats (vntr) polymorphism in the il- ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il- beta gene have been reported to influence the protein production: one is located in the promoter region at position Ϫ and the other is in exon at position ϩ . to analyze the contribution of il- alpha, il- beta and il- ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in italian patients with clinically definite ms and healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h o /cl Ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b- proline and arginine residues, hydroxy- -aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl , svedberg units (s f ) - and ldl , s f - ) by gc-ms. paired knee synovial fluid and plasma samples were collected from subjects with ra. additionally, plasma samples were collected from healthy controls. the ldl hava content in plasma was not different between the groups (ra, . Ϯ . vs controls, . Ϯ . mol/mol apob- , p ϭ . ). the ldl hava content in plasma was significantly higher in ra ( . Ϯ . vs . Ϯ . mol/mol apob- , p ϭ . ). furthermore, synovial fluid ldl and ldl in ra contained elevated hava levels when compared with plasma concentrations (ldl syn , . Ϯ . mol/mol apob- (p Ͻ . ) and ldl syn , . Ϯ . mol/mol apob- (p Ͻ . )). results suggest that proline and arginine residues of apob- are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob- to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl were prone to direct oxidation of apob- . correlation between hava content in plasma and synovial fluid ldl and ldl in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis- -hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm ; : ) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg )-lys-chyp} to increase the % by wt of the analogue. rats were exposed to % o for da to induce ph. on da , and after da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at and da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da , rvp (mmhg) was Ϯ and hyp (µg/vessel) was Ϯ in at. rvp and hyp increased to Ϯ * and Ϯ *, respectively, in hypoxic animals (n ϭ ; *p Ͻ . vs. at). on da , rvps were at Ϯ , ht Ϯ *, hc Ϯ * †; hyps were at Ϯ , ht Ϯ *, hc Ϯ * † (n ϭ ; *p Ͻ . vs. at; †p Ͻ . vs. ht). from da to , rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p belonging to the cyp family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp e catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p homologous to cyp e . the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp s and by introducing new cyp s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens and s. beernaert pediatrician, metabolic diseases and dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over % of infants born to mothers with blood phenylalanine (phe) concentrations above µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, years old, who showed phe blood levels of - µmol/l under a strict diet (total protein content of . g/kg body weight/day with . g/kg natural proteins and . g/kg proteins provided by the aminoacid mixture pku (milupa, germany); , cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of . g/kg bw proteins and , cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to . - meq/l (ref. value . - . meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku , carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at c° for total time period minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p Ͻ . ) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over volatile components were separated, however only the most important component for caramel flavour were reported. maltol and hydroxymethyl- -furfural (hmf) and . h-pyran- -one, , dihydro- , -dihydroxy- -methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca ϩ -independent mechanism involving phosphorylation of ser by a phosphatidylinositol -kinase (pi -kinase) dependent pathway. we investigated the involvement of pi kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser in enos were increased by d-glucose ( mm, min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi -kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi -kinase dependent, ca ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered ( - times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku ( g ϭ adh , g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e components of the pyruvate, oxoglutarate and branched-chain -oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a -fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde- phosphate dehydrogenase (gapd) [e.c. . . . ] from the cytosolic domain of the membrane protein band (cdb ) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k ϭ . m Ϫ s Ϫ and . m Ϫ s Ϫ , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb and gapd. the cys in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form Ϫsno on, and decrease the affinity between, gapd and cdb . in intact rbc, both the no-donors -morpholino-sydnonimine (sin- ) and peroxynitrite (onoo Ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo Ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and Ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin- on gapd translocation. the results demonstrate that cdb and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than - days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of - days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about , - , ml of bile fluid, , - , ml pancreatic juice and , - , ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than days. if artificial nutrition is necessary for more than days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated patients with ppa and with mma in a period of months by weekly capillary blood punctures performed by the parents. age of the patients were from . until years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment ( - mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c /c -acylcarnitine quotient. mean value in mma and ppa patients showed a range of . - . (normal . ϩ/Ϫ . , n ϭ ), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with , , , tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci , m. sokoĺowska , m. zapaĺa , and l. wĺodek institute of pharmacology, polish academy of sciences, kraków, and institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland , , , -tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and -hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out h after a single dose of tiq ( mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs , r. steinfeld , a. kohlschütter , j. zschocke , and k. ullrich department of pediatrics, university of hamburg, and university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. . . . ) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor ( r)-l-erythro- , , , tetrahydrobiopterin (bh ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh . indeed, two such patients in our hospital were also sensitive to daily oral doses of - mg bh /kg. the subsequent molecular genetic analysis revealed that patient was homozygous for the widespread mutation y c and patient was compound heterozygous for the mutations a d and k n. it is striking, that all mutations are located distant from the known bh -binding site and thus, should not be associated with bh -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf ) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of % gypsy rna was achieved with about ng of the protein in ml of the reaction mixture. the binding constant was &times; m- , which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg ) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: ) tlc is employed when starting the investigation, ) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients ( in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo . we cloned each gene encoding ybbq and yhae into e. coli jm . both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of kda, whereas yhae was a tetramer (native molecular mass, kda). ybbq showed the maximum activity at ph . for the oxidation of d-serine. whereas optimum ph of yhae was ph . . they catalyzed oxidation of glycerate and -hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate -dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi , Á. bódi , l. gráf , a. perczel , a. patthy , and g. orosz department of organic chemistry, biochemistry, eötvös university, budapest, agricultural biotechnology centre, gödölló´, and research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd- family of canonical serine protease inhibitors. in these peptides, there is a p -p Ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p -p Ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p -p Ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p -p Ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara , t. nakagawa , and m. minami department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec . . . , mst), inactivation kinetics with -chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of -mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is . ϫ Ϫ min Ϫ and k is . mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin ( ) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of chemistry and pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit ( mg/kg) and oral administraton of melatonin ( mg/kg) at or h after anit injection. animals administered with melatonin at and h after anit injection were sacrificed and h, respectively, after the injection. liver injury appeared h after anit injection and developed at h. melatonin administered at h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g r, g d and r h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g r showed comparable specific activity to wtht, whereas g d and r h mutants exhibited remarkable reduction in specific activity. since homoallelism for g d mutation and heteroallelism for g r and r h mutation were identified in typical and atypical nkh, respectively, these results suggest that g and r h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: . native gel electrophoresis. . cross-linking of proteins using dibromoacetone. . radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with % ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat , s. nagata , h. misono , and k. packdibamrung department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified fold to homogeneity from aeromonas hydrophila. molecular mass of , daltons was estimated for alanine dehydrogenase by sephadex g- chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed polypeptide band with molecular mass of , daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at °c for h. optimum ph for reductive amination and oxidative deamination were . and . , respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were . , , . , and . mm, respectively. an elevation of apolipoprotein (apo) b- concentrations is a particular feature of several metabolic disorders, such as type diabetes (t d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b- of very low density lipoprotein subfractions (vldl , svedberg units (s f ) - and vldl , s f - ) kinetic studies were performed in subjects with t d, igt, fchl, and healthy controls using a tracer of either l-[ring- c ]-phenylalanine or l-[ , , - h ]leucine. these studies showed direct hepatic vldl apob- secretion to be increased in patients with t d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl apob- secretion. in all patients vldl catabolism is not essentially impaired. vldl apob- secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl apob- secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t d, igt, and fchl. vldl overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl and vldl secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg c) whose product was not inhibited by cysteine, and introduced satg c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda , e. suzuki , and t. kurata institute of environmental science for human life, and department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of . mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp , oatp and oatp . these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec . . . , pbgs), two zinc ions are coordinated by cys , cys and cys , and his and cys , respectively. the fomer zinc ion, closer to catalytic site lys , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep b cell, his was replaced with arg (h r). to elucidate the role of his in catalysis, the kinetic properties of wild type and h r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep b cell, respectively. each cdna encoding pbgs with Ј non-coding region was inserted into pet- b(ϩ) vector and then the constract was transformed into e. coli strain bl (de ). the cells were cultured in lb medium containing mg/ml ampicillin and µm zn ion for h at °c. after addition of mm isopropyl--d(Ϫ)-thiogalactopyranoside, cells were further cultured for h at °c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex , serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from - years and raised on pasture were used in the study with as control and as nutritionally treated. twenty four hours prior to capture the elk were offered either kg of a cereal grain based dietary supplement or kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # ). the amino acid content of the nutritional therapy product was minimally . g per kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus g per kg weight of leu and g/ kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels ( . nmol/l) compared to the untreated controls ( . nmol/l; p Ͻ . ). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl- produced µg/ml methionine in shake flasks with methionine yield at . g methionine/g glucose and specific methionine production at . mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt- produced µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte- produced . g/l methionine. the methionine yield was . g methionine/g glucose and its specific productivity was . g methionine/g dcw. clinical biochemistry, laboratory , luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and minutes after oral administration of mg of acetylcysteine. in the group of healthy persons the plasma glucose level feel by . % over the minute period. in the diabetics on the contrary, the plasma glucose level observed minutes after administration of acetylcysteine was . % higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of ml of distilled water ϩ . ml % glucose ϩ . ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by ml . % solution of acetylcysteine. in the glucagon group the distilled water was replaced by . % solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after minutes of incubation. a % diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a . % increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at , and % levels of fortification and the fortified yoghurt was analyzed fresh, and after and days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain ( . units/g) for minutes (tb) and rice bran hydrolysates with alcalase ( . units/g) for minutes (te) to yoghurt can ex-ceed - %, while fortification with sesame hydrolysed with papain ( . units/g) for minutes (td) and soybean hydrolysed with papain ( . units/g) for minutes (tc) can not reach up to %. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: ) controls; ) rats subjected to hbo for min once and immediately sacrificed; ) rats treated as group but for consecutive days and immediately sacrificed; ) rats treated as group but sacrificed h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups - , while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups - . this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker growing in pregnant rats which were distributed into six groups. three groups received normal diet ( % protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with % leucine ( % protein plus % leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, % of leucine) for days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ -pyrroline- carboxylate dehydrogenase, ec . . . ) in a girl aged months presenting with seizures and encephalopathy. l-∆ pyrroline- -carboxylic acid accumulates in this disorder and there is a - -fold increase in plasma proline. surprisingly, she also had vitamin b deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b coenzyme) was de-activated by l-∆ -pyrroline- -carboxylic acid. with high resolution h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph . and °c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline- -carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline- -carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young ( -month-old), middle-aged ( month-old) and old ( -month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b -b ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm and h. s. sharma department of pathology, university hospital, lund university, lund, and laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at °c for h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name ( mg/kg, i.p.) and l-nmma ( mg/kg, i.p.) min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk- , and cgp , respectively, were studied in both saline-pretreated and mptp-pretreated c bl/ mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk- distinguished from amantadine and cgp . in the study of long-term effect of nmda antagonists mk- was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer and a. fredriksson department of psychology, university of göteborg, and department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa ( mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk- , the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce , phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α -adrenoceptor antagonist, yohimbine, but not the α adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block , f. träber , s. flacke , f. jessen , ch. pohl , and h. h. schild department of radiology, department of psychiatry, and department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than spectroscopic data sets of patients with mnd and more than data sets of ad patients were acquired within the last years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin- . this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that -hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur and mglur ) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur in snpr projection neurons and mglur in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello , m. parodi , and m. balestrino centro di neurofisiologia cerebrale, cnr, genova and dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca ϩ into neurons. pretreatment of tissue with creatine ( mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower ( . mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s- . while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura- am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca ϩ ] i increase in response to k ϩ ( mm) depolarization was % higher than in wild-type. ω-agatoxin iva . µm decreased ca ϩ -influx in neocortical wild-type synaptosomes by %, and abolished differences between gsnϩ/ϩ and Ϫ/Ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was % higher and lesion size after mca occlusion was % higher than in wild-type. it is concluded that presynaptic ca ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ ) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv . Ϯ . , mtt . Ϯ . , ttp . Ϯ . , p Ͻ . ). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to µm glutamate (glu) for min, induce significant cell death in cerebellar granule cell cultures, h after treatment. in contrast, the combined treatment with µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to µm glu alone for min, glucose deprivation for min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by %, % and %, respectively. furthermore, we found that exposure to either µm glu, glucose deprivation or µm glu ϩ glucose deprivation decreased incorporation of [ h]cho into phosphocholine and increased the intracellular content of free [ h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf - .) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu receptor subtype by -methyl- -phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu / receptor by (Ϫ)- -oxa- aminobicyclo[ . . ]hexane- , -dicarboxylic acid (ly ) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep ( - µm), perfused through a microdialysis probe affected neither the basal nor the veratridine ( µm)-stimulated striatal da release. however, mpep given intraperitoneally ( mg/kg) diminished either the basal or the veratridine-evoked da release. ly ( - µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine ( ϫ mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep ( ϫ mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly may be a consequence of activation of striatal mglu / receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja , h. h. schild , and j. flammer department of radiology, university of bonn, germany university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that h of exposure to reduced glucose ( . mm instead of mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations ( mm instead of . mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid , -dihydroxy-l-phenylalanine (l-dopa) was first synthetized in . in , the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in , of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between and , showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in , the central antireserpine effect of d,l-dopa was described in mice and confirmed in with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years / , d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in , the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen , a. tasker , and j. zimmer anatomy and neurobiology, sdu-odense university, odense, denmark department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from -days old rats and grown on semipermeable membranes for - weeks before exposure. dom ( . - µm) was added to the medium, alone or together with the glutamate receptor antagonists ns- , nbqx or mk- , for hrs followed by hrs in normal medium. neuronal degeneration was monitored and ec values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at , , and hrs. the lowest ec values, obtained at hrs, were: ca ( µm), dentate granule cells (dg) and ca ab ( µm),ca c ( µm). protective effects of µm nbqx at hrs were seen against µm dom in dg, ca and ca c and against µm dom in ca and ca c. µm ns and mk only displayed protective effects together with nbqx. mk thus significantly increased the protective effect of µm nbqx in ca against µm dom in combination with µm nbqx and µm ns . we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur / and glur , and a decrease in the expression of mglur and its accompanying scaffolding proteins homer bc. this was accompanied by an increase in the capacity of mglur / receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur / receptors. in addition, there is reduced activity in the cystine/glutamate exchanger wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with -hydroxydopamine ( -ohda; :g icv), there was a % reduction in striatal tissue da content in adulthood, and a to fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa ( mg/kg i.p.) suppressed ho* formation. however, when glutamate ( mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns .) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk- . functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk- and flt- , will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased -fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk / ) were rapidly activated, which peaked within six hours of nmda treatment. erk / activation is completely blocked by mk- and partially blocked by k a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk / . as an extension of this work, we discovered a single nucleotide polymorphism in the human nr gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o •Ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to -nitrotyrosine ( -nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o •Ϫ /no • system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, -nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o to o •Ϫ and parent -nt. one-electron oxidation of -nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o •Ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and -nt. the nitration/denitration processes can be pathologic, but these also may play: ) a signal transduction role; ) a role of "blocker" for radical-radical reactions (scavenging of no • , no • and co •Ϫ by tyro • ); or ) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o •Ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than % of patients with type diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p was detected in heart of dm-rats. substantial changes in the methylation status of the p dependent p waf /cip -gene and the cyclin d -gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p -dependent activation of cardiac cell death. this pathway includes de novo methylation of the p -inducible p waf /cip -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor (igf- ). igf- is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf- is upregulated in areas of brain damage and the igf- system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf- is reduced. we recently observed a neuroprotective effect of topical application of igf- in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf- , as well, is known to be decreased. therefore, exogenous substitution of gh and igf- might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in -hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below % of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a , adrenergic a , opiod, and serotonergic b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype (mglur ), mpep ( . - mg/kg ip), or the agonist of group ii mglurs, ly ( - mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur , (rs) aida ( . - µg/ . µl), but not of the agonist of group ii mglurs, r, r-apdc ( . - µg/ . µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats ( - g) and male c bl/ mice ( - g). aida (group i mglurs antagonist) given i.v. in the dose of µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur antagonist) given i.p., was not effective in the despair test in rats. however, in doses of . , and mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep ( mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu receptors in the ca field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu receptors in the ca field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. .po a. . ) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t - segments resulted in profound edema formation and cell damage in the adjacent t and t segments at h. pretreatment with h- / ( mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t and t segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h- / . these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h- / has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in , creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound ( biological and synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p and cdc . transcriptional activity of p was shown by increased p dna binding activity and by the concomitant induction of the cdk inhibitor p , the cell cycle regulator gadd and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of -oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh increases in cultured cerebellar neurons or in ca pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after years of therapy % of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in -methyl- -phenyl- , , , tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg mice overexpressing human -amyloid precursor protein (huapp k n/m l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. , - , . recently, we also found that the overexpression of p protein, an activator of the kinase cdk , results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg mice at -months of age (i.e. - months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: ) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. , - ); ) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and ) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional ( -d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip ( , , , -tetrahydroizokasazolo, , -c pyridin- -ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult - week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of and mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at - hours and recovered at - hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/ mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: /sag/ ).] and in long-term ethanol-experienced rats only at a dose mg/ kg i.p. control group (cg) received saline ml/kg i.p. as can be seen in fig. and table the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. ) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at Ϫ mv. in a first survey, each of the solutes was briefly applied in a concentration of mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and -indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat ), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. p a .) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a -min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son ( %; p Ͻ . vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to %; p Ͻ . vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en - ). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets- and collagen α (vi) chain precursor, encoded on chromosome , was investigated in fetal brain by western blot and two-dimensional electrophoresis ( -de). western blot detected app and ets- that migrated at ϳ and kda, respectively. subsequent densitometric analysis of app and ets- immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α (vi) chain precursor, a protein resolved as a single spot on d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the d image master software revealed a significant decrease in fetal ds (p Ͻ . ) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd , apo- ), caspase- , bcl- and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase- , bcl- and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl- and annexins together with unaltered caspase- expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for h followed by h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff (dna fragmentation factor ), and flip (fadd like interleukin- -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf- (apoptosis protease activating factor- ), procaspase- and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase- and - were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n -( -iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, -nitroindazole ( -ni) and the no scavenger, -( -carboxyphenyl)- , , , tetramethylimidazole- -oxyl -oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by vessel occlusion for min. l-nio ( mg/kg), -ni ( mg/kg) and ptio ( mg/kg) were administered intraperitoncally min before ischemia. ptio, -ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio Ͼ -ni Ͼ l-nio. l-nio, but not -ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and -ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. -ni recovered in part ps amplitude min after ischemia. -ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase , m. tanaka , k. tujioka , e. hirano , and h. yokogoshi department of home economics, aichi university of education, kariya, aichi, and laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether -estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of wk old female rats: group . ovariectomized to reduce the level of plasma estradiol; group . ovariectomized and treated with estradiol; and group . sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif- (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that -f-phe- -oh-pro-arg-gly-trp-nh (inn ) is superior in all the statistical parameters used. in comparative testing inn was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn show that it is effective in over % of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn has a rapid onset of action, - days (vs. - weeks for currently marketed products) with sustained effects for months following to doses over - weeks. h. iwama , , a. umino , a. hashimoto , k. takahashi , n. yamamoto , and t. nishikawa , section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm genes (h and h ) , which are related with the metastatic potential of some tumors. although ndpk/ nm has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm -h in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional ( -d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm -h was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm -h upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d- -hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d- hydroxyglutarate (d- ), which is structurally similar to lglutamate (ϭ -amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d- induces excitotoxic neuronal damage. here we report that d- decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura- measurement. furthermore, fluorescence microscopy using dihydrorhodamine- revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d- . n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d- is an agonist at nmda receptors. patch-clamp investigation confirmed that d- activated recombinant nmda receptors in hek cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d- . we conclude that excitoxic mechanisms contribute to the neuropathology of d- hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c bl/ mice of different ages ( , - , and - months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e and z mrna density in naïve, ad lib-fed mice. correlations were found between changes in e subunit mrna and agonist binding and z mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e and e mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e and e and/or z subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices ( mm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of kϩ ( mm) or veratridine ( mm) minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( mm) perfused for minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( mm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( mm) increased the basal release of glutamate (p Ͻ . , n ϭ - ). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ h]cgp to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ h]cgp affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ h]cgp binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ h]cgp binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to days) with mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing mm k ϩ also exhibited multiple changes in nr splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding Ј and Ј splice variants. nr - and nr - (each containing exon ) were decreased, whereas nr - and nr - (each lacking exon ) were increased, accordingly. the predominant expression of nr -b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: Ϫ mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of Ϫ pa under control conditions. neurones treated with mm k ϩ showed a significantly diminished response to nmda (max. response: Ϫ pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of mm k ϩ on neuronal survival and maturation. (supported by bmbf grant gg / ) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp , hsp , and hsp . however, no investigation has been carried out on changes in level of apg- , a member of hsp family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp and apg- in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg- level was significantly decreased in the frontal cortex, hippocampus, and striatum days after kainate administration, while hsp level was increased in these regions following the administration. decreased level of apg- returned to the control levels in the three regions days after kainate administration. no significant changes were observed in levels of both hsp and apg- in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp , apg- expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide- ( - ) amide (glp- ) and its specific receptor (glp- r) have been found in the rat hypothalamus. in this study the actions of glp- and its related peptides, exendin- (glp- r agonist), exendin ( - ) (glp- r antagonist) and glp- ( - )amide (major glp- metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. i-glp- binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp- Ͼ exending- Ͼ exendin ( - ) Ͼ glp- ( - )amide. intracerebroventricular (icv) glp- ( nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas nmoles of exendin ( - ) was ineffective. exendin- produced a statistically significant reduction in the levels of trp, glu, and tyr. glp- ( - )amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin ( - ) or glp- ( - )amide blocked the effects of glp- on the levels of the amino acids. these data are consistent with exendin- being a glp- r agonist and exendin ( - ) being a specific glp- r antagonist. glp- ( - )amide, a primary metabolite of glp- , appears to act as an endogenous antagonist at the glp- r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck ( fmol) and/or cck antagonists ( fmol) were injected days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck ns; cck ) or a mixed cck b and cck a receptor agonist (cck s) were injected. in contrast, cck , a cck a receptor agonist or cck - and cck - were uneffective. furthermore, the ansiogenic effects of cck s were prevented by the previous ( min) administration of l , (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than % of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and -mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after days with maximum myoclonic activity ( jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by % (p Ͻ . ) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate ( . µ mol), aspartate ( . µ mol) or nmda ( - n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. -amino- -phosphonovaleric acid (ap ) and -amino- -phosphonopheptanoic acid (ap ) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like ( s, s)-acpd the agonist of group ii, and l-ap the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(Ϫ) and divalent cations in the na(ϩ)/cl(Ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat- (hgat- ) gaba transporter cdna. our results showed that cl(Ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat- (loo et al., j biol. chem. : - , ) . the cl(Ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat- transported cl(Ϫ) across the membrane with a stoichiometry of na(ϩ) : cl(Ϫ) : gaba, the transported cl(Ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(Ϫ)/cl(Ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat- is also modulated by divalent cations. the uptake of [ h]-gaba was inhibited significantly when both ca( ϩ) and mg( ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca( ϩ) and mg( ϩ) from the efflux buffer. the gaba transporter inhibitor skf a blocked the enhanced efflux, suggesting that the hgat- operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha subunit gene in knockout mice (ko). in the ca region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha containing gaba a receptors contribute to functional postsynaptic receptors on ca pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure ( hours) twice in week during weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon - mg/kg, mephebut - mg/kg, gammoxin - mg/kg, pyracetam - mg/kg. the treated males were mated with intact females during days. after the mate the treated males and more in days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased . % (p Յ . ) and . % (p Յ . ) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa . % (p Յ . ), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p Յ . ) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p Ն . ) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced . %. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk- (dizocilpine) . mg/kg sc. control rats received tap water only. behaviour of month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf , quinpirole, apomorphine, haloperidol). our results indicate that mk- applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo ) and mesencephalic trigeminal neurons (me ); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo and me during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo . furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer , m. dierssen , m. fountoulakis , and g. lubec department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap and the channel associated protein of synapse (chapsyn ) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on -dimensional gel, identified and quantified. we quantified members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins - and the collapsin response mediator protein- (crmp- ) in ds and control cortex samples. drp- and crmp- levels were comparable in the control and ds samples. evaluation of drp- , drp- and drp- revealed significantly decreased levels of of the spots assigned to drp- and increased levels of one spot assigned to drp- and increased drp- in ds brain. we conclude that as early as from the th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases , and activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases , , and activity in the caudate/putamen. zvad significantly decreased the levels of caspases , and but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap to the goldfish telencephalon in avoidance conditioning. in experiment , fish received no injection or microinjections of saline or various doses of ap to their telencephalon minutes before three semiweekly training sessions. fish were tested without injec-tions in session . a one-way anova with multiple comparisons on the test scores showed that ap produced anterograde amnesia in a dose-dependent manner. in experiment , fish received several training sessions and a microinjection of various doses of ap minutes before testing. the test scores showed that ap did not decrease avoidance responses, suggesting that microinjection of ap did not impair performance processes. in experiment , fish received microinjections of ap or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session . a one-way anova on the test scores showed that ap did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto and e. a. newsholme health science laboratory, tezukayama university, nara, japan department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan ( um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan ( mm/ min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average g) were made by tryptophan free feeding for weeks, and the rat's running time increased (Ͼ min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [ h]d-serine into the synaptosomal p fraction from the rat cerebral cortex. the cortical p fraction was able to accumulate [ h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of µm and a vmax value of pmol/mg protein/min. depletion of na ϩ and cl Ϫ ions remarkably decreased d-serine uptake into the cortical p fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo , p. j. lea , s. a. gaziola , a. p. pellegrino , and s. m. g. molina departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: -oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: ( ) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. ( ) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. ( ) the opaque- mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and -fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque- mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque- maize gene has been shown to regulate ak and lor activity. ( ) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. ( ) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl , ranging from . - mm, for up to h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as % of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: -fold), n-trimethylglycine ( -fold), histidine ( -fold), tryptophane ( , -fold), putrescine ( , -fold), spermine ( , -fold) and trigonelline ( -fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass ( %) as a result of growth inhibition ( %). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or -aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a to -fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a -fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in to -fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. Ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr ) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of Ϫ mv at ph . and involves a -electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of , bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova , m. zeh , o. kreft , s. maimann , h. hesse , and r. höfgen max-planck-institut für molekulare pflanzenphysiologie, potsdam, and institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque- mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during days of germination quantity of zn in primary leaves increased from , to , % and decreased in soil: in upper layer from , to , , midde layer from , to , , lower layer from , to , mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b , but these results can also be inaccurate. vitamin b deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b deficiency. twenty-seven patients (mean age - . years) with low serum b were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of . %- . % of neutrophils examined. there was a significant correlation of homocysteine (r ϭ . , p Ͻ . ) and mma (r ϭ . , p Ͻ . ) with serum b levels but no correlation of appendage number (r ϭ . ) with serum b . there was no correlation of appendage number with homocysteine (r ϭ . ) or mma (r ϭ . ). these results suggest that b -deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d ϭ placebo, d ϭ mg, d ϭ mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m ϭ min. after stress/before testdrink, m ϭ min.-, m ϭ min.-, m ϭ min.-, m ϭ min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha , theta (frontal) and decreasing beta relative-power earlier in recovery with d . these findings were related to relaxing effects. after ingestion of l-theanine alpha -, beta -power at occipital regions decreased faster (m ) to placebo recovery levels (m /m ). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus , j. gabryś , j. konecki , and j. shani department of pharmacology and department of histology and embriology, medical university of silesia, zabrze, poland department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin- or of some of its analogs was found to cause a selective inhibition, up to %, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type- somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type- receptor antagonist. a type- specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between and amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin- and vvhemorphin- , could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin- and mainly lvv-hemorphin- could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of days. noteworthy, continuation of treatment for aperiod of days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. ) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately % of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. ) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in % of the female patients. moreover, - % of these patients omplained about a distorted sensory perception of objects. ) young soccer players were observed during a period of months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany percent of amino acids in green tea leaves are represented by l-theanine ( -n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine ( , and mg) were administered to healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as , , , , and minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: . decreasing skin resistances after exercise could be established in each volunteer. . esg-activation levels before exercise could not even be reached again after a period of regeneration of / hours. . maximal electrodermal activity did not appear immediately after exercise, but after minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero and h. pitkänen , neuromuscular research center, department of biology of physical activity, university of jyväskylä, and rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment ten of them were given leucine ( mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly ( %) in the placebo group (p; n ϭ ) during weeks but not when leucine was taken. also total amino acids (taas) decreased strongly ( %) during weeks when dally protein intake was . g/kg body weight. in experiment the subjects (n ϭ ) carried out a single strength training seasion (sts) and consumed a drink containing leucine mg/kg body weight. following sts leucine in serum increased by % (ns) when leucine was taken but decreased strongly ( %) in p, in experiment the subjects (n ϭ ) underwent at days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ s with a recovery of s between the runs) until exhaustion. the subjects consumed drinks containing leucine ( mg/kg body weight) or placebo min before the test runs. following mare the concentration of leucine strongly increased by % whereas isoleucine ( %) and valine ( %) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment ) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of track and field adult male power athletes. in experiment eleven of them performed a -week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) ( %), branched chain amino acids (bcaas) ( %), essential amino acids (eaas) ( %) and leucine ( %) were observed following training with the daily protein intake of . g/kg body weight. in experiment eleven subjects performed a short run session (srs) of ϫ ϫ m with recoveries of s and s, and a long run session (lrs) of s runs with recoveries of s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by % in srs and by % in lrs. leucine decreased by % following srs but only by % (ns) following lrs. the peak blood lactate concentrations after srs and lrs were . Ϯ . mmol/l and . Ϯ . mmol/l, respectively. in experiment sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during minutes. the taas decreased significantly by %, bcaas by % and leucine by % following sts, while the peak blood lactate concentration was . Ϯ . mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of . g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have , , -and , , -trisubstituted pyridine skeletons both with three carboxylic acids and a mass of (c h n o ), identified as -( -amino- -carboxybutyl)- , -di-( -amino- -carboxypropyl)pyridine and -( -amino- -carboxybutyl)- , -di-( -amino- carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il- production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il- production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by % hr after the race (p Ͻ . ). there was an apparent decrease (only close to significance, p Ͻ . ) in il- production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was % higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response ( . %) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma ( . %). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il- production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il- production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward and l. m. castell departments of biochemistry, university catholique de louvain, belgium oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt and crt (a na ϩ and clЈ dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor . more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss , t. barthel , r. schnittker , k. e. geiss , w. falke , and l. r. juneja university of paderborn, germany taiyo kagaku co., yokkaichi, japan, isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ healthy students consumed drinks containing either , or mg l-theanine in randomized double-blind trials in the min - after a near maximal bicycle step test. measurements were done directly after exercise (m ) and (m ), (m ), (m ), (m ) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m (catecholamines) or m (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec . . . ), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from - kda. they contain different types of subunits, named b , b , b , a , a , a , ba , ranging from - kda. in the rabbit eye lens two -crystallin subunits ( b and b ), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b -and b -crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines ( , diaminooctane, , -diaminononane, , -diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni , d. bertoldi , , e. candioli , l. martinelli , and a. tassoni istituto agrario, san michele a/adige, and dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme ) is related to an intrinsic gtp-ase activity of type transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b , in transmethylation reaction, the significance of ,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b may be in direct relation with the formation of ,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase- and parp, a substrate of caspase- . the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca ϩ , but that putrescine is also capable of inducing influx of extracellular ca ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: / evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and / determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh ( . iu, s.c., saizen, serono, switzerland) was administered once daily for or days, to randomly selected rats on the second postoperative day. animals were sacrificed , and days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are . µm and µm, respectively. amino acid residues, cys , trp , glu , trp and tyr are strongly involved in both activities, and that glu , tyr , cys , cys , cys and glu are moderately involved in the activities. mutations of tyr , trp and trp mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys and tyr mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has % sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are µm and µm, respectively. it was found that two glutamate residues (glu and glu ) and four tyrosine residues (tyr , tyr , tyr and tyr ) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. . . . ) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg ) carrying the ttg gene sequence to c bl /n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor- a (eif a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif a activity in human epidermoid lung h cancer cells. we have found that h exposure to , iu/ml ifnα induces % growth inhibition and % apoptosis in h cells. moreover, ifnα induced a -fold increase of ttgase activity and expression that already occurred after h of exposure to the cytokine. this effect was paralleled by a . -fold enhance of ttgase mrnas. ifnα induced also a % increased eif a expression while an about % decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif a activity. adometdc deficient mice k. nishimura , f. nakatsu , , k. kashiwagi , h. ohno , t. saito , and k. igarashi graduate school of pharmaceutical sciences, chiba university, chiba, graduate school of medicine, chiba university, chiba, and cancer research institute, kanazawa university, kanazawa, japan the amd gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by % in amd ϩ/Ϫ mice, adometdc activity reduced only by % and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd Ϫ/Ϫ mice, we intercrossed amd ϩ/Ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd Ϫ/Ϫ mice from heterozygous intercrosses over. amd Ϫ/Ϫ embryos died early in development, between e . and e . days post coitum. in culture of blastocysts at e . , the shapes of all cell lines were normal, but amd Ϫ/Ϫ cells appeared to arrest the cell proliferation at day after the onset of cell culture. the arrest of amd Ϫ/Ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd Ϫ/Ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h o and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h o formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m ) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h o in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k ), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis( -aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis( -aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis( aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by °c by the addition of . mm of tetrakis( -aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis( -aminopropyl)ammonium prevents depurination most effectively. tetrakis( aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde -phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an -aa peptide bearing the simplest q n domain (q ), and polyamines of different size and shape as q-and nh -donor, respectively. in the present study we report that out of the lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm - , institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, -(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms ( , s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g- cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were . mm in the reaction mixture. bis(uracilyl)-analogs and -( -oxyethyl)ami-no- --d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ was stimulated . -fold and that of σ was stimulated . -fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ synthesis, whose translation is altered by polyamines. stimulation of σ synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( , , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than %. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol- -yl, isoxazol- -yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)- -amino- -( hydroxy- -methylisoxazol- -yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing h-pyran- -one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m ( ) tetrahedron : ). as a continuation of our investigation in this field we report here a detailed study of the transformation of h-pyran- -one derivatives with hydroxylamine ( , x ϭ o) and various hydrazines ( , x ϭ nr ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives . in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines also occured during the reaction course. some attempts to hydrogenate compounds towards α-amino acid derivaties by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin and were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position and were studied. in position several hydroxy-and serine related amino acids were incorporated, whereas in position the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser -endomorphin Ϫ . quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted h-pyran- -ones containing α, -didehydroamino acid unit in their structure as dienes in [ ϩ ]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of h-pyran- -ones upon the cycloaddition of acetylene derivatives the first intermediate formed ( ) still contains the carbon dioxide bridge. in many cases easily expels co and substituted benzene derivative is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co from the adduct there are two possible paths: so formed cyclohexa- , -diene ( ) is either aromatized into benzene derivative ( ) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts ( ) are formed. since co -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co -containing adducts ( ), while with alkenes we have isolated, depending on the structure pattern of the compound , all three types of products: aromatized , co containing and double adducts . especially the type is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc or orl . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc receptor and for full biological activities. noc( - )-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc( - )-nh , hoping that these derivatives retain the ability to bind lc receptor and are resistant against biological degradation: phe-nh-co--ala-noc( - )-nh phe--ala-nh-co-phe-noc( - )-nh phe-gly-nh-co--hphe-noc( - )-nh the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rЈ)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions Ñà ϩ or mg ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax and mgx where one metal ion binds together several (up to ) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca ϩ or mg ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to %). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose- p, the product of the reaction catalysed by glucosamine- p synthase is d-glucosamine -phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active from d-serine will be described and first results in the biological evaluation on glucosamine -phosphate synthase will be included. o. melnyk , d. bonnet , e. loing , l. bourel , and h. gras-masse -umr , -sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal - portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l- -(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a Јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur . as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly and its c epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[ , , ]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p , p and p Ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new Јdeoxynucleoside analogues modified at Ј-and Ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky and f. bordusa , max-planck-society, research unit "enzymology of protein folding", halle, and institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: ( ) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v protease (v protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev , a. k. dadayan , b. v. vaskovsky , and n. f. myasoedov institute of molecular genetics, russian academy of sciences, and shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ h] peptide analysis using h nmr spectroscopy was carried out, and the modified fragment [ h]actc - (met-glu-his-phe-gly-pro), with molar activity of ci/mmol and [ h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ amino-isobutyric acid) with molar activity of ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y , e. kontny , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il- trigger production of il- and il- also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il- and il- production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps ( h) in the presence of tau or tau-cl ( - µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il- and il- were inhibited by tau-cl with ic Ϸ µm and µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly ( . times) at low ( - µm) while reducing it (ic Ϸ µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (Ͼ µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl- ) to investigate the activation of a swelling sensitive [ h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, µm) increased transiently the rate coefficient for [ h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor -isobutyl- -methyl xanthine ( µm) increased the swelling activated efflux rate coefficient . times above the control level and the camp analogue dibutyryl-camp ( µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph Ͻ , is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: - °c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph of the aqueous solution decreases which correlates with a decrease of oxidation capacity of . % per day, probably originated by the elimination reaction r-ch -ncl ae r-chϭncl ϩ h ϩ ϩ cl Ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of mm ndct at ph and against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny , m. kurowska , j. kowalczewski , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for hours with rhpdgf or rhtnf-α. tau or tau-cl were added at - µm concentrations. cell proliferation was determined by incorporation of h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at µm concentration tau-cl inhibited (by Ϸ %) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p , p ) nor anti-apoptotic bcl- protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny , k. szczepań ska , j. kowalczewski , m. kurowska , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il- and il- by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil- . tau or tau-cl were added at - µm concentration. after . - h or h the dna binding activity of nfkb and ap- (emsa) and the expression of il- and il- mrnas (rt-pcr) was examined, respectively. results. il- raised nfkb and ap- activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il- Ͼ il- ) and the activity of transcription factors (nfkb Ͼ ap- ). conclusion. tau-cl inhibits transcription of il- and il- genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using h and c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within h, % of the total intracellular hypotaurine and . % of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, ) . ges, when administered to rats in their diet in the drinking water as a - . % solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within - weeks the levels of taurine reach their nadir ( - % of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in , koppel and associates (geggel et al., n. eng. j. med. : - , ) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. : - , . upon administering . % guanidinoethanesulfonic acid (ges) in the drinking water of rats for weeks, the taurine levels in cardiac tissue decline by %. however, the phosphorylation of a ϳ kdalton protein in the mitochondrial fraction of the heart tissue increased by % (j. mol. cell. cardiol. : - , ) . reversal of these effects could be accomplished by feeding the rat . % taurine in the drinking water for weeks. the ϳ kdalton protein was isolated by -dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ kdalton was pyruvate dehydrogenase (amino acids : - , ) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges ( %) for weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp ( ì) and radioactive atp for minutes. animals were also treated with taurine ( %) in their drinking water for - weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity Ϯ sem (normalized to mg protein) control Ϯ (n ϭ ) ges-treated Ϯ (n ϭ ) p Ͻ . (paired -test) control Ϯ (n ϭ ) taurine-treated Ϯ (n ϭ ) p Ͼ . control Ϯ (n ϭ ) ges followed by taurine Ϯ (n ϭ ) p Ͼ . these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume - % less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine ( - mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine ( mg/kg) also suppressed water intake. the taurine precursor hypotaurine ( - mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine ( - mg/kg ip), which suppressed ethanol intake and preference by approximately % without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi -kinase (phosphatidylinositol -kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl- . increases in the extracellular osmolality by either addition of mm taurine or mm mannitol to the incubation medium activates similar pathways. however, pi kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl- and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg human liver cells. the intracellular taurine content of hepg cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing nm angiotensin ii undergo hypertrophy, a process blocked by mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is : - . in the present study, we determined urinary sulfate and taurine in urine samples of healthy japanese women after h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, . Ϯ . ; free sulfate, . Ϯ . ; ester sulfate, . Ϯ . ; taurine, . Ϯ . ; urea, . Ϯ . . the ratio of total sulfate and taurine was : . . this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, . . this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi and h. oda laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine ( . - g/kg) were supplemented to hc for wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol α-hydroxylase (cyp a ) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp a gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ % of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ %), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ % in the presence of pdc and reduced in an equal degree (ϳ %) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no p a and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the and kda forms of mitogen-activated protein kinase (mapk) and the stressactivated kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p mapk. this kinase currently exists in four different isoforms which are p α, , γ and δ. the involvement of individual isoforms of p in enhancing the expression of carrier proteins for l-arginine will be discussed. gw is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. . bioorg. med. chem. lett., : , - ) . the systems associated with transport of this compound have been investigated using the macrophage cell line j . prior to each study, j cells were seeded in -well culture plates and allowed to adhere for h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution ( µl; °c) containing l-[ c]gw ( µciml Ϫ ) in the presence of either . mm or . - mm unlabelled substrate. in parallel studies transport ( µciml Ϫ , . mm) was monitored in the presence of mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of . mm of l-[ c]gw occurred in a time-dependent manner and was linear for up to min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of . mm and v max of . pmol·µg protein Ϫ min Ϫ . at mm, -methylaminoisobutyric acid (meaib), lalanine, l-valine and - -amino-bicyclo-( , , )-heptane- carboxylic acid (bch) caused little or no inhibition of l-[ c]gw ( . mm) uptake. in contrast, transport of l-[ c]gw was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, -diazo- -oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of . mm l-[ c]gw was inhibited in a concentration dependent manner by l-arginine (ki ϭ . mm), l-leucine (ki ϭ . ), don (ki ϭ . mm) and l-glutamine (ki ϭ . mm). taken together, these data suggest that gw may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw ; if so, the monophasic substrate kinetics imply that the two systems handle gw with similar affinity. other systems such as b ,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw is largely (ϳ %) na ϩ -independent. similarly, b ,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw does not interact with l-glutamine and thus unlikely to be involved in transport of gw , at least in j cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with - putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of h-l-alanine and h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and f hc) and seven light subunits are known. rbat and the light subunits b ,ϩ at and y ϩ lat are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b ,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat- , - a, - b, - and - ) with cat- a and - b being splice variants. in humans, only the "old" members of the family have been characterized (hcat- , - a and - b). hcat- and - b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat- a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat- a and hcat- b differ only in a region of amino acids. cat- , so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat- expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat- has recently been isolated, however, the transport activity of hcat- has not been characterized. when optimally aligned, the amino acid sequence of hcat- shows only about % identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat- , - (a or b) and - are about % identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat- and hcat- a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat- a compared to the high affinity cat-isoforms. to characterize the human cat- , we cloned a cdna encoding hcat- . when expressed in xenopus laevis oocytes, hcat- had a similar transport activity and affinity for l-arginine as hcat- or - b. hcat- mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat- is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat- also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat- , but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat- with the green fluorescent protein attached to the c-terminus. therefore, hcat- might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; . mg/ g b. wt. i. p.), sodium dichromate (cr; mg/ g b. wt. s. c.) and tl so (tl, mg/ g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in -(both sexes) and -day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times ( st day after cr in both age groups and in -day-old rats after cp and rd day after cp in adult rats; nd [ -day-old rats] and th - th day [ -day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious -day-old rats cr nephrotoxicity is not detectable after mg/ g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in -day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata , a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata and ata , have been cloned and functionally characterized. ata is expressed primarily in the brain and placenta whereas ata is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata . it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata cdna from a skeletal muscle cdna library using rat ata cdna as the probe. rat ata consists of amino acids and exhibits a high degree of homology in amino acid sequence to rat ata ( % identity) and rat ata ( % identity). interestingly, this new transporter also has a comparable degree of homology to sn and sn , the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata -expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k . for alanine (i.e., concentration of the amino acid yielding half-maximal current) is . Ϯ . mm and that the na ϩ : alanine stoichiometry is : . subsequently, we have cloned the human homolog of rat ata from a liver cell line (hepg ) cdna library. human ata also contains amino acids and shows % identity in amino acid sequence with rat ata . the sequence identity of human ata with human ata and human ata is % and %, respectively. the homology of human ata with human sn and sn is also similar ( % and % identity, respectively). the gene coding for human ata contains exons and is located on chromosome p . in the human, ata is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata for cationic amino acids is higher than for neutral amino acids. however, the human ata -mediated cationic amino acid transport is na ϩ -independent. in this respect, ata is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata and ata have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata also differs from ata and ata in substrate affinity. ata and ata interact with meaib with a k t of ϳ . mm whereas the affinity of ata for this model substrate is comparatively at least -fold lower (k t , ϳ mm). but, ata interacts with arginine with a k t value of . mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: ( ) cr, cp and tl were more nephrotoxic in -day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. ( ) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. ( ) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. ( ) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr -family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept that will be useful in further studies on structure and functions. starting from lys[z(no )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept . the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept with k i values as low as nm. this is the highest affinity of a ligand of pept ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept . ata is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ %) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic ϭ µm; ok hc , ic ϭ µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa ( µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b ,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b ,ϩ and lat , the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter protein (cat ). the regulation of translation of the cat mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif a. translation of the transporter mrna increases when eif a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif a are required for increased activity. we propose that eif a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some min following cd receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept , one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept is encoded by the pep- gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep- transporter in vivo, a c. elegans pep- mutant was constructed. the animals deficient in pep- show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep- gene in the mutant. the observed starved phenotype in pep- mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. , - , ) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a a , a b and a -adenosine receptor subtype, whereas negligible levels were detected for the a -receptor. adenosine ( µm, min) induced increases in l-arginine transport and no production were ca ϩ and camp independent and stimulated transport was abolished in cells depolarised with mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek / (pd , u ) but unaffected by inhibitors of pkc (calphosin c) and pi- kinase (ly ). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli , s. lord-fontaine , e. przybytkowski , and d. a. averill-bates department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr- gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h o and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c , at and °c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h o and aldehyde(s) relative to their drug-sensitive counterparts, auxb cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from -hydroxymethyl- methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts from the corresponding diol. treatment of with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams . activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi , m. a. s. di leo , s. caputo , n. gentiloni silveri , and g. ghirlanda department of pharmacology, university of sassari, and department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting % of diabetics and progressing to blindness in about %. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type and type diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic ( months) taurine ( % and %) supplementation in diabetic rats in comparison with vitamin e ( and ui). dietary taurine supplementation, for months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of -month duration, we found that cd, lp are significantly increased and they remained elevated for months. while, the na/k atpase is significantly decreased during the whole experimental time ( months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being % taurine more effective than % diets. the morphological examination reveals that in rats feed with % taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch (ch ) n xco iran applicated be ( mg/kg - days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p Ͻ . ; p Ͻ . ) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o Ϫ ), hydrogen peroxide production (h o ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o Ϫ , h o formation and mpo. taurine decreased o Ϫ and h o production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with -nitroso- -naphthol in the presence of nitric acid - -benzyo- -(alanyl)- -phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting -nitroso- -naphthol with -(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that , -benzo- phenoxazone derivative is not produced but a hydrogenated form of , -benzo- -phenoxazone which is probably , -benzo- -(α-hydroxy -methylaminopropyl)- -hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three ( ) breast cancer cell lines: mda-mb- , mda-n and hs- t. the % growth inhibitory (gi ) concentrations for these three cell lines were . ϫ Ϫ , . ϫ Ϫ and . ϫ Ϫ molar respectively. a. bocheva and t. pajpanova institute of molecular biology, bulgarian academy of sciences, and institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva , s. pancheva , and t. pajpanova institute of physiology, bulgarian academy of sciences, and institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh ) that was discovered in hypotalamus.the mif- exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a (txa ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue -ro-c h -coϳ, {where rϭh or ch co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at g for min at °c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp was used. the ic values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg , d-phe , d-trp , , leu ]sp (antagonist d) and the c-terminal analog [arg , d-trp , , mephe ]sp - (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp , glu(bu t ) ]sp - and [glp , glu(bu t ) ]sp - showed significant inhibition in the proliferation of the cancer cell lines hela and t d.in the present study the c-terminal analogs of sp [glp , d-trp , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) ]sp - ( ), [glp , trp , mephe , glu(bu t ) ]sp - ( ), [glp , mephe , d-trp , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) -oh]sp - ( ), [glp , d-trp , cys(acm) -oh]sp - ( ), [glp , d-trp , mephe , cys(acm) -oh]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , cys(acm) -oh]sp - ( ) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs - are peptide amides whereas the analogs - are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at Ј, Ј-positions j. velkov , i. stankova , a. ivanova , and a. tadjer department of chemistry, south-west university "neophit rilski", blagoevgrad, and department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives ( Ј, Ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, Ј, Ј-bis-o-n-α-benzyloxycarbonyl-valyl , Ј, Ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl, Ј, Јbis-o-n-α-benzyloxycarbonyl-phenylalanyl, Ј, Ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am ) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept substrate from a non-substrate?computer modelling of a large number of pept substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept , and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/ mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/ mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution ( . mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw human colon carcinoma cells and c rat glioma cells, stimulated human lymphocytes and macrophages. about ϫ cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). . mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately %, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f- -fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c- -methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c- ( min) in contrast to f- ( min). amino acid analogous labeled with f- like f- -fluoro-α-methyltyrosine (fmt), f- -fluoro-ethyltyrosine (fet), f- -fluoro-phenylanaline, f- -fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i- -iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i- allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo , , v. fiorito , a. pietrosanto , a. pepe , g. andria , and g. sebastio telethon foundation, rome, and department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim ) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim ), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the f heavy chain ( f hc) and a light chain represented by either the solute carrier family a, member (slc a ) or (slc a ). after excluding the f hc as the causative gene of lpi, we identified slc a as the lpi gene and characterized mutations in twenty-five patients from families ( italian, japanese, moroccan, greek, and pakistani; independent alleles) affected by lpi. thirty-two of the independent alleles ( . %) were characterized and fourteen mutations were identified. only five mutations (namely insatca, w x, delctct, ivs ϩ gaea, s r) were identified in more than one independent family. most mutations are located in the slc a coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc a gene in lpi, which, in addition to slc a , is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either f hc/ slc a or f hc/slc a , is still unknown. hypothetically, the activation of f hc/slc a in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n. cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal ( Ϯ vs . ϫ pmol/ cell/ min) and histaminestimulated ( Ϯ vs Ϯ pmol/ / min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of Ϫ Ϯ and Ϫ Ϯ mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions ( . m nacl, ph . ) were obtained in the temperature interval - c degrees. the measurements of the dielectric parameters were carried out at the frequency of . hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of - , - and - c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both and kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc - or pomc - with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three kda n-pomc fractions enhanced prl mrna levels by - %, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation ( . Ϯ . %) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all and kda isoforms activated the mc- and mc- receptor in cell lines in which these receptors were transfected. thus, att cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc- and mc- receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices ( µm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of k ϩ ( mm) or veratridine ( µm) minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( µm) perfused for minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( µm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( µm) increased the basal release of glutamate (p Ͻ . , n ϭ - ).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or na ϩ channels.glucagon-like peptide- ( - ) amide (glp ) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp on serotonin ( -ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp ( . µm) produced a significant decrease in levels of -ht by % after minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of hydroxyindolacetic acid ( -hiaa), the principal metabolite of -ht, and tryptophan the amino acid precursor of -ht, were also decreased significantly by % and % respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba ( %) and glu ( %) by glp was not significant.the results suggest that decreased synaptosomal levels of -ht and -hiaa caused by glp are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso treatment ( . Ϯ . vs control . Ϯ . µmol/mg prot.) and ( . Ϯ . vs control . Ϯ . nmol/mg protein p Ͻ . ) respectively. it was associated with significantly increased iron content ( . Ϯ . µg/mg prot. vs control . Ϯ . p Ͻ . ). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso and spermine, iron content was significantly decreased ( . Ϯ . p Ͻ . compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso treated only ( . Ϯ . ), but mda level didn't change ( . Ϯ . ). in addition, treatment with spermine alone resulted in increase of mda level ( . Ϯ . vs control p Ͻ . ), iron content didn't change ( . Ϯ . ), but carbonyl groups were decreased ( . Ϯ . vs control p Ͻ . ). feso treatment increased gsh level ( . Ϯ . nmol/mg prot. vs control Ϯ . ; p Ͻ . ) while in combination with spermine this increase was more profound ( . Ϯ . ; p Ͻ . vs control, p Ͻ . vs feso ). spermine alone produced similar increase of gsh level ( . Ϯ . , p Ͻ . vs control; p Ͼ . vs feso ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be ( mg/kg - days), second key: cord- -c d nk x authors: mikasa, keiichi; aoki, nobuki; aoki, yosuke; abe, shuichi; iwata, satoshi; ouchi, kazunobu; kasahara, kei; kadota, junichi; kishida, naoki; kobayashi, osamu; sakata, hiroshi; seki, masahumi; tsukada, hiroki; tokue, yutaka; nakamura-uchiyama, fukumi; higa, futoshi; maeda, koichi; yanagihara, katsunori; yoshida, koichiro title: jaid/jsc guidelines for the treatment of respiratory infectious diseases: the japanese association for infectious diseases/japanese society of chemotherapy – the jaid/jsc guide to clinical management of infectious disease/guideline-preparing committee respiratory infectious disease wg date: - - journal: journal of infection and chemotherapy doi: . /j.jiac. . . sha: doc_id: cord_uid: c d nk x nan the japanese association for infectious diseases (jaid) and japanese society of chemotherapy (jsc) announced the "guide for the use of antimicrobial drugs" in and the "guidelines for the use of antimicrobial drugs" in . subsequently, the "the jaid/ jsc guide to clinical management of infectious diseases " was published. with its revision, guidelines were newly prepared. concerning respiratory infectious diseases, in japan, the japanese respiratory society published guidelines for the management of community-acquired pneumonia, hospital-acquired pneumonia, respiratory tract infection, and -/nursing and healthcare-associated pneumonia. furthermore, the japanese society of pediatric pulmonology and japanese society for pediatric infectious diseases announced the "guidelines for the management of respiratory infectious diseases in children in japan". internationally, many guidelines, including those established by the american thoracic society and infectious diseases society of america, have been published from various countries. thereafter, clinical research on respiratory infectious diseases has advanced, leading to the accumulation of many outcomes regarding epidemiology, clinical diagnosis, and treatment. however, the types of microorganisms that cause respiratory infectious diseases have increased with the number of resistant bacteria. in addition, conditions have also varied with causative microorganisms through the recent compromised host's severe status. the place of treatment varies: from the outpatient clinic to the icu. physicians responsible for treatment also vary: practitioners, hospital doctors, pulmonologists, emergency physicians, board certified member of jaid, japanese antimicrobial chemotherapy physician. there are a large number of options of antimicrobial drugs that are available, including new drugs; therapeutic strategies are confused. on the other hand, recently, the entity of pk-pd has been commonly recognized, and the importance of scientifically using antimicrobial drugs has been emphasized. in addition, the japanese society of chemotherapy established a system for antimicrobial chemotherapy-certified physicians, and promoted the widespread, adequate use of antimicrobial drugs. based on these, the two societies prepared the jaid/jsc guidelines for the treatment of respiratory infectious diseases. if specific treatment guidelines can be presented, this may contribute to an improvement in the treatment responses of respiratory infectious diseases, a reduction in health expenditure, and the prevention of resistant bacteria. the guidelines were prepared based on the ebm so that they reflected the management of respiratory infectious diseases in japan and covered all such diseases in adults and children. to prepare the guidelines, a committee was established in , and a draft was published on homepage based on an approval from the boards of directors at the two societies through a review-based consensus. opinions were collected from the two societies' members. in japan, there have been no such guidelines covering respiratory infectious diseases. in the future, with further advances in research, the contents of the guidelines must be revised. however, we successfully provided treatment guidelines that are the most advanced at present. the guidelines were prepared for all clinicians to understand the treatment of respiratory infectious diseases and manage them with antimicrobial drugs adequately. they do not limit treatment by individual physicians or affect their rights to select it. the guidelines may be commonly applied for respiratory infectious disease management/research/education in japan, improving the quality of respiratory infectious disease management, preventing an increase in the number of resistant bacteria, and contributing to national health. we hope that the guidelines will be utilized by a large number of clinicians in respiratory infectious disease management. lastly, we thank the committee members and secretariat staff for their cooperation. . descriptions on the recommendation grade and evidence level . definition of firstand second-choice drugs . precautions -in this article, with respect to the administration method (especially doses) of antimicrobial drugs, they are recommended based on sufficient doses. considering the products adopted at each medical institution, antibiograms, severity, underlying disease, age, and presence or absence of organ disorder, the dose should be increased or decreased if necessary. -the spectra of third-generation cephems for intravenous injection, ctx and ctrx, are similar, but ctx, which is excreted in the kidney, should be primarily used when liver dysfunction is present, and ctrx, which is excreted in bile, should be primarily used when renal dysfunction is present. -as quinolones exhibit antitubercular actions, patients with pulmonary tuberculosis should be excluded for use. . a list of antimicrobial drug abbreviations and doses for neonates are presented at the end of this volume. . . community-acquired pneumonia . . . empiric therapy ---executive summary---patient with bacterial pneumonia should be treated primarily with high-dose penicillin (aii). in elderly patients and those with underlying lung diseases, the use of respiratory quinolones may be considered positively (bii). in case of atypical pneumonia, a macrolide or tetracycline is the first choice. respiratory quinolones should be reserved as alternative drugs (bii), but may be used depending on local circumstances about drug resistance (ciii). in case of whether pneumonia or atypical pneumonia dose not diagnose, comobination with high-dose penicillin and a macrolide or tetracycline should be attempted first (bii). respiratory quinolones should be reserved as alternative drugs (bii). in severer cases requiring treatment in the icu, a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment (aii). ---explanation---community-acquired pneumonia refers to hospital-acquired pneumonia that develops h or more after admission or pneumonia that develops in healthy adults on social activities other than medical practice-/nursing-associated pneumonia [ e ] . as signs and symptoms, cough, sputum, thoracic pain, and dyspnea appear, and this disease acutely occurs with systemic symptoms such as fever and general malaise [ e ] . however, these symptoms are not marked in some elderly patients. furthermore, atypical pneumonia including mycoplasma is characterized by a small amount of sputum, and can be differentiated (tables and ) [ , ] . concerning examination, gram staining and culture of sputum are used to identify causative microorganisms and select subsequent treatment strategies [ , ] (aii). kits for rapid diagnosis with urine or nasal swab are also used for auxiliary diagnosis [ , ] (aii). a blood test shows inflammatory findings such as leukocytosis and an increase in the crp level, facilitating a certain assessment of the disease [ , ] . on thoracic imaging, consolidation or a ground glasslike shadow is observed [ e ] (ii). when patients are in an immunosuppressive state related to an underlying disease, a causative microorganism test should be performed, considering the possibility of opportunistic infection [ e , , ] (a). in elderly patients, aspiration pneumonia is frequently observed, and the management of this disorder is necessary (refer to the section " . aspiration pneumonia" on page. ). in the presence of renal dysfunction, the type and dose of an antimicrobial drug must be carefully selected [ , ] (aii). bacterial pneumonia should be differentiated from atypical pneumonia in accordance with "the jrs guidelines for the management of community-acquired pneumonia in adults in " (edited by the committee to prepare guidelines regarding respiratory infectious diseases, japanese respiratory society) (tables and ) [ ] . although legionella pneumonia is routinely classified as atypical pneumonia, various types of atypical pneumonia do not include legionella pneumonia in this differentiation method. a. bacterial pneumonia ( ) outpatient treatment bacterial pneumonia is primarily caused by sterptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis [ e , , ] (ii). basically, these types of pneumonia should be treated by orally administering high-dose penicillin [ e ] (aii). in japan, macrolide-resistant s. pneumoniae is detected in most cases; therefore, macrolides are not recommended as the first choice, differing form those in europe and the united states [ , , , , ] (aii). for outpatient treatment, b-lactamase inhibitorcontaining penicillin is commonly used. therapy with cva/ampc or sbtpc ( tablets/ e times a day) is recommended with respect to the efficacy and suppression of resistant bacteria [ , , ] (aii). however, such high-dose prescriptions are not always accepted by health insurance system in japan, and the following prescriptions (examples) should also be considered. in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [ , , ] (bii). however, many new quinolones also have antimicrobial activities against mycobacterium tuberculosis; therefore, the presence or absence of active tuberculosis must be strictly checked before administration [ ] (aii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. considering s. pneumoniae, h. influenzae, and m. catarrhalis, high-dose penicillin or cephems, which are effective for these microorganisms, should be selected [ e ] (aii). if more potent treatment is required, respiratory quinolone injection should be used [ , ] (bii). ---drugs to be recommended---( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day * concerning cva/ampc and sbtpc, up to mg of ampc or up to mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral ( / mg), tablet/ times a day þ ampc, oral ( mg), tablet/ times a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day -tflx, oral, mg/twice a day ( ) hospital treatment a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day <> second choice -lvfx, intravenous drip, mg/once a day b. atypical pneumonia ( ) outpatient treatment atypical pneumonia is primarily caused by mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila [ e , , , , ] (ii). the oral administration of a macrolide or tetracycline is the first choice [ , , , ] (aii). to suppress resistant bacteria, respiratory quinolones should be reserved as alternative drugs [ , , , , ] (bii). however, recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue in japan. respiratory quinolones must be used as the first choice depending on local circumstances about drug resistance [ ] (ciii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [ e , , , ] (bii). ---drugs to be recommended---( ) outpatient treatment a first choices -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day -mino, oral, mg twice a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day table items used to differentiate between bacterial and atypical pneumonia [ ] . . under years of age . no or minor underlying diseases . stubborn cough . poor chest auscultatory findings . no sputum, or no identified aetiological agent by rapid diagnosis . a peripheral white blood cell count below , /ml table criteria for differentiation [ ] . in cases using the items in -tflx, oral, mg/twice a day ( ) hospital treatment -azm, intravenous drip, mg/once a day -mino, intravenous drip, mg/twice a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day. -pzfx, intravenous drip, to mg/twice a day c. cases in which whether the disease is bacterial pneumonia or atypical pneumonia is unclear ( ) outpatient treatment in this case, combination therapy with high-dose penicillin and a macrolide or tetracycline should be selected as the first choice to cover both bacterial and atypical pneumonia [ e , , , , , ] (bii). as respiratory quinolones cover both bacterial and atypical pneumonia, they are convenient, but should be reserved as alternative drugs from the perspective of suppression of resistant bacteria [ e , , , , ] (bii). however, in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [ , , ] (bii). recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue. respiratory quinolones may be used as the first choice depending on local circumstances about drug resistance [ ] (ciii). ( ) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [ e , , ] (bii). ( ) severer cases requiring treatment in the icu in severer cases requiring treatment in the icu, s. pneumoniae should be initially considered, and a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment primarily to cover latent atypical bacteria (in particular, when l. pneumophila is not covered, the condition may become fatal) [ e , , , ] (aii). in particular, combination therapy with a macrolide is recommended from immunological aspects to suppress excessive inflammation related to cytokines [ ] (cii). as the possibility that causative microorganisms may be enteric bacteria including esbl-producing bacteria cannot be ruled out, carbapenem injection should be used as a first-choice drug in patients with a background factor for which esbl-producing bacteria are frequently detected [ , ] (bii). the sensitivity of a urinary antigen kit to s. pneumoniae and legionella spp. is approximately %. therefore, even when the patient is negative for these bacteria in the initial phase, the possibility of pneumonia related to these bacteria should not be ruled out [ e , , ] (ii). ---drugs to be recommended---( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day * concerning cva/ampc and sbtpc, up to mg of ampc or up to mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral ( / mg), tablet/ times a day þ ampc, oral ( mg), tablet/ times a day þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day -mino, oral, mg/twice a day <> second choices -lvfx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/ e times a day -mflx, oral, mg/once a day -tflx, oral, mg/twice a day ( ) hospital treatment a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day þ one of the followings: -azm, intravenous drip, mg/once a day -mino, intravenous drip, mg/twice a day -cam, oral, mg/twice a day <> second choices -lvfx, intravenous drip, mg/once a day -pzfx, intravenous drip, to mg/twice a day ( ) severer cases requiring treatment in the icu -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -bipm, intravenous drip, . e . g/ e times a day -drpm, intravenous drip, . e g/ times a day þ one of the followings: -azm, intravenous drip, mg/once a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day -mino, intravenous drip, mg/twice a day ---executive summary----when causative microorganisms are identified based on the results of microbial examination of good-quality sputum, blood culture, and urinary antigen (s. pneumoniae, l. pneumophila) tests and drug susceptibility testing of the causative agents, definitive therapy should be performed if possible [ , ] (biii). -the place of treatment and drugs should be selected in accordance with the severity of the disease [ , ] (aii). -antimicrobial drugs should be selected in reference to the susceptibility of isolated bacteria to antimicrobial drugs or a drugsusceptibility tendency in the area [ , , , ] when these data are available (aii). -the administration period of an antimicrobial drug is determined in accordance with the improvement of symptoms and laboratory data, with a target of e days [ , ] (biii). -when the patient is infected with l. pneumophila or c. pneumoniae, the optimal administration period is about days [ ] (biv). a. streptococcus pneumoniae -the clinical and laboratory standards institute (clsi) has established higher criteria for breakpoints for penicillin susceptibility on the administration of parenteral antimicrobial drugs for s. pneumoniae infections other than meningitis [ ] , based on the following findings: patients with severe pneumonia due to s. pneumoniae with a low pcg susceptibility (mic: . e mg/ml) showed no difference in responses to pcg and outcome [ , ] (ii). for the treatment of pneumococcal pneumonia, the dose of penicillin should be increased [ , ] (a). -in japan, most s. pneumoniae isolates are macrolide resistant [ , ] . -respiratory quinolones have potent anti-pneumococcal activities (iii). the clinical effects of such quinolones are similar to those of high-dose ampc [ ] (ii). -in japan, quinolone-resistant s. pneumoniae is detected in e % of the isolates [ ] . as quinolone resistance may be readily induced by point mutations of dna gyrase or topoisomerase genes [ ] , quinolones must be used adequately (aiii). b. haemophilus influenzae -the abpc-resistant mechanism of h. influenzae involves blactamase production and/or pbp mutation. previously, blactamase production was primarily involved, but, recently, pbp mutation-mediated b-lactamase-negative abpc-resistant (blnar) strains have been increasingly detected. abpcresistant strains with both b-lactamase production and pbp mutation are classified as b-lactamase-positive cva/abpcresistant (blpacr) strains. -according to a national survey in japan, ( . %) and ( . %) of h. influenzae strains were blnar and b-lactamase-producing strains, respectively [ ] . -blnar strains are also resistant to firstand secondgeneration cephems. -pipc exhibits an antimicrobial activity against blnar strains. however, it is ineffective for blpacr strains. c. klebsiella spp., escherichia coli, proteus spp. -the proportion of extended spectrum b-lactamase (esbl)producing bacteria has slightly increased among isolates from respiratory samples. -according to a national survey in japan, esbl-producing bacteria account for . e . % of respiratory sample-derived klebsiella spp. strains [ , ] . -most esbl-producing strains are simultaneously resistant to quinolones [ ] . antimicrobials should be selected according to the drug susceptibility of isolated bacteria. -in japan, carbapenemase-producing strains are extremely rare. d. mycoplasma pneumoniae -in the field of pediatrics, the detection rate of macrolideresistant m. pneumoniae has markedly increased. in adults, that of macrolide-resistant m. pneumoniae also increase [ , ] . -tetracyclines exhibit potent clinical effects on macrolideresistant m. pneumoniae [ ] . -respiratory quinolones have good activities against m. pneumoniae [ , ] . e. legionella spp. -it should be noted that pneumonia related to legionella spp. other than l. pneumophila sg cannot be diagnosed using legionella urinary antigen testing. -as neither b-lactams nor aminoglycosides have antimicrobial activities against legionella spp., which proliferates within host cells, they are clinically ineffective. -quinolones, macrolides, and tetracyclines have been confirmed to exhibit clinical effects on legionella spp. previously, em was the first choice for this infection, but many recent studies have showed the clinical efficacies of lvfx and azm [ , ] . -rfp is effective when combined with em. the combination of em and rfp is useful. a study suggested the effects of combination therapy with lvfx and a macrolide [ ] (ciii). -although there are no marked differences in antimicrobial drug susceptibility among legionella spp., clinical reviews to verify this are limited [ ] . f. chlamydophila pneumoniae -only a few studies have supported the clinical effects of antimicrobial drugs against c. pneumoniae pulmonary infections. -tetracyclines, macrolides, and quinolones may be effective. these drugs are recommended primarily based on the results of basic studies [ , ] . g. staphylococcus aureus -with respect to staphylococcus aureus in japan, there has been an increase in the number of methicillin-resistant strains even in patients with community-acquired pneumonia. in particular, recently, municipal-onset-type mrsa (ca-mrsa) with panton-valentine-leucocidine (pvl) has been detected in japan, raising an issue [ ] . -in cases of mssa infection (bacteremia), the clinical effects of cez are superior to those of vcm [ ] . -as the susceptibility of mrsa to oral antimicrobial drugs differs among isolates, drugs should be selected according to its drug susceptibility results. h. streptococcus spp. -among various types of streptococcus, the streptococcus anginosus group is frequently detected, and characterized by strong abscess-forming features [ ] . streptococcus pyogenes and streptococcus agalactiae may also cause pneumonia. the former may lead to serious pulmonary infection [ ] (v). -there is no penicillin resistance, but macrolide resistance is observed at a low frequency [ ] . -the anti-streptococcus activities of quinolones vary. among quinolones, grnx, mflx, and stfx have relatively potent antimicrobial activities [ , ] . i. moraxella catarrhalis -the number of b-lactamaseproducing strains has increased since the 's. currently, most strains produce b-lactamase [ , ] . b-lactamase produced by m. catarrhalis decomposes penicillin. -in japan, macrolide-or quinolone-resistance have not been reported [ ] . j. anaerobes -most anaerobes that cause pneumonia exist in the oral cavity. peptostreptococcus spp., prevotella spp., and fusobacterium spp. are involved. mixed infection with microaerophilic streptococci is often observed. -in many cases, infection with anaerobes may be associated with aspiration. -most oral anaerobes (prevotella spp., fusobacterium spp., and porphyromonas spp.) are susceptible to combination drugs consisting of penicillin and a b-lactamase inhibitor, cldm and mnz [ ] . k. pseudomonas aeruginosa -in patients with chronic respiratory tract infection, pseudomonas aeruginosa colonizes in the airway, and may cause community-acquired pneumonia [ ] . -as the susceptibility of p. aeruginosa to antimicrobial drugs differs among clinical isolates, drugs should be selected according to its drug susceptibility results. ---drugs to be recommended----the drug susceptibility of each clinical isolate should be classified in accordance with the clsi criteria [ ] . -establishment of prescriptions recommended in this article * antimicrobials have been approved for specific diseases and specific causative agents by japanese ministry of health and welfare. the approvals are based on the results of clinical studies with good clinical practice. as a general rule, the recommendations in this section refer to this (aii). however, recent trends in drug susceptibility are also considered. * the recent drug susceptibility results of the nationwide studies in japan were referred [ , ] . * the recommendations without the approvals by japanese ministry are graded by evidence levels. [ -when the susceptibility of identified causative microorganisms is clarified, or after the treatment responsiveness is evaluated, whether or not de-escalation is possible should be reviewed [ e ] (aii). ---explanation---definition: hospital-acquired pneumonia is defined as "pneumonia that newly develops h or more after admission". in many cases, treatment is difficult due to unfavorable patient conditions such as the presence of an underlying disease, immune capacity, and general condition [ e ]. laboratory findings: patients meeting of items, fever, an abnormal leukocyte count, and purulent secretes, in addition to the appearance of an abnormal shadow of the chest should be diagnosed with hospital-acquired pneumonia [ e ]. ) ventilator-associated pneumonia (vap): vap refers to pneumonia that newly develops h or more after endotracheal intubation/ventilator initiation. its onset within e days after endotracheal intubation is classified as early-type, and its subsequent onset as late-type [ , , , ] . ) hospital-acquired pneumonia other than vap: several types of hospital-acquired pneumonia other than vap include ( ) immunodeficiency (for example, neutropenia during anticancer therapy, cell-mediated immunodeficiency related to the administration of steroids or immunosuppressive drugs) and ( ) aspiration pneumonia including latent aspiration (refer to the section " . aspiration pneumonia" on page ). appropriate management and selection of antimicrobial drugs in accordance with individual conditions are necessary [ ] . with respect to microorganisms that are expected, refer to the section " . hospital-acquired pneumonia--- . . empiric therapy: cases in which gram staining is available" (p. ). ---drugs to be recommended--a. cases in which there is no risk of resistant bacteria antimicrobial drugs should be selected, targeting streptococcus pneumoniae, h. influenzae, and klebsiella spp. as causative microorganisms [ e ] (biii). although it is difficult to estimate/identify causative microorganisms using sputum samples, bacteria that are not isolated/cultured from goodquality sputum may not be causative microorganisms. if resistant bacteria such as mrsa and p. aeruginosa are not detected on sputum culture and there is no deterioration of clinical symptoms, an initial drug should be continued [ ] (biii). in patients in whom aspiration episodes are clear, those in whom oral hygiene is not maintained, or those with consciousness disorder, drugs with anti-anaerobe activities should be selected, considering the involvement of anaerobes [ ] (biii). if an adequate antimicrobial drug is administered, the treatment period may be e days, excluding mrsa and p. aeruginosa [ , ] (bii). a first choices -sbt/abpc, intravenous drip, g/ e times a day -ctx, intravenous drip, e g/ times a day -ctrx, intravenous drip, g/once a day or g/twice a day * if the involvement of anaerobes is suspected, sbt/abpc should be selected. <> second choice -lvfx, intravenous drip, mg/once a day (as its antimicrobial activity against anaerobes is weak, monotherapy with this drug should be avoided in patients with aspiration pneumonia.). b. cases in which there is a risk of multi-drug-resistant bacteria (table ) [ ] to cover multi-drug-resistant bacteria including p. aeruginosa, broad-spectrum antimicrobial drugs with anti-p. aeruginosa activities should be selected [ e ] (aiii). considering the frequency of esbl in each institution, carbapenems should be considered even when enteric bacteria, including klebsiella spp. and escherichia coli, are suspected (biv). if p. aeruginosa is not isolated on good-quality sputum culture, a treatment option should be de-escalated to drugs for cases in which there is no risk of resistant bacteria [ e ] (aii). if aspiration is suspected, or if the involvement of grampositive bacteria is suggested, combination therapy with cldm must be considered (biv). if there is a risk of mrsa carrier (table ) , combination therapy with anti-mrsa drugs should also be considered. the mean administration period of antimicrobial drugs with respect to causative bacteria in patients with an improvement was approximately days. however, that for resistant bacteria such as p. aeruginosa and mrsa was approximately days [ ] (bii). if appropriate antimicrobial drugs can be administered after clarifying causative bacteria, a treatment period of approximately days is recommended [ , , ] (bii). a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . g/ times a day or g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day <> second choices -cfpm, intravenous drip, e g/ e times a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day if the involvement of anaerobes is suspected, one of the following options should be combined with one of the above regimens: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day c. severe cases one of the following options must be combined with one of the regimens for cases in which there is a risk of multi-drugresistant bacteria. when comparing the results between patients undergoing appropriate and inappropriate treatments, the prognosis of the latter was significantly poorer [ , ] (bii). however, a study reported that the prognosis in a group with compliance with recommended drug selection was significantly poorer than in a non-compliance group in patients in whom infection with drug-resistant bacteria in the icu was suspected even among those in whom the etiology was bacteriologically investigated [ ] (bii). therefore, it must be considered that, even when resistant bacteria are etiologically involved, the administration of an appropriate antimicrobial drug that covers them does not always improve the prognosis. -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . g/ times a day or g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day one of the following options should be combined with one of the above regimens: should be estimated based on the results of the clinical microbiological culture (cmc: gram staining and culture) of a lower airway sample immediately before the start of treatment, and not based on bacteria isolated on active surveillance culture (asc), which was conducted as a strategy to prevent/ control infection prior to onset [ ] . -microorganisms that cause pneumonia or (colonization of) the lower airway should be estimated based on the presence or absence of neutrophils or phagocytosis (excluding those patients with neutropenia or functional impairment of neutrophil) [ ] (bii). ---explanation---[gram staining] diagnostic accuracy of hospital-acquired pneumonia is improved by confirming neutrophils and bacterial cells using gram staining of airway samples. this observation has also been confirmed through an increase in the likelihood ratio of hospital-acquired pneumonia in patients with a clinical pulmonary infection score (cpis) of points or higher [ ] . as bacteria isolated from the lower airways of inpatients are common colonizers in many cases, gram staining is also useful for discerning colonization from infection by evaluating the presence or absence of neutrophil and phagocytosis. therefore, it is desirable to combine bacterial culture with gram staining [ , e ] . antimicrobial-drug selection based on gram staining findings leads to appropriate empiric therapy in two-thirds of patients with hospital-acquired pneumonia, and it can be continued as definitive therapy in many cases [ ] . if there are no bacterial cells on gram staining of lower airway sample in whom an antimicrobial regimen was not changed within the past h, it is unlikely that the focus of infection/inflammation is within the lungs (lower airway) [ ] . in this case, the possibility of pneumonia mimic, such as pleural effusion, atelectasis, and pulmonary edema, is suggested if the lung field opacity still remains in chest x-ray. if there is no other infectious focus, the discontinuation of an antimicrobial drug may be warranted [ , , ] . a study has reported that the culture results of asc performed as a strategy of routine infection control measure prior to the development of nosocomial pneumonia accurately predicted the causative pathogen in only % of cases [ ] . therefore, it is necessary to submit airway samples for clinical microbiological culture (cmc) immediately before the start of presumptive treatment. [causative microorganisms and their origin] microorganisms that cause hospital-acquired pneumonia are derived from the oropharynx, airway (including the nasal cavity and nasal sinus), digestive tract, and environment. gastrointestinal tract-derived causative microorganisms are enteric bacteria (primarily, klebsiella spp., e. coli and others such as proteus spp., enterobacter spp., serratia spp., morganella spp., and citrobacter spp.). those derived from the upper airway include s. pneumoniae, h. influenzae, moraxella catarrhalis, s. aureus (namely methicillin sensitive strain), and oral anaerobes. those derived from the environment include methicillin-sensitive s. aureus, pseudomonas spp., acinetobacter spp., and stenotrophomonas spp. [ , , , ] . as the above bacteria derived from the airway and gastrointestinal tract basically exert strong virulence to the airway, they can be considered a core pathogen group of hospital-acquired pneumonia. their potential for developing airway inflammatory response is generally believed stronger than those caused by environmental pathogen [ , ] . table risk factors for multi-drug-resistant bacteria. . previous use of antimicrobial drugs within days . interval of days or more from admission . admission from an area/hospital in which resistant bacteria are frequent . immunosuppressive state or treatment table risk factors for carrying mrsa [ ] . conditions under which anti-mrsa drug therapy should be considered (including gram stain) mrsa---" (p. ). ( ) diplococcus consisting of a pair of two cocci (gpdc: grampositive diplococci) s. pneumoniae should initially be suspected. enterococcus is also a gpdc in microscopic appearance, but is basically considered a non-pulmonary pathogen [ ] . <> cases in which there have been no previous treatment with antimicrobial drugs or risks of penicillin-resistant pneumococcus -pcg, intravenous drip, , , to , , units/ e times a day -abpc, intravenous drip, g/ e times a day <> cases in which previous treatment with antimicrobial drugs or a risk of prsp is present -ctrx, intravenous drip, g/twice a day or g/once a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day -vcm, intravenous drip, g/twice a day (tdm should be conducted so that a trough is e mg/ml [ ] .) ( ) gram-positive coccus in either short or long chain (gpc in chain) aor b-hemolytic streptococci is indicated. -pcg, intravenous drip, , , to , , units/ e times a day -abpc, intravenous drip, g/ e times a day ( ) gram-positive bacillus with a rod-like morphology (gpr: gram-positive rod) corynebacterium spp. may be indicated. -vcm, intravenous drip, g/twice a day (tdm should be conducted so that a trough is e mg/ml [ ] .) c. gram-negative bacteria ---executive summary----when gram-negative bacteria are observed, h. influenzae, m. catarrhalis, enterobacteriaceae, p. aeruginosa, acinetobacter spp., and stenotrophomonas spp. may be indicated [ e , , ] (bii). -it is difficult to estimate the type of bacteria based on the morphology on gram staining in comparison with grampositive bacteria. -gram-negative bacteria frequently detected as causative microorganisms include enteric bacteria and p. aeruginosa. -it is encouraged important to recognize the basic antimicrobial drug susceptibility pattern of each type (group) of bacteria to make sure that the empiric antimicrobial therapy is appropriate (table ). ---drugs to be recommended--- ( ) cases of early-onset hospital-acquired pneumonia in which there have been no previous administration of antimicrobial drugs or risk of resistant bacteria aero-respiratory pathogen, such as h. influenzae and m. catarrhalis, and enteric bacteria, such as klebsiella spp., are indicated. -sbt/abpc, intravenous drip, g/ e times a day -ctrx, intravenous drip, g/twice a day or g/once a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day ( ) cases of late-onset hospital-acquired pneumonia or ventilator-associated pneumonia in which the risk of resistant bacteria is high an antimicrobial drug with anti-pseudomonal activity that targets non-glucose-fermentative gram-negative rod should be administered [ , , ] -to treat polymicrobial infection, the administration of an antimicrobial drug with an activity against obligate anaerobe is not always necessary [ , ] . -in non-severe cases, the administration of antimicrobial agents with anti-mrsa activity may be withheld in the initial phase even when staphylococcus-like bacterial cells are observed [ ] . ---explanation---when several types of bacteria differing in gram staining and morphology are observed, the condition is commonly interpreted as aspiration pneumonia, suggesting the involvement of anaerobes. however, the number of hospital-acquired pneumonia (including vap) caused by anaerobes have been reported relatively smaller than generally anticipated. [ ] , polymicrobial infection does not always require the prompt antimicrobial therapy that covers anaerobes. even though when aspiration pneumonia is suspected, sbt/abpc is frequently prescribed assuming anaerobic infection, which actually works good on many occasions, it has to be acknowledged that sbt/abpc exert good antimicrobial activity not solely against anaerobes, but also aero-enteric pathogen of pneumonia such as streptococcus pneumonia, oral streptococci, h. influenzae, m. catarrhalis, and klebsiella pneumonia. inpatients are often exposed to gram-negative bacteria residing in the hospital environment. furthermore, there are many opportunities to undergo antimicrobial drug therapy that affects the indigenous microflora. for such reasons, gram-negative bacillus (enteric bacteria or p. aeruginosa) frequently colonize within the oropharyngeal region of the elderly patients or long-term bedbound patients, many of whom need airway suctioning or have tracheostomy that may serve as portal of entry of environmental pathogen. oropharyngeal microflora primarily consisting of these gram-negative bacteria can be aspirated into the airway after surgery requiring sedation or anesthesia, or during or after endoscopic examination [ , , ] . briefly, anaerobes may be an occasional pathoen in polymicrobial infection as seen on gram staining of patients with suspected aspiration pneumonia, but s. pneumoniae, h. influenzae, s. aureus, klebsiella spp., p. aeruginosa, and acinetobacter spp. are more commonly involved in many cases, being similar to the microorganisms that are thought to be the major pathogen of hospital-acquired pneumonia. this is in contrast with the community-onset aspiration pneumonia, represented by lung abscess, in that anaerobes are primarily involved [ , ] . anaerobes involved in hospital-acquired pneumonia include facultative anaerobic a-hemolytic streptococci in the oral cavity and obligate anaerobes. oral obligate anaerobes include grampositive coccus (peptostreptococcus sp.), gram-negative coccus (veillonella sp.), and gram-negative bacillus "oral pigmented" bacteroides (bacteroides melaninogenicus), prevotella sp., porphyromonas sp., and fusobacterium sp.). many of these types of bacteria are susceptible to b-lactams that do not contain a b-lactamase inhibitor, new quinolones, macrolides, and tetracyclines. therefore, patients with hospital-acquired pneumonia may be basically treated by standard empiric therapy for hospital-acquired pneumonia even when aspiration pneumonia related to several types of bacteria is suspected [ ] . ---drugs to be recommended---( ) cases in which it is not necessary to consider the involvement of multi-drug-resistant bacteria, or early hospitalacquired pneumonia the involvement of oral streptococcus, oral anaerobes, s. pneumoniae, h. influenzae, and enteric bacteria should be considered. -sbt/abpc, intravenous drip, g/ e times a day -ctrx, intravenous drip, g/once a day or g/twice a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day ( ) late-onset hospital-acquired pneumonia or cases in which there is a risk of multi-drug-resistant bacteria in addition to the above pathogens, the involvement of non-glucose-fermentative gram negative bacteria or esblproducing enteric bacteria must be considered. -cfpm, intravenous drip, e g/ e times a day -czop, intravenous drip, e g/ e times a day -taz/pipc, intravenous drip, . g/ e times a day table basic susceptibility of various pathogen groups to antimicrobial drugs. ---explanation---if drug susceptibility test is not conducted for some reasons after the identification of causative microorganisms, an antimicrobial drug should be selected with reference to the susceptibility pattern (local sensitivity) of the identified bacteria at each institution. if the local sensitivity is not obtained, a drug should be selected based on the basic susceptibility of various pathogens to antimicrobial drugs (table ) [ ] . in the treatment of hospital-acquired pneumonia, the duration of antimicrobial therapy generally tends to be longer than required for the following reasons: opacity on chest x-ray often remains for reasons other than pneumonia even after the start of antimicrobial drug treatment; and there may be a large number of latent nonpneumonia (or non-infectious-disease) factors that may cause increase in body temperature or crp level in inpatients [ ] . however, if appropriate antimicrobial drug treatment is performed, it is possible to complete treatment in week [ ] . in strains such as enterobacter spp., serratia spp., citrobacter spp., and morganella spp. (table gnr b ), the expression of intrinsic antimicrobial-drugresistance genes encoded in chromosome genes is induced during antimicrobial drug treatment, a phenomenon which is basically rarely seen in e. coli, klebsiella spp., h. influenzae, and m. catarrhalis (table gnr a ) ( table ) [ , , ] . therefore, if adequately chosen treatment parameters are improved, antimicrobial treatment could be completed with careful follow up of patients' condition. although it is useful to recognize these pathogens, abbreviated as space (serratia, pseudomonas, acinetobacter, citrobacter, and enterobacter), as a representative microorganism group that causes hospital-acquired pneumonia, the space group is essentially a common colonizer. therefore it is important to bear in mind that antimicrobial drug is not always indicated upon the isolation of space to avoid selection of antimicrobial resistant bacteria related to unnecessary or long-term antimicrobial therapy [ , ] . lzd should be selected [ , ] (ai). the therapeutic efficacy of lzd is similar to those of glycopeptides [ , ] . the penetration of lzd into the alveolar epitheliumlining fluid and intra-alveolar sputum is more favorable. therefore, use of lzd should be encouraged in cases of restricted sputum expectoration, such as vap [ ] (bii). exclusive use of a single drug may accelerate the acquisition of resistance to the agent [ e ] (ci). as dap is inactivated by pulmonary surfactants, its use should be avoided for mrsa pneumonia. -glycopeptides should be selected as first-line drug for pneumonia caused by corynebacterium sp [ ] (aii). ---explanation---there is no significant difference in the therapeutic efficacy for mrsa pneumonia between glycopeptides and lzd. several studies reported that the overall clinical efficacy of lzd, including the incidence of side effects, was superior to vcm in patients with hospital-acquired pneumonia caused by mrsa [ , ] . however, since the dosing of vcm in these studies have been considered suboptimal, further study is needed [ , ] . some investigators have recommended that, when mrsa is susceptible to cldm or mino on a susceptibility test, lzd, a protein synthesis inhibitor should be administered given the possible involvement of the panton-valentine leukocidin [ , ] . if a prompt improvement is achieved by the intravenous drip of lzd mg q h, or if the patient's condition is not critical, switch from the intravenous administration to an oral preparation of lzd, which shows high bioavailability [ ] , is encouraged. as dap is inactivated by pulmonary surfactants, it should not be used to treat mrsa pneumonia. this may not apply to the treatment of septic pulmonary embolism [ ] . ---drugs to be recommended---( ) mrsa a first choices -vcm, intravenous drip, g/twice a day -teic, intravenous drip, mg for the first days/ twice a day for loading, mg/once a day from day * tdm should be conducted so that the trough levels of vcm and teic range from to mg/ml [ ] . -lzd, intravenous drip or oral administration, mg/ twice a day <> second choices -abk, intravenous drip, mg/once a day (a trough level was established as mg/ml using tdm.) -st combination drug (smx at mg/tmp at mg), oral administration, tablets/twice a day or intravenous drip, mg/twice a day -cldm, intravenous drip, mg/ e times a day (the results of drug susceptibility testing must be confirmed). [non-extended-spectrum b-lactamase (esbl)-producing bacteria] ( ) hospital treatment " (p. ). ( ) e. coli, klebsiella spp., proteus mirabilis (esbl-producing bacteria) refer to the section " . community-acquired pneumonia--- . . definitive therapy--- [ ] klebsiella spp. [esbl-producing bacteria] ( ) hospital treatment" (p. ). ( ) enterobacter spp., serratia spp., citrobacter spp., morganella spp., proteus vulgaris a third-generation cephems or quinolones should be administered [ , , ] (aii). -ctrx, intravenous drip, g/once a day or g/twice a day -ctx, intravenous drip, e g/ e times a day -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, mg/twice a day <> if a strain is estimated to constantly express cephalosporinase (highly resistant to b-lactamase inhibitorcontaining b-lactams, oxyimino [¼ rd generation] cephalosporin and cephamycin, through plasmid genes) on an antimicrobial drug susceptibility test, fourthgeneration cephems or carbapenems should be administered. -cfpm, intravenous drip, e g/ times a day -czop, intravenous drip, e g/ times a day -mepm, intravenous drip, g/ times a day -drpm, intravenous drip, . e g/ times a day ( ) p. aeruginosa anti-pseudomonal penicillins, third-generation or later cephems, carbapenems, or new quinolones should be administered [ , ] refer to the section " . community-acquired pneumonia--- . . definitive therapy--- [ ] h. influenza (abpcsusceptible), [ ] h. influenza (b-lactamase-producing), [ ] h. influenza (b-lactamase-negative ampicillin-resistant (blnar), and [ ] h. influenza (b-lactamase-positive amoxicillin clavulanate-resistant (blpacr)" (p. e ). ---explanation---according to some investigators, enteric bacteria are classified into types: sensitive, gram-negative rods, such as e. coli, klebsiella pneumoniae, and p. mirabilis, which are susceptible to first-generation cephalosporin, and resistant, gram-negative rods, such as enterobacter spp., serratia spp., and citrobacter spp., which show an intrinsic or inducible resistance to third-generation cephalosporin through chromosomal ampc genes [ , , ] . in addition, the number of extended spectrum of b-lactamase (esbl)-producing strains of e. coli, klebsiella, and proteus sp. that are resistant to all cephalosporin has increased. among resistant gnrs, such as enterobacter spp., strains that constantly produce ampctype b-lactamase (cephalosporinase) (plasmid type) must also be considered [ , ] . concerning non-fermentative bacteria, their intrinsic susceptibility to antimicrobial agents differs among p. aeruginosa, stenotrophomonas spp., and acinetobacter spp. a study indicated that, in patients with p. aeruginosa pneumonia, monotherapy with a new quinolone might show unfavorable bacteria-eradicating effects or lead to recrudescence [ ] . in some patients, combination therapy with a b-lactam (pipc, caz, cfpm, or carbapenems), which has an anti-pseudomonal activity, and aminoglycoside or new quinolone may be considered [ , , ] . most strains of stenotrophomonas spp. are susceptible to mino or an st combination drug. m. catarrhalis and acinetobacter spp. are the frequent types of gram-negative coccus detected in patients with early and late hospital-acquired pneumonia, respectively. many strains of the former produce b-lactamase. the latter is a gnr existing in the hospital environment, and may be resistant to many antimicrobial drugs. however, in japan, the multi-drug resistance of this type of bacteria has not widely distributed. carbapenems and new quinolones should be selected. however, the vast majority of acinetobaccter strains are susceptible to sbt/abpc. in particular, sbt has an antimicrobial activity against this type of bacteria, and their susceptibility to sbt/abpc should routinely be confirmed. primary test drugs for an antimicrobial susceptibility of this type of bacteria (drugs appropriate for a routine examination panel) are sbt/abpc, caz, ipm/cs, mepm, gm, tob, lvfx, and cpfx [ ] . pan-sensitive strains of h. influenzae are b-lactamase (bl)-negative, abpc-sensitive (blnas) strains. however, there are various resistance patterns: bl-producing, abpcresistant (blpar), bl-negative, abpc-resistant (blnar), and bl-producing, ampc/cva-resistant (blpacr) strains. blnas strains can be treated with abpc, but sbt/abpc therapy is required to control blpar strains. the administration of ctrx or new quinolones is necessary for blnar or blpacr. a randomized-controlled trial with multivariate analysis has shown that factors for favorable bacteriological effects included the absence of p. aeruginosa-related pneumonia (< . ), a higher body weight (< . ), a low apache ii score (severity) ( . ), and cpfx therapy ( . ) [ ] . conditions suggesting the use of new quinolones include allergy to b-lactams, the presence of or concern for nephropathy (an aminoglycoside cannot be combined with a b-lactam), necessity of covering obligate intracellular pathogen, or situations in which switching to an oral preparation is indicated [ ] . an in vitro study indicated that the alveolar epithelial lining fluid (elf) concentration of lvfx reached as high as its serum concentration. furthermore, a prospective open-label study reported that switching of intravenous drip to oral administration decreased the elf concentration, but it was within the range at which many pathogens are deemed sensitive based on the cumulative data of minimum inhibitory concentrations for causative microorganisms [ ] . ---executive summary----nursing and healthcare-associated pneumonia (nhcap) is a category independently defined in japan based on medical circumstances. -the attending physician proposes a treatment category (groups a to d) by evaluating what treatment is necessary as the most important item based on the patient's and his/her family's will ( fig. ) [ ] . -risk factors for resistant bacteria are categorized into two items, and initial treatment options are recommended, assuming target causative microorganisms (civ). -in patients in whom the general condition is unfavorable due to complications or in terminal-stage patients, initial treatment options are recommended considering side effects from the perspective of innocent properties (civ). -in group d, in which intensive care is required, combination therapy with broad-spectrum (involving resistant bacteria and legionella) and potent antimicrobial drugs is recommended (bi). ---explanation--- in , the japanese respiratory society issued the "guidelines for the management of nursing and healthcare-associated pneumonia (nhcap)" [ ] , considering medical circumstances in japan with reference to the entity of healthcare-associated pneumonia (hcap) proposed in the untied states [ ] . the definition of nhcap is shown in table . as this committee has no objection to the entity itself, the selection of drugs will be explained based on evidence to avoid duplications with the above guidelines. the mortality rate and frequency of resistant bacteria in patients with nhcap are intermediate between community-acquired pneumonia (cap) and hospital-acquired pneumonia (hap). however, nhcap may be primarily regarded as being similar to geriatric pneumonia [ , ] . there is no fact that the rate at which resistant bacteria are isolated increases with severity [ ] . even when pneumonia is not severe, the host's activities of daily living (adl) and an underlying disease/immunodeficiency reduce the prognosis in many cases [ ] . as this type of pneumonia develops in a variety of uneven populations, it is difficult to simply determine severity classification. therefore, considering various conditions, the entity of "treatment category", involving the ethical aspects of geriatric care, was introduced based on evaluation by the attending physician who knows the patient well ( fig. ). frequent basic conditions or concomitant diseases for nhcap in japan include an advanced age, central nervous diseases, aspiration, a reduction in adl, and tubal feeding. their factors are aspiration pneumonia itself or risk factors, and hcap in japan may overlap with aspiration pneumonia [ ] . on the other hand, in nhcap patients, mrsa, p. aeruginosa, and anaerobes are more frequently isolated in comparison with cap patients. it is necessary to switch therapeutic strategies, considering these causative microorganisms. refer to the next section " . aspiration pneumonia" (p. ). [type and frequency of causative microorganisms] concerning causative microorganisms in nhcap patients, resistant bacteria are frequently detected, differing from cap patients. however, with respect to microorganisms that cause hcap, the distribution and frequency of streptococcus pneumoniae and h. influenzae, which are frequently isolated in cap patients, as well as mrsa, p. aeruginosa, and gram-negative bacillus, which are frequently detected in hap patients, differ among countries, areas, and institutions due to their variety (iii). concerning causative microorganisms, a study reported that there was no marked difference between nhcap and cap [ ] . on the other hand, a study in the united states indicated that s. aureus was frequently detected [ ] , and another study in italy, where the rapid aging of society is advanced, as described for japan, reported that aspiration pneumonia, h. influenzae, s. aureus, and gram-negative bacillus were more frequent than in patients with cap [ ] . as a result, the rate of resistant bacteria increased, and inappropriate antimicrobial drugs were selected in a high proportion of patients. in addition, the mortality rate was higher than in cap patients, suggesting the association between the two factors. representative causative microorganisms with respect to the presence or absence of risk factors for resistant bacteria are presented in table [ ] . of these, resistant bacteria, which are not targeted in cap patients, were isolated in approximately %. however, the value was lower than in hap patients. this is a current status of japan (iii). however, we must consider that patients in whom isolated bacteria are unclear account for approximately %, with the involvement of aspiration as a background factor [ ] . in addition to bacteria commonly isolated in cap patients, the frequency of enteric bacteria and anaerobes has increased [ ] . [rules of antimicrobial drug therapy] risk factors for resistant bacteria in nhcap patients include "the previous use of antimicrobial drugs for days or more within days" and "tubal feeding" ( [ ] . risk factors for involvement by drug-resistant pathogens. -if no antibiotic therapy in the preceding days or current tube feeding, the patient can be assumed to have no risk of involvement by drug-resistant pathogens. -however, if past medical history indicates isolation of mrsa, the patient should be assumed to have risk of involvement by mrsa. * ) inappropriate when aspiration pneumonia is suspected, because it has insufficient activity against anaerobic bacteria. * ) because of insufficient activity against anaerobic bacteria, when used to treat suspected aspiration pneumonia, it should be used in combination with an antibiotic that has activity against anaerobic bacteria (e.g., mnz, cldm, sbt/abpc). definition of nhcap [ ] . . pneumonia diagnosed in a resident of an extended care facility or nursing home . pneumonia diagnosed in a person who has been discharged from a hospital within the preceding days . pneumonia diagnosed in an elderly or disable d person who is receiving nursing care . pneumonia diagnosed in a person who is receiving regular endovascular treatment as an outpatient (dialysis, antibiotic therapy, chemotherapy, immunosuppressant therapy) standards for nursing care patients whose performance statues is ps (capable of only limited self-care, confined to bed or a chair more than % of their waking hours) or more. item incudes patients on psychiatric wards. not isolated in those who had made favorable daily life activities without a history of antimicrobial drug therapy [ ] . another study indicated that tubal feeding was an independent risk factor for infection with p. aeruginosa (odds ratio: . ) [ ] (ii). this is the reason why treatment category c was established in the guidelines. briefly, patients who do not meet the above two items are regarded as having no risk factor for resistant bacteria, and assigned to group b. patients meeting or items or those in whom mrsa was previously isolated are assigned to group c. respective drugs to be recommended were separately established. patients in whom outpatient treatment is considered to be appropriate are assigned to group a, and those in whom the attending physician considers ventilator or icu management necessary to group d. drugs to be recommended were added, and a treatment category algorithm ( fig. ) [ ] was prepared. concerning hcap treatment in europe and the united states, there is a gap between drugs used in clinical practice and those recommended in guidelines [ ] (ii). therefore, treatment category-based empiric therapy in japan may be acceptable in clinical practice; future investigation is necessary. there is no evidence regarding the administration period of antimicrobial drugs. an administration period of e days, which is routinely adopted in the highest percentage of patients, is appropriate (biv). when administering antimicrobial drugs for a longer period, equivalent-spectrum antimicrobial drugs should be selected, or de-escalation of antimicrobial drugs should be performed. in this case, fever, crp, and leukocyte counts are often used as indices of the treatment response. in cases of aspiration pneumonia in which aspiration recurs during treatment despite the efficacy of antimicrobial drugs, it is necessary to evaluate whether the effects of antimicrobial drugs are not obtained or recurrence occurs. ---drugs to be recommended--a. empiric therapy ( fig. ) [ ] ( ) cases in which there is no risk of resistant bacteria and outpatient treatment is performed (group a) according to a study, chlamydophila spp. and m. pneumoniae accounted for . and . % of patients in whom the type of microorganisms that cause nhcap was clarified in japan, respectively [ ] . the results suggested that chlamydophila spp. is a target of treatment, as described for cap. therefore, in group a, combination therapy with a b-lactam and macrolide or monotherapy with a respiratory quinolone should be performed (bii). in group d, an anti-p. aeruginosa drug should be combined with cpfx, pzfx, or azm for injection, considering legionella or chlamydophila spp. pneumonia. however, concerning combination therapy with a macrolide (cii) in patients without "severe pneumonia requiring intensive care", as described below, the evidence level is not always high from the perspectives of medical economics, side effects, and resistant bacteria [ ] . some studies examined the mortality rate with respect to the presence or absence of treatment covering atypical pathogens, and reported that the mortality rate was significantly lower in the presence of such treatment [ ] . a recent meta-analysis also showed a difference [ ] . respiratory quinolones were established as an option (bii) based on many references describing that their effects are similar to or more potent than those of combination therapy with a b-lactam and macrolide. however, this must be further examined, considering factors such as severity and the presence or absence of concomitant sepsis [ ] . furthermore, the prevalence of penicillinresistant pneumococcus, which has been internationally emphasized as an issue, and macrolide-resistant pneumococcus, which has been markedly observed in japan, was also a background factor for establishing respiratory quinolones as an option [ ] . the previous use of antimicrobial drugs, which is often observed in patients with nhcap, is considered to be a risk factor for resistant pneumococcus [ ] . a study reported that penicillin or em resistance in hcap patients was more advanced than in cap patients [ ] . a study indicated that the efficacy of oral therapy with lvfx was similar to that of ctrx injection therapy in patients with cap [ ] . another study reported that, among patients with non-severe hcap, oral lvfx was useful in those with no description of causative microorganisms [ ] . however, when aspiration pneumonia is suspected, grnx or mflx should be selected, because the effects of lvfx on anaerobes are weak. furthermore, several studies suggested the usefulness of mflx, which is not influenced by the kidney function and does not require dose regulation, in elderly patients with nhcap [ , ] . as treatment is completed with a single, high dose, compliance is favorable. azm sustained-release preparations [ e ], which simultaneously cover bacteria and atypical pathogens, and stfx, which shows a favorable mic for anaerobes, may also be recommended [ ] . a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day table risk factors for involvement by drug-resistant pathogens a in nhcap [ ] . history of antibiotic therapy for or more days in the preceding days current tube feeding the risk of mrsa should be taken into account whenever there is past history of mrsa isolation. when attempting to predict the isolation of drug-resistant pathogens based on the presence of these risk factors, it should be borne in mind that their sensitivity and negative predictive value are high, but their specificity and positive predictive value are low. a drug-resistant pathogens include pseudomonas aeruginosa, mrsa, acinetobacter, esbl-producing enteric bacteria, and stenotrophomonas maltophilia. table possible pathogens isolated from nhcap patients [ ] . when an nhcap patient has no risk factors for involvement by drug-resistant pathogens pneumococcus mssa gram-negative enteric bacteria (including klebsiella and e. coli) haemophilus influenzae oral streptococci atypical pathogens (particularly chlamydophila) when an nhcap patient has a risk factor for involvement by drug-resistant pathogens (the following will be considered in addition to the above-mentioned pathogens) pseudomonas aeruginosa mrsa acinetobacter esbl-producing enteric bacteria þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day <> second choices -mflx, oral, mg/once a day -grnx, oral, mg/once a day -stfx, oral, mg/l to times a day or -ctrx * , intravenous drip, g/once a day or g/twice a day -ctx * , intravenous drip, e g/ e times a day þ one of the followings: -azm sustained-release preparation, oral, g/single dose -cam, oral, mg/twice a day * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. ( ) cases in which there is no risk of resistant bacteria and hospital treatment is performed (group b) in this category, we recommend monotherapy from the perspectives of causative microorganisms resembling those for cap and side effect-based "innocent properties". a study reported that initial treatment with narrow-spectrum antimicrobial drugs did not always lead to a poor prognosis [ ] . in particular, according to two articles [ , ] , it is not necessary to consider resistant bacteria in hcap patients in whom causative microorganisms are unclear; treatment in accordance with cap treatment is sufficient (bii). however, an advanced age, central nervous diseases, aspiration, and a reduction in adl are the clinical characteristics of aspiration pneumonia. the condition of nhcap in japan overlaps with aspiration pneumonia [ ] . therefore, in group b, to which patients admitted for the first time, with no recent use of antimicrobial drugs, correspond, antimicrobial therapy with b-lactamase inhibitor-containing penicillins is appropriate, as described for cap. however, when aspiration pneumonia is suspected, ctrx and lvfx should be avoided (biv). for the management of enteric bacteria, candidate drugs for group b, papm/bp, may be selected. actually, it is not necessary to consider p. aeruginosa in patients with cap or non-icu hap. a study indicated that ertapenem, which is not effective for p. aeruginosa, was useful [ ] , as demonstrated for papm/bp. however, another study reported that the widespread use of ertapenem induced the cross resistance of p. aeruginosa to other carbapenems; the use of papm/bp alone should be avoided [ ] (biv). in elderly persons, with a high risk of aspiration, who are repeatedly admitted and discharged, klebsiella is often involved. taz/pipc is more useful according to a study [ ] (bii). in patients in whom gram-negative bacillus is detected on gram staining of sputum or those in whom the involvement of enteric bacteria is suspected, papm/bp or taz/pipc should be selected (biv). -sbt/abpc, intravenous drip, g/ e times a day -ctrx * , intravenous drip, g/once a day or g/twice a day -ctx * , intravenous drip, e g/ e times a day -lvfx * , intravenous drip, mg/once a day -papm/bp, intravenous drip, . e g/ e times a day * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. ( ) cases in which there is a risk of resistant bacteria and hospital treatment is performed (group c) the target microorganisms include p. aeruginosa, mrsa, and acinetobacter spp. in addition to frequent microorganisms that cause respiratory infection [ , , , ] . as antimicrobial drugs, taz/pipc, with an antimicrobial activity against p. aeruginosa, fourth-generation cephems, carbapenems, and quinolones (cpfx, pzfx) are recommended. taz/ pipc exhibits effects similar to those of ipm/cs and mepm in patients with nursing and healthcare-associated pneumonia [ ] (bii). pzfx also has an antimicrobial activity against s. pneumoniae when used at a high dose ( g/day). when pneumonia related to atypical pathogens such as chlamydophila spp. is suggested, quinolones should be selected. as the antimicrobial activities of fourth-generation cephems and quinolones against anaerobes are weak, these drugs should be combined with mnz, cldm, or sbt/abpc. recently, the resistance of the bacteroides fragilis group to cldm has advanced [ ] . therefore, in europe and the united states, mnz is selected as a first-choice antimicrobial drug against anaerobes. however, the rate at which the b. fragilis group is involved in oral anaerobes is low, and combination therapy with cldm may be selected [ , ] . therefore, for combination therapy with fourth-generation cephems, we recommend the two drugs. when there is a risk of mrsa, such as previous admission, they should be combined with vcm, teic, or lzd. if there is no abscess formation, abk is also effective. a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day <> second choices -cfpm* , intravenous drip, e g/ e times a day -cpr * , intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day or -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day *in addition to the above drugs, if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. ( ) severe cases requiring intensive care (group d) to cover l. pneumophila and atypical pathogens, which are rare as causative microorganisms but may cause severe conditions, group-c antimicrobial drugs should be combined with cpfx, pzfx, or azm injection (bi). concerning the usefulness of combination therapy with a b-lactam and macrolide injection for severe pneumonia, evidence has been accumulated [ ] . a study indicated that, in severe community-acquired pneumonia patients with sepsis or requiring icu management, combination therapy with a blactam and macrolide led to a more favorable prognosis compared to that with a quinolone (i), suggesting that antiinflammatory actions are involved in the mechanism [ ] . in addition, another study reported that, among pneumonia patients with acute pulmonary disorder, both the ventilator withdrawal and survival rates in a macrolide-treated group were higher than in a non-macrolide-treated group [ ] (i). several meta-analyses also support them [ , ] . a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day þ one of the followings: -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day -azm, intravenous drip, mg/once a day <> second choices -cfpm * , intravenous drip, e g/ e times a day -cpr * , intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day þ one of the followings: -cpfx * , intravenous drip, mg/twice a day -pzfx * , intravenous drip, mg/twice a day -azm, intravenous drip, mg/once a day in addition to the above drugs, *if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". * ) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. antimicrobial drugs against identified causative microorganisms should be selected in accordance with the section " . hospital-acquired pneumonia" (p. ). ---executive summary----as oral indigenous bacteria, including anaerobes, cause aspiration pneumonia, b-lactamase inhibitor-containing penicillins are appropriate (bii). -in cases of nosocomial onset, it is necessary to cover gramnegative bacillus, including p. aeruginosa. -in cases of severe ventilator-associated pneumonia (vap), the selection of broad-spectrum antimicrobial drugs or combination therapy with them should not be hesitated (ai). -the detection rate of esbl-producing gram-negative bacillus has increased, and antimicrobial drugs should be carefully selected. -it is important to prevent subclinical aspiration through oral care and the prevention of gastroesophageal reflux, such as head lifting (bii). -to prevent aspiration pneumonia, it is also important to improve the nutritional status and avoid the overuse of sleeping pills/ sedatives (bii). ---explanation---[characteristic and classification of diseases] aspiration pneumonia occurs with a background factor, dysphagia, which is frequently observed in the presence of a reduction in adl or systemic functions, especially cerebrovascular disorder. its onset is associated with dietary ingestion in elderly persons [ ] . currently, aspiration pneumonia is accurately defined only in the guidelines for the management of hospitalacquired pneumonia (hap) in adults, which were prepared by the japanese respiratory society [ ] . the guidelines present conditions that may cause dysphagia, which were proposed by the japanese study group on aspiration pulmonary disease (table , modified) [ ] . in our guidelines, we primarily explain antimicrobial drugs to be selected for patients with such conditions. the above definition is also adopted in the guidelines for the management of nursing and healthcare-associated pneumonia (nhcap) [ ] . in elderly persons admitted to long-term care beds or nursing homes, risk factors include dysphagia and tubal feeding according to international data on pneumonia that develops in nursing homes [ e ]. in japan, frequent underlying diseases in patients with nhcap also include central nervous diseases and dementia, which are closely associated with aspiration. the proportion of patients after percutaneous endoscopic gastrostomy (peg) is high [ ] . however, among various types of community-acquired pneumonia, a diagnosis of aspiration pneumonia is made based on onset factor-based classification, and is not equal to nhcap diagnosed primarily based on the place of onset or grade of nursing. according to data in spain, aspiration pneumonia accounts for . % of patients with healthcareassociated pneumonia (hcap) requiring admission. this percentage was markedly higher than in those with communityacquired pneumonia (cap) requiring admission ( . %), but corresponded to no more than / [ ] . on the other hand, a multicenter cooperative study involving inpatients with pneumonia in japan, where the rapid aging of society is advanced, reported that . % of patients who were admitted with cap had aspiration pneumonia. even in patients with cap, which is not classified as nhcap, the involvement of aspiration cannot be ignored [ ] . furthermore, the study indicated the involvement of aspiration in . % of patients, aged over years, with cap/ hap [ ] . in the future, the significance of distinguishing aspiration pneumonia among patients with nhcap or hap and changing therapeutic strategies should be examined. however, nhcap more markedly affects adl compared to cap, and the aspect of elderly pneumonia is emphasized; it may be significant to positively diagnose aspiration pneumonia and establish therapeutic strategies different from those for cap [ ] . concerning hap, a reduction in the immune function is a background factor. hap has two aspects: pneumonia with a high risk of resistant bacteria and that in which central nervous diseaserelated aspiration is involved. in the guidelines for the management of hospital-acquired pneumonia in adults, which were prepared by the japanese respiratory society, mendelson syndrome and vap are categorized as a group, and classifications, involving diffuse deglutition-related bronchiolitis, in which there are no findings of pneumonia, are proposed. in addition, a flow chart for diagnosis is presented [ ] (fig. ) . with respect to the condition and treatment of vap, refer to a review described by chastre et al. [ ] management other than antimicrobial drug therapy should also be considered, and bundle (table )-based prevention should be performed [ ] (aii). [type and frequency of causative microorganisms] streptococcus pneumoniae, s. aureus, and enterobacteriacae have been reported. a study indicated that k. pneumoniae was frequent [ ] . the involvement of oral indigenous bacteria, such as streptococcus anginosus group. and anaerobes, has been suggested [ , ] . in cases of nosocomial onset, gram-negative bacillus, including p. aeruginosa, must also be considered. concerning e. coli, klebsiella spp., and proteus spp., the number of esbl-producing strains may increase in the future. [rules of antimicrobial drug therapy] if appropriate antimicrobial drug therapy is not selected under a diagnosis of aspiration-related pneumonia, insufficient treatment may lead to a fatal condition, or excessive treatment may increase the number of resistant bacteria, showing negative effects. there may be differences in options for empiric therapy between patients with vap (most patients show severe conditions) and those with diffuse deglutition-related bronchiolitis, in whom the start of treatment is not accelerated. on the other hand, approaches to prevent pneumonia after aspiration or avoid aspiration are important. oral care, head lifting, and improvement in the nutritional status must be considered, and the overuse of sleeping pills/ sedatives should be avoided (bii). the best option for standard-type aspiration pneumonia is an antimicrobial drug that exists an antimicrobial activity against both aerobes and anaerobes. sbt/abpc and taz/pipc are effective for anaerobes frequently isolated in the respiratory system, such as fusobacterium spp., prevotella spp., and peptostreptococcus spp. [ , ] . as the resistance rates of these types of bacteria to the two regimens are low, these regimens are also recommended as first choices in the guidelines established by the japanese association for anaerobic infection research [ ] . however, a study reported that the previous administration of antimicrobial drugs and adl were correlated with the frequency of enterobacteriacae-or p. aeruginosa-related pneumonia [ ] . a retrospective study involving patients with aspiration pneumonia showed that the frequency of k. pneumoniae-related pneumonia was % [ ] . based on these studies, drugs to be selected should be changed in accordance with the previous administration of antimicrobial chemotherapeutic drugs in patients admitted to general or medical wards. among patients with hospital-acquired pneumonia, broad-spectrum drugs should be selected in those with severe aspiration pneumonia or vap (bii). when causative microorganisms are identified and an improvement in the condition is achieved, de-escalation should be performed. ---drugs to be recommended--a. empiric therapy ➀ no risk of resistant bacteria drugs with potent antimicrobial activities against oral anaerobes are presented. however, no article has provided high-level evidence regarding aspiration pneumonia. as the following drugs affect the intestinal flora, antimicrobial drugassociated diarrhea may occur. if symptom improvement is delayed, patients must be promptly admitted, and drip table conditions that may cause dysphagia [ ] [ ] . bundles for the prevention of ventilator-associated pneumonia. ( ) upper body lifting the head should be lifted at e . ( ) discontinuation of sedatives a sedative should be discontinued once a day to evaluate whether or not extubation is possible. ( in addition to the above items, methods to prevent aspiration pneumonia include oral care, the administration of drugs that improve the deglutition function, such as ace inhibitors and cilostazol, improvement in the nutritional status, eating/swallowing rehabilitation, and anti-pneumococcus vaccination. infusion therapy should be performed (outpatient treatment should not be prolonged). ( ) outpatient treatment a first choices -cva/ampc, oral ( / mg), tablets/ e times a day -sbtpc, oral ( mg), tablets/ e times a day <> second choices -mflx, oral, mg/once a day -stfx, oral, mg/ e times a day -grnx, oral, mg/once a day ( ) hospital treatment when a diagnosis of aspiration pneumonia is made, sbt/abpc is most frequently used in japan [ ] . kaneko et al. reported that the sensitivity of oral anaerobes that may cause aspiration pneumonia, such as peptostreptococcus spp., prevotella spp., and fusobacterium spp., to sbt/abpc was %, similar to that to taz/pipc [ ] . the effects of cldm on aspiration pneumonia or a pulmonary abscess are similar to those of sbt/abpc (bi) [ ] . sbt/abpc and cldm showed similar effects and tolerance on aspiration pneumonia ( . and . %, respectively) [ ] . oral anaerobes, excluding bacteroides spp., are still susceptible to cldm. a randomized clinical trial (rct) indicated that cldm was more potent than cephems [ ] . a first choice -sbt/abpc, intravenous drip, g/ e times a day <> second choice -cldm, intravenous drip, mg/ e times a day ➁ cases in which there is a risk of resistant bacteria or severe cases in cases in which there is a risk of resistant bacteria or severe cases, drugs should be selected in accordance with options for group c for nhcap. tubal feeding is a risk factor for aspiration, and is also a risk factor for resistant bacteria [ ] . when the involvement of enterobacteriacae, such as k. pneumoniae and e. coli, is suggested, empiric therapy should be selected in accordance with cases in which there is a risk of resistant bacteria [ ] . in japan, the proportion of esbl-producing bacteria on sputum culture in patients with respiratory infectious diseases is % or less [ ] , but the number of esbl-producing bacteria has slightly increased; this must be considered in the future [ , ] . a study reported that the clinical effects of taz/pipc on non-esblproducing k. pneumoniae were more potent than those of sbt/abpc; caution is needed [ ] . it must be considered that, in cases of aspiration pneumonia classified as hcap, enterobacteriacae is isolated at a frequency that cannot be ignored. concerning aspiration pneumonia that occurs in hospitals, some reviews proposed that antimicrobial drugs should be selected, regarding the condition as hospitalacquired pneumonia; empiric therapy may be selected in accordance with the guidelines for the management of hospital-acquired pneumonia, which were published by the japanese respiratory society [ , ] . according to a study, bipm, which does not cause kidney dysfunction in elderly patients, is also effective (civ); therefore, it is presented as an option for cases in which there is a risk of resistant bacteria [ ] . the mortality rate in patients with vap is high. if causative microorganisms cannot be initially covered, the mortality rate may increase [ ] . therefore, drugs should be selected, regarding the condition as severe aspiration pneumonia. three studies reported that, in a group in which taz/pipc was selected as a drug to be combined with aminoglycosides for vap treatment, the mortality rate was lower than in a group in which caz was selected [ e ]. in particular, when pneumonia was caused by p. aeruginosa, the clinical effects of taz/pipc were more potent than those of ipm/cs (bii). for monotherapy, information on the culture of protected specimen brush (psb) samples or broncho-alveolar lavage (bal) fluid is strongly recommended [ , ] . on the other hand, an observational study indicated that threedrug therapy (two anti-p. aeruginosa drugs þ an anti-mrsa drug) deteriorated the prognosis; an rct should be conducted in the future [ ] . therefore, if there is a risk of resistant bacteria, at least broad-spectrum antimicrobial drugs must be used for empiric therapy. however, assuming causative microorganisms with reference to gram staining reactions, minimum necessary antimicrobial drugs should be selected based on local factors (antimicrobial drug susceptibility pattern of each type of bacteria in each hospital). recently, the entity of ventilator-associated tracheobronchitis (vat) was proposed, and the disadvantages of aggressive treatment have been discussed [ ] . in a multicenter cooperative study, patients with vat, which occurred in the icu, were divided into two groups with and without antimicrobial drug therapy, and the results were compared. in the former, the incidence of vap was significantly lower, and the mechanical ventilation-free period was significantly longer. in addition, the icu mortality rate was significantly lower. on the other hand, there was no significant difference in the appearance of resistant bacteria between the two groups [ ] . a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day -bipm, intravenous drip, . e . g/ e times a day <> second choices -cfpm, intravenous drip, e g/ e times a day -cpr, intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day or -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day -sbt/abpc, intravenous drip, . e g/ e times a day *if mrsa infection is suspected, antimicrobial drugs should be administered in accordance with the section "mrsa pneumonia" in addition to the above drugs. [administration period of antimicrobial drugs] it is recommended that the treatment period of hospitalacquired pneumonia should be e days. however, the treatment period should be days in patients with pneumonia related to non-glucose-fermenting bacteria such as p. aeruginosa [ ] (bii). concerning vap, a study reported that there was no difference in the clinical effects between and days [ ] . to control identified causative microorganisms, antimicrobial drugs should be selected in accordance with the section "hospital-acquired pneumonia". if mrsa infection is suspected, antimicrobial drugs should be selected in accordance with the section "mrsa pneumonia". (bii). -in severe or refractory cases in which the efficacy of initial treatment is not sufficient, combination therapy with an antifungal drug should also be considered [ e ] (bii). -as the effects of combination therapy with an azole and amph-b preparation antagonize in some strains, a combination of these drugs should be avoided [ ] (aiii). -for the target treatment of this disease, an antifungal drug of which the class is different from that of a drug used for preventive administration should be used (biii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in severe immunocompromised hosts such as patients received chemotherapy for leukemia or hematopoietic stem cell/organ transplantation. symptoms such as fever that does not respond to broadspectrum antimicrobial drugs, cough, dyspnea, sputum, and bloody sputum/hemoptysis are observed. -laboratory findings: chest x-ray shows an infiltrative shadow (typically, a wedge shadow involving the pleura as the base). on thoracic ct, infiltrative and nodular shadows (with a halo sign in some cases) are observed. in the recovery phase of neutrophils, an air crescent sign is noted. an increase in the inflammatory marker such as the crp level, aspergillus galactomannan antigen-positive reactions, and an increase in the ( / )-b-d-glucan level are useful for diagnosis. however, neither the sensitivity nor specificity is sufficient. the results should be carefully evaluated. -causative microorganisms: aspergillus fumigatus is frequently detected, but, recently, an increase in the number of patients with non-fumigatus aspergillus-related ipa has been indicated. -specific condition: lesions are sometimes formed in the nasal sinus and brain; caution is needed. -early diagnosis: early treatment is important for successful treatment for this disease. ---drugs to be recommended---a study reported that, in a group in which vrcz was used for the initial treatment of ipa, the results of treatment were more favorable than in a group in which d-amph was used [ ] . furthermore, another study indicated that therapy with l-amb at mg/kg/day was safer than that at mg/kg/ day, although there was no significant difference in the clinical efficacy [ ] . cpfg, mcfg, and itcz also have anti-aspergillus activities, and can be used. it is important to consider different strategies in accordance with the appearance of the host's allergy or adverse events and interactions with drugs used to treat an underlying disease. b. chronic progressive pulmonary aspergillosis (cppa) ---executive summary----in japan, various disease types such as aspergilloma with infiltration and enlargement of an existing cavity are included. cppa includes various diseases such as chronic necrotizing pulmonary aspergillosis (cnpa), chronic cavitary pulmonary aspergillosis (ccpa), and chronic fibrosing pulmonary aspergillosis (cfpa). it refers to a series of syndrome for which the administration of antifungal drugs is essential. -treatment should be started with injection. if symptoms and findings are stabilized, injection should be switched to oral drugs. -initial treatment with mcfg or cpfg should be performed [ , ] (ai). -initial treatment with itcz, vrcz, or l-amb can also be selected in accordance with the host's underlying disease or drugs used to treat the underlying disease. -for maintenance therapy, itcz and vrcz oral preparations are recommended (aiii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in hosts with organic diseases such as a cavity or cystic disease of the lung or bronchus. symptoms such as fever, sputum, bloody sputum/ hemoptysis, and dyspnea are observed. -laboratory findings: chest x-ray and ct show an infiltrative shadow, enlargement of a cavity, thickening of the cavity wall/pleura, and a niveau in the cavity. there is an increase in the crp level in many patients. most patients are positive for anti-aspergillus precipitating antibody. neither aspergillus galactomannan antigen nor b-d-glucan is a clue to diagnosis. -causative microorganisms: a. fumigatus is frequently detected. non-fumigatus aspergillus-related cppa is also often observed. ---drugs to be recommended---a clinical study in japan indicated that there was no marked difference in the efficacy of treatment between mcfg-and vrcz-treated groups, whereas mcfg was safer [ ] . another study reported that there was no difference in treatment results between mcfg and cpfg [ ] . in the phase of severe symptoms such as fever and bloody sputum, treatment should be started using these injections. -pulmonary aspergilloma is classified into two types: simple and complex aspergilloma based on the grade of difficulty in resection. the former refers to aspergilloma formation in a focus with a thin wall, such as a cyst, without accessory lesions at the periphery. the latter refers to aspergilloma formation in a cavity derived from a strongly destructed existing structure of the lung, such as old pulmonary tuberculosis and bronchiectasis, with marked destructive lesions or pleural adhesion at the periphery of the cavity. ---drugs to be recommended---resection should be selected as a first choice. when resection is impossible, medical treatment can be considered. in preparation, such as l-amb, and -fc should be performed for weeks or more. subsequently, treatment should be continued using flcz or f-flcz. -in japan, cryptococcus gattii infection has also been reported. if possible, causative fungus must be isolated/identified. ---explanation---[characteristics of the disease] -symptoms: this disease is often asymptomatic, and is detected on a health checkup in many cases. -laboratory findings: chest x-ray and ct show solitary or multiple nodular and infiltrative shadows. -some cavities are observed in a lot of cases. there is no enhancement of the inflammatory marker in many cases, but glucuronoxylomannan antigen-positive reactions are detected. -causative microorganisms: this disease is caused by cryptococcus neoformans. recently, infection with c. gattii has been reported in vancouver, canada and the north area of the west coast of the united states of america; caution is needed. -c. gattii is primarily distributed in the tropical and subtropical zones. infection in humans has been considered to be rare. however, since , patients infected with c. gattii have been reported in the pacific coast of north america. even healthy adults are infected with c. gattii, and the mortality rate is high. ---drugs to be recommended---although there is no evidence regarding pulmonary cryptococcosis, flcz tablets, which have a potent activity against cryptococcus, are frequently selected when the patient's condition is stable in the absence of an underlying disease. azoles other than this drug can also be selected. [ ] (aii). -if the lesion is localized, resection should be considered. -combination therapy with an iron chelating agent and l-amb should be avoided [ ] (ai). ---explanation--- recently, an increase in the incidence of infection with cunninghamella has also been indicated. -specific condition: nasal/brain-type, dermal, and disseminated zygomycosis is observed. ---drugs to be recommended---currently, only amph preparations may be clinically useful for treating zygomycosis among antifungal drugs that are available in clinical practice in japan. as high-dose therapy must be started as early as possible, not d-amph but l-amb should be selected. -l-amb, intravenous drip, mg/kg/once a day precautions for the use of antifungal drugs (confirm the package inserts.) ➀ vrcz vision disorder, liver dysfunction, and neurological/ mental adverse events may occur. combination therapy with rfp, rbt, efavirenz, ritonavir, carbamazepine, long-acting barbiturate, pimozide, quinidine sulfate, ergot alkaloid, or triazolam is contraindicated. this drug is also contraindicated for pregnant women. as a rule, it is contraindicated for patients with a ccr of < ml/ min (injection only). as the blood concentration of vrcz may vary, tdm should be conducted. in patients with mild to moderate liver dysfunction, the dose should be regulated. ➁ itcz hepatopathy and congestive heart failure may occur. combination therapy with pimozide, quinidine, bepridil, simvastatin, triazolam, azelnidipine, ergotamine, nisoldipine, dihydroergotamine, vardenafil, eplerenone, blonanserin, sildenafil, tadalafil, aliskiren, dabigatran(itcz oral only), rivaroxaban, ergometrine, or methylergometrine is contraindicated. this drug is contraindicated for patients with severe liver diseases, pregnant women. patients with a ccr of < ml/min are also contraindicated (injection only). ➂ flcz hepatopathy and a prolongation of qt may occur. combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➃ f-flcz combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➄ l-amb adverse events such as nephropathy, hypopotassiumemia, and fever may occur. this drug is contraindicated during leukocyte transfusion. ➅ cpfg this drug is safe, but hepatopathy may occur. caution is needed for combination therapy with cyclosporin, tacrolimus, rfp, efavirenz, nevirapine, phenytoin, dexamethasone, or carbamazepine. ➆ mcfg this drug is safe, but hepatopathy may occur. ➇ -fc anorexia and myelopathy may occur. combination therapy with tegafur-/gimeracil-/oteracil potassium-containing drugs is contraindicated. even after the discontinuation of tegafur-/gimeracil-/oteracil potassium-containing drugs, combination therapy should be avoided within days. this drug is also contraindicated for pregnant women. ---drugs to be recommended---st combination drugs are used as a gold standard of pcp treatment. however, there have been a large number of patients in whom treatment was discontinued due to their side effects. recently, atovaquone also became available in japan as a second-choice drug. in patients with mild pcp, atovaquone tablets were as effective as st combination drugs. in those with moderate pcp, st combination drugs were more effective, but there was no significant difference due to a small number of subjects. in atovaquone-treated patients, the incidence of adverse events for which administration was discontinued was lower than in st combination drug-treated patients, suggesting that the tolerance is high [ ] . the administration period should be days in hivinfected patients and days in non-hiv-infected patients. a target daily dose of trimethoprim should be e mg/kg. -st combination drug, oral, to tablets/ times a day -st combination drug, intravenous drip, e mg as trimethoprim/ times a day (infused over e h) <> second choices -pentamidine, intravenous drip, mg/kg/once a day (infused over e h) -atovaquone oral suspension, ml ( mg as atovaquone)/twice a day for days (orally administered after meals) * adjuvant therapy in patients with a pao of < mmhg or a-ado of > mmhg in room air, one of the above drugs should be combined with a corticosteroid from the start of treatment. however, the dose may be reduced or administration may be discontinued in the early phase in accordance with symptoms. when the respiratory state is extremely unfavorable, pulse therapy should also be considered. prednisolone days e : oral, e mg/twice a day days e : oral, e mg/twice a day days e : oral, . e mg/twice a day ---precautions for each drug---(confirm the package inserts.) ➀ st combination drug (baktar tablets) fever, exanthema, digestive symptoms, hepatopathy, nephropathy, and blood disorder may occur. this drug may interact with methotrexate, sulfadoxine, pyrimethamine, diaphenylsulfone, sulfonyl amide/sulfonylurea oral drugs for diabetes, warfarin, phenytoin, cyclosporin, zidovudine, digoxin, tricyclic antidepressants, and lamivudine. this drug is contraindicated for neonates, low-birthweight infants, pregnant women, and patients with g- -pd deficiency. in patients with renal dysfunction, dose reduction must be considered. side effects such as hypoglycemia, hypotension, nephropathy, taste disorder, numbness of the tongue/lips, ventricular arrhythmia, exanthema, and fever may occur. combination therapy with zalcitabine, pfa, or amiodarone is contraindicated. this drug is contraindicated for patients with severe ventilatory disturbance. ➂ atovaquone nausea/vomiting, exanthema, and diarrhea may occur. this drug should be carefully administered to patients with severe kidney or liver dysfunction. this drug may interact with rfp, rbt, tetracycline, metoclopramide, zidovudine, acetaminophen, benzodiazepines, aciclovir, opioid analgesic drugs, cephalosporin antibiotics, antidiarrheal drugs/laxatives, and indinavir. h. cytomegalovirus (cmv) pneumonia ---executive summary----in the field of transplantation, preemptive treatment with gcv should be conducted through cmv antigenemia test monitoring. -the efficacy of preemptive treatment with vgcv or pfa is similar to that of gcv. -if a diagnosis of cmv pneumonia is made, treatment with gcv should be promptly started [ ] (aii). -vgcv and pfa are recognized as alternative drugs for gcv [ , ] (bii). -combination therapy with an antiviral drug and high-dose immunoglobulin should be performed [ ] (aiii). ---explanation--- ---antimicrobial drugs to be recommended---a first-choice drug for cmv pneumonia treatment is gcv, which has been frequently used. pfa has been used to treat cmv infection in aids patients, but experience on its use is limited in patients after hematopoietic stem cell transplantation. ( ) initial administration a first choice -gcv, intravenous drip, mg/kg (over h or more)/ every h for e weeks þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days <> second choices -pfa, intravenous drip, mg/kg (over h or more)/ times a day, every h for e weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days or -pfa, intravenous drip, mg/kg (over h or more)/ twice a day, every h for e weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, . e g/once a day for the first days ( ) maintenance administration a first choices -gcv, intravenous drip, mg/kg (over h or more)/once a day, days a week or -gcv, intravenous drip, mg/kg (over h or more)/once a day, days a week * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. <> second choice -pfa, intravenous drip, e mg/kg (over h or more)/once a day (in clinical practice in japan, there have been few case reports on once-a-day administration at mg/ kg as maintenance therapy. a dose exceeding mg/ kg should be avoided. for administration at mg/kg, twice-a-day administration at mg/kg is commonly selected.) * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. ---precautions for each drug---(confirm the package inserts.) ➀ gcv severe leukopenia, neutropenia, anemia, thrombopenia, pancytopenia, aplastic anemia, and bone marrow suppression may occur. an animal experiment showed that this drug induced transient or irreversible spermatogenic dysfunction and reduced fertility. in humans, this drug may cause spermatogenic dysfunction. an animal experiment demonstrated the teratogenicity, mutagenicity, and carcinogenicity of this drug. in the presence of renal hypofunction, it is necessary to regulate the dose. this drug is contraindicated for patients with marked bone marrow suppression (neutrophil count: < /mm or platelet count: < , /mm ) and pregnant women. it may interact with didanosine, zidovudine, ipm/cs, bone marrow-suppressing and kidney function-affecting drugs, zalcitabine, st combination drugs, cyclosporin, probenecid, and mycophenolate mofetil. ➁ vgcv this is a prodrug of gcv. ➂ pfa acute renal failure, shock, heart failure, thrombophlebitis, and convulsion may occur. it is necessary to regulate the dose in accordance with the kidney function. combination therapy with pentamidine is contraindicated. this drug is contraindicated for patients with a ccr of < . ml/min/kg. ---executive summary---for the treatment of community-acquired pneumonia in children, antimicrobial drugs should be selected, considering age and severity. ---explanation--- patients with acute respiratory infectious disease symptoms, such as fever, nasal discharge, pharyngeal pain, and cough, and the appearance of a new infiltrative shadow in the lung on imaging examinations such as chest x-ray and ct are regarded as having pneumonia [ ] . in patients with pneumonia, thoracic auscultation findings often include accessory murmurs and the attenuation of respiratory sounds. most patients with respiratory infectious diseases consult hospitals with fever and cough. the lesion site of the airway is estimated based on symptoms and physical findings (fig. ) [ ] . in addition to thoracic findings, it is necessary to check the presence or absence of dyspnea signs, such as tachypnea, nasal alar breathing, retractive breathing, shoulder breathing, orthopnea, groaning, and cyanosis. to consider the need of antimicrobial-drug administration and options of antimicrobial drugs, pneumonia is classified into three types: bacterial, viral, and atypical pneumonia based on causative microorganisms [ ] . [type and frequency of causative microorganisms] microorganisms that cause childhood community-acquired pneumonia differ among ages. according to the data on investigation of causative microorganisms based on lavage sputum culture in japan, bacterial and viral pneumonia is frequent in infants/children aged year or younger. in those aged e years, the incidences of bacterial, viral, and atypical pneumonia are similar. in those aged over years, the incidence of atypical pneumonia is the highest [ ] (fig. ) insufficient. however, a review reported similar findings [ ] ( table ) [ ] . in children, it is not easy to investigate causative microorganisms. in institutions in which it is impossible to investigate causative microorganisms, treatment must be performed based on the statistical frequency of causative microorganisms described below. however, in those in which investigation is possible, the etiology should be investigated if possible. [rules of antimicrobial drug therapy] it is important to improve the efficacy of treatment by accurately predicting causative microorganisms and selecting an appropriate antimicrobial drug and its administration method. whether or not an antimicrobial drug should be indicated must be comprehensively evaluated by differentiating bacterial, viral, and atypical pneumonia with reference to age, severity, clinical symptoms, physical findings, laboratory data, and x-ray findings [ ] . as a rule, a single antimicrobial drug should be selected. when the type of microorganisms that caused pneumonia is identified, an antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics. [clinical symptoms, physical findings] wet cough and tachypnea are frequently observed in children with bacterial pneumonia. the proportion of labored breathingfree patients is high in children with mycoplasma pneumonia [ , ] . auscultation findings include intermittent accessory murmurs (rales) regardless of the type of pneumonia. in children with mycoplasma pneumonia, the proportion of those in whom auscultation findings are not marked is significantly higher than in other groups. in children with chlamydia pneumonia, fever is mild, and cough is protracted. thus, clinical symptoms and physical findings show characteristics related to causative microorganisms, but it is difficult to identify causative microorganisms based on clinical symptoms and physical findings alone in individual patients [ e ] . concerning laboratory findings on admission in children with bacterial and viral pneumonia, there are significant differences in the leukocyte count, crp level, and erythrocyte sedimentation rate between the two groups (p < . ). however, measurements overlapping in about one-third of patients are presented [ ] (fig. ) . briefly, it is impossible to accurately differentiate bacterial from viral pneumonia based on inflammatory responses reflected by the leukocyte count, crp level, and erythrocyte sedimentation rate. mycoplasma pneumonia is characterized by increases in the crp level and erythrocyte sedimentation rate, but many patients show normal leukocyte counts or a slight decrease in this parameter. furthermore, it is difficult to differentiate mycoplasma from viral pneumonia based on laboratory data [ ] . [chest x-ray] chest x-ray findings show characteristics related to causative microorganisms to some degree, but it is difficult to identify causative microorganisms in individual patients [ ] . it is important to evaluate the severity of pneumonia, for selecting outpatient or hospital treatment and reviewing the necessity of antimicrobial drugs and route of administration (oral or intravenous). the classification of severity in the guidelines for the management of respiratory infectious diseases in children in japan is presented [ ] (table ) . causative microorganisms with respect to age in children with community-acquired pneumonia [ ] . the severity classification of childhood pneumonia has not been reached in japan or internationally. this should be examined in the future. [standards for outpatient/hospital treatment] as a rule, outpatient treatment should be performed in mildstatus patients evaluated according to the severity classification, and hospital treatment in mild-status patients with dehydration. in addition, it is necessary to determine admission when outpatient treatment does not lead to an improvement in symptoms or considering social adaptation [ ] (table ) . [initial antimicrobial drug therapy] when examining children with pneumonia, treatment must be started without any precise information about causative microorganism in many cases. basically, empiric therapy should be performed, considering the severity of pneumonia and causative microorganisms. -the type of causative microorganisms depends on age and severity. therefore, the necessity of antimicrobial drugs should be examined, and selected, considering age and severity. in addition, bacterial, viral, or atypical pneumonia should be differentiated, and comprehensively evaluated in reference to clinical symptoms, physical findings, laboratory findings, and xray findings [ ] . in particular, acute bronchitis/pneumonia related to intermediately susceptible h. influenzae (mic: mg/ml) can be managed with oral ampc or abpc intravenous injection therapies [ ] . recently, the number of abpc-susceptible strains has annually decreased [ , ] . the proportion of blnar strains (mic: mg/ table age-related distribution of microorganisms that cause pneumonia in children [ ] . immediately after birth to . inflammatory response on admission in children with pneumonia [ ] . ml or more) has increased, raising an issue with respect to drug selection. when the involvement of blnar strains is suspected, high-dose ampc or new oral cephems may be necessary at outpatient clinics [ ] . the efficacy of outpatient antimicrobial drugs for blnar strains, which will increase in the future, should be carefully monitored. concerning hospital treatment, the clinical effects of abpc intravenous injection for e days until the results of a susceptibility test were clarified were investigated, and approximately % of patients responded to this therapy. there was no exacerbation in any patient [ ] . in non-responders or patients in whom clinical effects are insufficient, it is necessary to switch the antimicrobial drug. pipc, ctx, and ctrx have stable antimicrobial activities. when reviewing the clinical effects of pipc on childhood bronchopulmonary infection, the response rate was %; the results were satisfactory [ ] . -concerning treatment for m. catarrhalis pneumonia, m. catarrhalis produces b-lactamase. however, when examining the clinical course, ampc is effective [ , ] . this is because the enzymatic activity of b-lactamase produced by m. catarrhalis is low [ ] . (table ) . [evaluation of the treatment response and administration period] the administration period of antimicrobial drugs is shown in table [ ] . classification of the severity of community-acquired pneumonia in children [ ] . moderate severe . increases in the number of resistant strains of s. pneumoniae and h. influenzae derived from respiratory infectious diseases [ ] . to treat community-acquired pneumonia, antimicrobial drugs should be administered for e days. the treatment response should be evaluated after e days. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days [ ] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients; therefore, it is difficult to establish standardized criteria. as m. pneumoniae and c. pneumoniae slowly proliferate, the treatment period is prolonged (table ). in patients infected with general bacteria, the administration of antimicrobial drugs can be discontinued days after pyretolysis [ ] . however, further antimicrobial drug therapy is necessary to treat s. aureus pneumonia. [management for non-responders to antimicrobial drugs] when there are no therapeutic effects of antimicrobial drugs on pneumonia, whether or not a diagnosis of pneumonia is correct should be initially investigated [ ] . the possibility of diseases other than pneumonia, with a pneumonia-like shadow, must be reviewed (table ). if it can be ruled out, whether or not the expected type of pathogenic microorganisms is correct should be examined. if the type of causative microorganisms is the same as expected, the possibility of resistant microorganisms should be considered. new therapeutic strategies should be devised carefully and promptly. when the condition further exacerbates despite treatment switching, additional examination should be conducted. [ ] . concepts regarding the diagnosis and treatment of childhood mycoplasma pneumonia [ ] . . as it is often difficult to make a diagnosis of mycoplasma pneumoniae infection based on serum antibody titer-positive findings in the acute stage alone, the mycoplasma pneumoniae nucleic acid identification test (lamp method) should be conducted to make a definitive diagnosis in the acute stage. . as first-choice drugs for mycoplasma pneumonia, macrolides should be used. . the effects of macrolides can be evaluated based on pyretolysis within e days after administration. . to treat pneumonia that does not respond to macrolides, the administration of tosufloxacin or tetracyclines should be considered if necessary. however, as a rule, tetracyclines are contraindicated for children aged years or younger. . these antimicrobial drugs should be administered in accordance with administration periods recommended for individual drugs. . in patients with severe pneumonia, systemic steroid therapy must be considered. however, easy steroid administration should be avoided. however, mycoplasma pneumoniae is not included in bacterial types for which this preparation may be indicated. ---drugs to be recommended--- a first choices ) cases in which there is no risk of resistant bacteria -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day ) cases in which infection with resistant bacteria is suspected i) two years or younger, ii) pretreatment with an antimicrobial drug (within weeks), iii) concomitant development of otitis media, iv) history of pneumonia/repeated otitis media -ampc, oral, e mg/kg/ times a day -cva/ampc ( : preparation), oral, . mg/ kg/twice a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day ) cases in which onset/recurrence/recrudescence is observed despite previous treatment ) -tbpm-pi, oral, e mg/kg/twice a day -tflx, oral, mg/kg/twice a day <> second choices -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day ( ) admission (moderate, general ward) a first choices -abpc, intravenous injection or drip, mg/kg/ times a day -pipc, intravenous injection or drip, mg/kg/ times a day -sbt/abpc, intravenous injection or drip, mg/kg/ times a day * when m. pneumoniae, chlamydia trachomatis, or c. pneumoniae infection is strongly suspected, one of the above regimens should be combined with a macrolide [with respect to the administration method/ dose, refer to the section "➁ six years or older---( ) outpatient clinic (mild)".]. <> second choices -ctx, intravenous injection or drip, mg/kg/ times a day -ctrx, intravenous injection or drip, e mg/kg/ once to twice a day ( e mg/kg/day) ( ) admission (severe, icu) -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -taz/pipc, intravenous drip, . mg/kg/ times a day * when legionellosis cannot be ruled out, one of the above regimens should be combined with a macrolide [with respect to the administration method/dose, refer to the section "➁ six years or older---( ) admission (moderate, general ward)".]. ➁ six years or older ( ) outpatient clinic (mild) a first choices -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day <> second choices -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day -mino, oral, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or nonresponders to other drugs.) ( a first choices ) cases in which bacterial pneumonia is suspected -abpc, intravenous injection or drip, mg/kg/ times a day -pipc, intravenous injection or drip, mg/kg/ times a day -sbt/abpc, intravenous injection or drip, mg/ kg/ times a day -ctx, intravenous injection or drip, mg/kg/ times a day -ctrx, intravenous injection or drip, e mg/ kg/once to twice a day ( e mg/kg/day) ) cases in which atypical pneumonia is suspected -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day -em, intravenous drip, mg/kg/ e times a day -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) ) cases in which it is impossible to differentiate bacterial from atypical pneumonia one drug each should be selected from choices ) and ), and combined. -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day for days -cam, oral, . mg/kg/twice a day -em, intravenous drip, mg/kg/ e times a day -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) ---executive summary---for the treatment of hospital-acquired pneumonia in children, antimicrobial drugs should be selected, considering severity and the involvement of resistant bacteria. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for communityacquired pneumonia (biii). hospital-acquired pneumonia is defined as pneumonia that newly develops h or more after admission. ventilator-associated pneumonia is defined as pneumonia that develops h or more after endotracheal intubation [ ] . these conditions may become severe due to an underlying disease, reduced immune capacity, or the deterioration of the general condition, and are caused by drugresistant microorganisms in many cases; treatment is difficult in most cases. not only microorganisms acquired in the community but also those existing in the hospital environment cause hospital-acquired pneumonia in children, as reported in the adult field. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause hospital-acquired pneumonia [ ] . in addition, not only general bacteria but also fungus and viruses sometimes cause hospital-acquired pneumonia in patients with immunodeficiency. among patients with nosocomial infection, causative microorganisms differ, and drug-resistant microorganisms are involved in many cases. in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, lavage or aspiration sputum culture should be conducted to investigate the etiology, if possible [ , ] . [rules of antimicrobial drug therapy] basically, empiric therapy should be performed, considering the severity of pneumonia, an underlying disease, and causative microorganisms. in particular, the involvement of drug-resistant microorganisms, such as mrsa, extended-spectrum b-lactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [ ] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. however, when complications such as severe immunodeficiency, pulmonary suppuration, lung abscess, and pleuritis are absent, antimicrobial drugs should be administered for days after pyretolysis ( e days) [ ] . considering an underlying disease or the immune state, flexible management must be performed. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days [ ] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . concerning multi-drug-resistant microorganisms, it is important to promote standard preventive strategies and those to control nosocomial infection, such as the prevention of droplet/contact infection, thoroughly. furthermore, oral care and devised postures (if there is no medical contraindication, the head should be lifted at e .) are necessary to prevent vap [ ] . in this article, the classification of severity (table , p. ) in the guidelines for the management of respiratory infectious diseases in children in japan was used [ ] (refer to the section " . pneumonia (children), . community-acquired pneumonia".). ---drugs to be recommended--- a first choices -caz, intravenous injection or drip, mg/kg/ times a day -czop, intravenous injection or drip, mg/kg/ e times a day -cpr, intravenous injection or drip, mg/kg/ e times a day if necessary, one of the above regimens should be combined with one of the following drugs: ) cases in which anaerobe infection is suspected (such as aspiration pneumonia) -cldm, intravenous drip, in children with immunodeficiency-/blood disease-related pneumonia, antimicrobial drugs should be selected, considering an underlying disease, the grade of immunodeficiency, and involvement of various causative microorganisms. initial antimicrobial drug therapy should be started by combining two drugs if necessary, considering various causative microorganisms, differing from that for community-acquired pneumonia (biii). [characteristics and classification of the disease] as immunodeficiency-/blood disease-related pneumonia in children often develops in hospitals, it has the characteristics of hospital-acquired pneumonia in many cases. it may become severe due to the patient's unfavorable conditions, such as an underlying disease, reduced immune capacity, and the deterioration of the general condition. even non-pathogenic microorganisms may cause pneumonia in many cases. furthermore, drug-resistant microorganisms often cause pneumonia; treatment is difficult in many cases [ , ] . to achieve multidisciplinary, comprehensive treatment to save children, it is necessary to cooperate with other special fields. [type and frequency of causative microorganisms] not only microorganisms acquired in the community in the presence of various immunodeficiency states but also nonpathogenic microorganisms existing in the hospital environment cause immunodeficiency-/blood disease-related pneumonia in children. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause this type of pneumonia. in addition, not only general bacteria but also fungus and viruses often cause this type of pneumonia. furthermore, drugresistant microorganisms are involved in many cases, as indicated for hospital-acquired pneumonia [ , ] . [type of immunodeficiency, causative microorganisms to be monitored, and precautions for diagnosis] ➀ humoral immunodeficiency: as bacterial opsonization and complement activation are affected, patients with humoral immunodeficiency are prone to be infected with general bacteria. among immunodeficiency patients with hyper-igm-emia, pneumocystis pneumonia should be considered in those with conditions related to cd ligand abnormalities. ➁ cellular immunodeficiency: in addition to infection with general bacteria, infection with intracellular parasitic bacteria, fungus, or protozoa may become severe, and be protracted. as the differentiation and induction of b and killer t cells by cd -positive lymphocytes are affected, the eradication of virus-infected cells is inhibited (table ) [ ] . ➂ neutrophil abnormalities: neutrophil abnormalities are classified into two types: neutropenia and neutrophil functional disorders. patients with a peripheral blood absolute neutrophil count (anc) of < /ml or those in whom the anc is estimated to reach < /ml within h are regarded as having neutropenia [ ] . of these, the risk is higher in patients with an anc of /ml or less in whom the period is estimated to exceed days. many patients with neutropenia do not show purulent sputum or abnormal findings on chest x-ray even in the presence of pneumonia. therefore, when fever persists, thoracic ct should be performed in the early stage. all microorganisms including general bacteria (gram-positive bacteria, gramnegative bacteria), fungus, and viruses may cause pneumonia. in particular, in addition to neutropenia early after homologous hematopoietic stem cell transplantation, infection-prone features associated with humoral/cellular immunodeficiency related to the administration of immunosuppressives persist over a long period. furthermore, the concomitant development of acute/chronic graft-versus-host disease (gvhd) is a risk factor for the onset of pneumonia. in addition, non-infectious pulmonary disorder related to drugs/radiation for pretreatment may occur, and it is important to differentiate it from infectious diseases (fig. ) [ , ] . a representative neutrophil functional disorder, chronic granulomatosis, induces active oxygen production disorder of neutrophils, affecting bactericidal actions. therefore, patients with this disorder are prone to be infected with non-h o -producing catalase-positive bacteria (s. aureus, k. pneumoniae, e. coli, candida spp., aspergillus spp.). ➃ complement deficiency: patients with complement deficiency are prone to be infected with bacteria with capsules, such as s. pneumoniae, h. influenzae (capsule strains), and neisseria meningitidis [ ] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, various cultures should be conducted to investigate the etiology, if possible. in addition, testing of various antigens, such as urinary s. pneumoniae/legionella antigens, b-dglucan, aspergillus antigen, cryptococcus antigen, candida antigen, and cytomegalovirus antigen, and tests using nucleic acid amplification methods, such as the pcr method, should be utilized, if possible. [rules of antimicrobial drug therapy] initial antimicrobial drug therapy should be performed, considering the severity of pneumonia and an underlying disease. for the treatment of immunodeficiency-/blood disease-related pneumonia in children, antimicrobial drug therapy should also be basically selected, considering causative microorganisms. as described for nosocomial infection, the involvement of drugresistant microorganisms, such as mrsa, extended-spectrum blactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. initial antimicrobial drug therapy should be started by combining two drugs, if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [ , ] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. in children, disease progression is often prompt, and the first evaluation should be performed after days in younger and severe-status children, and not after days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . monitoring culture of the airway is useful for treating immunodeficiency-/blood disease-related pneumonia in children [ ] . general preventive methods are presented in table [ ] . in addition, long-term low-dose macrolide therapy or the intermittent administration of b-lactams to prevent p. aeruginosa fixation is effective in some patients with chronic bronchitis [ , ] . in those with chronic granulomatosis, the oral administration of itcz ( e mg/kg/day, maximum: mg/day) or subcutaneous injection of ifn-g ( , domestic standard units/m , e times a week) is useful for preventing infection [ ] . in this article, the classification of severity (table , p. ) in the guidelines for the management of respiratory infectious diseases in children in japan was used (refer to the section " . pneumonia (children), . community-acquired pneumonia".). ---drugs to be recommended--- ( ) pneumonia related to mild immunodeficiency in the initial phase after admission -sbt/abpc, intravenous injection or drip, mg/kg/ e times a day -ctx, intravenous injection or drip, mg/kg/ e times a day -ctrx, intravenous injection or drip, mg/kg/twice a day ( ) pneumonia related to moderate/severe immunodeficiency -caz, intravenous injection or drip, mg/kg/ e times a day -czop, intravenous injection or drip, mg/kg/ e times a day -cpr, intravenous injection or drip, mg/kg/ e times a day in severe cases, -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -drpm, intravenous drip, mg/kg/ times a day -taz/pipc, intravenous drip, . mg/kg/ times a day -vcm, intravenous drip, mg/kg/ times a day -abk, intravenous drip, e mg/kg/once a day -teic, intravenous drip, mg/kg/every h, times, subsequently: e mg/kg/once a day when there is no response, -amk, intravenous drip, e . mg/kg/twice a day -tob, intravenous drip, . mg/kg/ times a day if necessary, an antifungal drug (mcfg) and st combination drug should be additionally administered. when aspergillus infection is suspected, treatment should be started with vrcz or l-amb instead of mcfg. in patients with cellular immunodeficiency, one of the above drugs should be combined with mcfg and an st combination drug in the early stage. in those with neutrophil abnormalities, one of the above drugs should be combined with mcfg in the early stage. when legionellosis cannot be ruled out in the severest cases, one of the above regimens should be combined with a macrolide (with respect to the administration method/dose, refer to the section " . community-acquired pneumonia---➁ six years or older---( ) admission (moderate, general ward)" (p. ).). in the presence of gcv resistance, the intravenous drip of pfa (foscarnet), mg/kg/ times a day should be performed. ---executive summary---in the treatment of neonatal pneumonia, antimicrobial drugs should be selected after differentiating congenital from acquired pneumonia. initial antimicrobial drug therapy should be started by combining two drugs, regarding the condition as severe systemic infection and considering various causative microorganisms. the administration method and dose should be selected based on the age (days) and birth weight (biii). [characteristics and classification of the disease] the incidence of neonatal pneumonia is not high. according to sakata, pneumonia occurred in only ( . %) of approximately , neonates who were admitted during a -year period [ ] . neonatal pneumonia is frequently observed as a portion of systemic infection represented by sepsis, and not as a single condition. it is classified into two types: congenital and acquired pneumonia [ ] . in most cases, neonatal pneumonia does not cause any typical symptoms such as fever or cough in comparison with infantile/ childhood pneumonia. the neonatal protective capacity against infection is physiologically immature, often leading to a severe condition. in the neonatal phase, artificial respiration management is performed in many cases, and ventilator-associated pneumonia (vap) is frequently observed [ e ]. in addition, many neonates are admitted to the neonatal intensive care unit (nicu) for a long period; therefore, the incidence of nosocomial infection is high. [type and frequency of causative microorganisms] several types of congenital pneumonia include transplacental infection, intrauterine infection related to aspiration of infected amniotic fluid on premature rupture, and birth canal infection with vaginal discharge on delivery. these types of congenital pneumonia are primarily caused by various viruses (cytomegalovirus, herpes simplex virus), bacteria (streptococcus agalactiae (gbs), e. coli, listeria monocytogenes), chlamydia, and fungus. the clinical onset of perinatal pneumonia is frequently observed immediately to days after birth [ ] . postnatal pneumonia usually develops weeks or more after birth. it is caused by viruses and bacteria. viral infection may occur when rs virus, parainfluenza virus, or adenovirus prevails in the ward. however, it rarely leads to the onset of pneumonia. frequent causative bacteria include s. aureus, e. coli, p. aeruginosa, acinetobacter spp., enterobacter spp., and legionella spp., which exist in the environment. these often cause nosocomial infection [ ] . in premature babies, gram staining of tracheal aspirate samples is useful for selecting antimicrobial drugs to be administered in the initial phase [ ] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, blood or bronchial lavage fluid culture should be conducted to investigate the etiology, if possible. [rules of antimicrobial drug therapy] as many neonates have systemic infection in an immunodeficiency state, the same administration method and dose as selected for the treatment of sepsis should be used [ ] . therefore, the severity of pneumonia is not considered. when bacterial pneumonia is suspected, combination therapy with abpc and ctx is commonly selected as initial treatment. considering l. monocytogenes infection, combination therapy with abpc is recommended. after causative microorganisms are identified, antimicrobial drugs should be selected with reference to their drug susceptibility. in cases of vap, antimicrobial drug options should be customized based on records on the drug susceptibility (antibiograms) of microorganisms that cause nosocomial infection at each institution. in neonates, there are marked age-related differences in the pharmacokinetics of antimicrobial drugs; therefore, these drugs must be carefully administered in the neonatal phase. concretely, the same dose as established in the field of pediatrics should be used, and the administration interval should be prolonged in accordance with age [ ] . in cases of severe asphyxia or acute renal failure, the administration interval must be further prolonged. it should be shortened with an improvement in the kidney function [ e ] . a consensus regarding the administration period of antimicrobial drugs has not been reached. however, the standard administration period is days. with respect to the administration method/dose, refer to the section "xi. doses for neonates". in neonates, disease progression is often prompt, and the treatment response should be evaluated after days, and not after days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [ ] . ---drugs to be recommended--- ---executive summary----pyothorax refers to a condition in which pus accumulates in the thoracic cavity. usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: < . (biii). -in patients with acute pyothorax related to community-acquired pneumonia, treatment should be performed in accordance with that for community-acquired pneumonia, considering microorganisms that cause community-acquired pneumonia, such as streptococcus pneumoniae (biii). -in patients with slowly progressing pyothorax, mixed infection with oral aerobes/anaerobes is frequently observed. combination therapy with pcg or abpc and cldm or mnz, which show anti-anaerobe activities, or therapy with a single antimicrobial drug with an anti-anaerobe activity, such as sbt/abpc, should be selected (biii). -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem, combination therapy with a fourthgeneration cephalosporin and cldm or mnz, and combination therapy with a quinolone and cldm or mnz should be considered, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, anaerobes, and acinetobacter (biii). -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them (aiii). -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, the use of aminoglycosides should be avoided as a rule (biii). -if a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be started, and drainage must be performed (aii). if possible, the attending physician should consult a surgeon in the early stage (aiii). -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary (biii). in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgical interventions such as thoracotomy or decortication is performed in some cases (biii). ---explanation--- [diagnosis] -pyothorax is defined as a condition in which pus accumulates in the thoracic cavity, but this definition has no diagnostic objectivity. for this reason, usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: < . [ , ] . [causative microorganisms] -pleural effusion appears in e % of patients with bacterial pneumonia, but leads to pyothorax in . e %. other etiological factors for pyothorax include surgery, trauma, and esophageal perforation. -microorganisms that cause pyothorax depend on its etiology and course. in the presence of bacterial pneumonia, pyothorax is caused by the same microorganisms as caused bacterial pneumonia. furthermore, acute pyothorax is frequently caused by s. pneumoniae and s. aureus. however, in many patients with chronic pyothorax, mixed infection primarily with anaerobes is involved. among anaerobes, fusobacterium spp. (especially fusobacterium nucleatum), prevotella spp., peptostreptococcus spp., and bacteroides spp. are frequently detected [ e ]. according to recent studies, the detection rate of the streptococcus anginosus group is high [ , ] . -in many cases, pyothorax with a relatively slow course is associated with m. tuberculosis. it must be considered that pulmonary lesions do not always concurrently exist with tuberculous pleuritis. [treatment] -no randomized comparative study regarding antimicrobial drug treatment for pyothorax has been conducted. an antimicrobial drug with an activity against microorganisms expected or obtained on culture should be selected and administered. in acuteonset patients in whom there is no risk of resistant bacteria, for example, those with pyothorax accompanying communityacquired pneumonia, antimicrobial drugs such as pcg and abpc should be initially selected, considering s. pneumoniae. these drugs simultaneously cover fusobacterium, peptostreptococcus, and the viridans streptococcus group. however, prevotella and bacteroides produce b-lactamase; therefore, when the results of culture are not obtained, combination therapy with antimicrobial drugs with activities against anaerobes, such as cldm and mnz, or therapy with such a single drug, such as sbt/abpc, should be selected. -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem should be selected as a first choice, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, and acinetobacter. a fourth-generation cephalosporin should be combined with cldm, or a quinolone should be combined with cldm or sbt/abpc. -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them. however, the culture of anaerobes is difficult, or is not conducted in some cases. therefore, this should be confirmed to the laboratory. when only one type of bacteria are detected on a culture test despite several types of bacteria detected on gram staining of pleural effusion, anaerobes must also be considered. -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, for pyothorax treatment, the use of aminoglycosides should be generally avoided [ e ]. [treatment period] -the pyothorax treatment period has not been established. however, when pneumonia promptly responds to treatment and thoracic drainage is successfully achieved in patients with pneumonia-related pyothorax, a treatment period of e days is required. in patients in whom drainage is unsuccessful, those with marked pleural thickening, or those with encapsulated/septum-like pyothorax, a treatment period of about weeks is often required. [treatments other than antimicrobial drug therapy] -after a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be promptly started, and drainage is necessary. when the pleural fluid is purulent and viscous in the absence of a multilocular pattern, chest-tube insertion is routinely performed. the position of insertion should be confirmed using thoracic ct within h after insertion. wozniak et al. performed multivariate analysis involving patients with pyothorax and indicated that failure in the first drainage was strongly correlated with mortality, suggesting the necessity of early consultation with surgeons [ ] . -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary. in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgerical interventions such as thoracotomy or decortication is performed in some cases [ ] . ---drugs to be recommended--- a first choice -sbt/abpc, intravenous drip, g/ e times a day <> second choices -pcg, intravenous drip, , , to , , units/ times a day -abpc, intravenous drip, g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day ➁ cases in which there is a risk of multi-drug-resistant bacteria a first choices -taz/pipc, intravenous drip, . g/ e times a day -ipm/cs, intravenous drip, . e g/ e times a day -mepm, intravenous drip, g/ e times a day -drpm, intravenous drip, . e g/ times a day <> second choices (i) -cfpm, intravenous drip, e g/ e times a day -czop, intravenous drip, e g/ e times a day -cpr, intravenous drip, e g/ e times a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -mnz, intravenous drip, mg/ times a day <> second choices (ii) -lvfx, intravenous drip, mg/once a day -cpfx, intravenous drip, mg/twice a day -pzfx, intravenous drip, to mg/twice a day þ one of the followings: -cldm, intravenous drip, mg/ e times a day -sbt/abpc, intravenous drip, g/ e times a day -mnz, intravenous drip, mg/ times a day * with respect to the risk of multi-drug-resistant bacteria, refer to the section " . hospital-acquired pneumonia" table (p. ). * in particular, mrsa infection must be considered in patients with nosocomial onset or the previous administration of antimicrobial drugs. when staphylococcus infection is suspected on gram staining of pleural effusion, an anti-mrsa drug should be used as an empiric therapy. if mssa is identified as causative bacteria, deescalation should be performed. . definitive therapy -antimicrobial drugs against causative microorganisms identified should be selected in accordance with the section " . ---executive summary---for the treatment of pyothorax in children, antimicrobial drugs should be administered after investigating the etiology using thoracentesis or blood culture, if possible. in addition to antimicrobial drug therapy, treatment for retention fluid (pleural effusion drainage, continuous drainage) must also be considered (biii). [characteristics and classification of the disease] pleuritis refers to inflammation of the pleura. fluid (pleural effusion) is retained in the pleural cavity. pleuritis is classified into types: fibrinous (dry) pleuritis, exudative (wet) pleuritis, and purulent pleuritis (pyothorax) based on conditions [ ] . on auscultation, the attenuation of respiratory sounds, as well as pleural friction rubs, are heard. percussion dullness is noted. in the chronic stage, localized pleural thickening in various shapes is observed. when pus is obtained on thoracentesis, a definitive diagnosis of pyothorax is made. when the protein level in nonpurulent pleural effusion is high, tuberculous pleuritis should be differentiated [ ] . [type and frequency of causative microorganisms] previously, pyothorax was associated with s. aureus in many cases. however, recently, such cases have been rare. according to data form a national survey in the former half of the 's, there were only a few patients with s. pneumoniae-or anaeroberelated pyothorax [ ] . in many cases, pleuritis follows m. pneumoniae-or virus-related pneumonia. decubitus-view imaging shows the retention of pleural effusion in approximately % of patients with mycoplasma pneumonia [ ] . if the drug susceptibility of causative microorganisms is clarified, it contributes to successful treatment. therefore, pleural effusion obtained by thoracentesis should be cultured to investigate the etiology, if possible. when bacterial infection-related pyothorax is suspected, the intravenous injection or drip of sbt/abpc, ctx, or ctrx should be selected in community-onset patients without an underlying disease. on the other hand, combination therapy with sbt/abpc (intravenous injection or drip) and cldm (intravenous drip) or carbapenem therapy (intravenous drip) should be started in those with an underlying disease or nosocomial onset, considering s. pneumoniae, anaerobes, h. influenzae, and s. aureus [ ] . based on the gram staining reactions of pleural effusion, causative microorganisms should be estimated, and antimicrobial drugs must be reviewed. if necessary, tuberculosis should also be investigated. the administration period of antimicrobial drugs must be longer than that for pneumonia. as the type of causative microorganisms, patient background, and state of retention-fluid drainage differ among individual patients, it is difficult to establish standardized criteria. however, target administration periods for s. pyogenes (gas)-, s. pneumoniae-, and s. aureus-related pyothorax are , , and days or more, respectively. the treatment response should be evaluated e days after the start of administration. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until causative microorganisms and their drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a targetfocused antimicrobial drug should be selected through deescalation, considering drug susceptibility and pharmacokinetics [ ] . for the treatment of pyothorax, treatment for retention fluid (pleural effusion drainage) is also a basic procedure in addition to antimicrobial drug therapy. if necessary, continuous drainage should be performed (table ) [ ] . if pleural thickening leads to underexpanded lung, decortication should be indicated. the widespread application of thoracoscopic surgery has facilitated minimally invasive surgery [ ] . ---drugs to be recommended--- ( ) community onset (without an underlying disease) -sbt/abpc, intravenous injection or drip, mg/kg/ times a day -ctx, intravenous injection or drip, mg/kg/ e times a day -ctrx, intravenous injection or drip, mg/kg/twice a day ( ) community-acquired infection (with an underlying disease), nosocomial onset -cldm, intravenous drip, mg/kg/ times a day þ -sbt/abpc, intravenous injection or drip, mg/kg/ times a day or one of the following drugs alone should be administered: -taz/pipc, intravenous drip, . mg/kg/ times a day -papm/bp, intravenous drip, mg/kg/ times a day -mepm, intravenous drip, mg/kg/ times a day -drpm, intravenous drip, mg/kg/ times a day . definitive therapy refer to the section " . community-acquired pneumonia--- . . definitive therapy" (p. ) or " . hospital-acquired pneumonia--- . . definitive therapy" (p. ). table indications for continuous drainage [ ] . ➀ cases in which pleural effusion obtained on thoracentesis is purulent ➁ cases in which clinical effects are not achieved by antimicrobial drug therapy alone (within h) ➂ cases in which retention fluid affects the respiratory function ---executive summary----as initial treatment, four drugs (inh, rfp, and pza þ eb or sm) should be administered for months. subsequently, as a rule, standard treatment (a), in which two drugs, inh and rfp, are administered for months, should be performed as maintenance treatment (ai). -when pza cannot be used for initial treatment for some reason, inh, rfp, and eb or sm, should be administered for months as initial treatment. subsequently, standard treatment (b), in which two drugs, inh and rfp, are administered for months, should be performed as maintenance treatment (aii). -in patients in whom chest x-ray shows a cavity on initial consultation to during initial treatment and the septum culture is still positive at the completion of initial treatment, maintenance treatment should be prolonged over months. in addition, the prolongation of maintenance treatment should also be considered in patients with severe tuberculosis, such as military tuberculosis and tuberculosis of the central nervous system, those with immune depression, and those with relapse of tuberculosis (aii). -for the treatment of latent tuberculosis infection, inh should be administered for or months (ai). when m. tuberculosis, as the source of infection, is resistant to inh, or when the oral administration of inh is difficult due to side effects, rfp should be used as a second-choice drug for or months (ai). ---explanation--- [ , ] . in particular, this method is named "standard treatment (a)" in the guidelines for the management of tuberculosis in in japan. in this article, this name is also used. -the secondary assessment of a study examining the efficacy of rifapentine and inh showed that factors for unsuccessful treatment/recurrence included a cavity on chest x-ray at the start of treatment and positive findings on culture at the completion of initial treatment for months [ ] . similarly, when the treatment period was extended from to months in patients with silicotuberculosis, in whom the unsuccessful treatment/recurrence rates are high, the recurrence rate decreased from to %. therefore, various guidelines recommend that maintenance treatment should be prolonged over months in patients with a cavity and those showing positive findings on septum culture at the completion of initial treatment [ , ] . -in , combs et al. compared the results of treatment between a group treated with inh, rfp, and pza for months and, then, with inh and rfp for months and that treated with inh and rfp for months, and reported that the efficacy and incidence of side effects were similar [ ] . based on such a study, the following regimen is recommended as standard treatment (b) in the guidelines for the management of tuberculosis in in japan: when pza cannot be used for some reason, inh, rfp, and eb or sm, are administered for the first months, and, subsequently, two drugs, inh and rfp, are administered for months. -patients who are infected with m. tuberculosis, but do not develop tuberculosis are regarded as having latent tuberculosis infection (ltbi). inh administration for ltbi decreases the incidence of tuberculosis by e % [ ] . previously, this was called preventive therapy, but is currently termed ltbi treatment. the decrease in the incidence of tuberculosis is correlated with compliance with inh, and more marked preventive effects may be achieved when compliance is higher [ e ]. in a study involving inh administration to ltbi patients with an old shadow on chest x-ray, this therapy inhibited the onset of tuberculosis in and % of patients treated for and months, respectively (there was no significant difference between the two groups) [ ] . based on the data, some studies recommended that the period of standard inh administration for ltbi should be months [ e ]. however, a consensus regarding an effective administration period ( or months) has not been reached from various aspects including the efficacy, compliance, expenses, and incidence of side effects. actually, the administration period should be determined based on compliance and the incidence of side effects. -in ltbi treatment, rfp should be used for or months as an alternative drug when the oral administration of inh is impossible for some reason. the preventive effects of rfp on the onset of tuberculosis in ltbi patients may be similar to those of inh. furthermore, the incidence of liver dysfunction is lower than that related to inh [ e ]. however, for the use of rfp, drug interactions must be considered. ---drugs to be recommended---a first choice -inh, oral, mg/kg/once a day (maximum: mg/ day) þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ pza, oral, mg/kg/once a day (maximum: mg/day) þ eb, oral, mg/kg/once a day (maximum: mg/day) or sm, intramuscular injection, mg/kg/once a day (maximum: mg/day)/ e times a week. * the above drugs should be administered for months, and, subsequently, two drugs, inh and rfp, should be administered for months. <> second choice -inh, oral, mg/kg/once a day (maximum: mg/ day) þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ eb, oral, mg/kg/once a day (maximum: mg/day) or sm, intramuscular injection, mg/kg/once a day (maximum: mg/day)/ e times a week. * the above drugs should be administered for months, and, subsequently, two drugs, inh and rfp, should be administered for months. [ ] . in japan, tanaka et al. conducted combination therapy with cam ( mg/kg) and eb/rfp/km in patients, and indicated that . % of those who underwent initial treatment became negative for mac [ ] . concerning cam, many studies have reported a correlation between the in vitro drug susceptibility and treatment response [ e ]. furthermore, a study indicated that azm was as effective as cam [ ] . -rbt, which was published in the drug price in nhi scheme in japan in , was also approved for tuberculosis and non-tuberculous mycobacterium infection, including pulmonary mac infection. the drug interactions of rbt are less marked than those of rfp, and rbt is used as a first-choice drug for disseminated mac infection in hiv-infected patients [ ] . on the other hand, rbt induces side effects such as uveitis. in elderly patients, in whom pulmonary mac infection frequently develops, various side effects, such as gastrointestinal disorder, make long-term therapy difficult [ ] . in addition, no study has indicated that rbt is more effective than rfp for pulmonary mac infection in non-hiv-infected patients. therefore, rfp should be selected as a first-choice drug for pulmonary mac infection in non-hiv-infected patients. -kobashi et al. divided patients with pulmonary mac infection into two groups: a group treated with cam, rfp, eb, and sm (intramuscularly injected at mg/kg times a week for months) and a group treated with saline, and conducted a randomized, double-blind, comparative study [ ] . in the sm-treated group, the rate at which the culture of sputum became negative was higher than in the salinetreated group ( . vs. . %, respectively). there has been no high-quality study demonstrating the usefulness of combination therapy with aminoglycosides other than their study. however, various guidelines recommend that combination therapy with sm, amk, or km should be performed for e months in the initial phase of treatment in patients with a cavity or severe nodular/bronchodilatation type infection based on experience [ , , ] . guidelines in the united states recommend sm or amk, and comment that no study has showed which of two drugs, sm and amk, is more effective, although sm has been more frequently used [ ] . in japan, sm or km is recommended [ ] . -based on the results of these studies, the non-tuberculous mycobacterium infection control committee, japanese society for tuberculosis recommends the following doses and administration methods for chemotherapy for pulmonary mac infection in the "opinions regarding chemotherapy for pulmonary non-tuberculous mycobacterium infection---revision in ": rfp: mg/kg (up to mg)/day, once a day, eb: mg/kg (up to mg)/day, once a day, cam: e mg/day ( e mg/kg), once a day or two divided doses ( mg: two divided doses), and sm or km: mg/kg or less (up to mg), intramuscularly injected or times a week [ ] . -a cooperative statement on non-tuberculous mycobacterium infection by the american thoracic society/infectious diseases society of america recommends therapy with cam at to mg/day or azm at mg/day, eb at mg/kg/day, and rfp at mg/kg/day (up to mg) for patients with a cavity or severe nodular/bronchodilatation type infection. in addition, it is recommended that combination therapy with sm or amk ( e mg/kg, e times a week, patients aged over years: mg or less) for e months in the initial phase should be considered [ , ] . -the treatment period is established as about year after the culture becomes negative in the above guidelines, but this is not based on evidence. in the guidelines for the management of non-tuberculous mycobacterium infection, which were published by the british thoracic society, the treatment period of pulmonary mac infection is established as years. in the future, an optimal treatment period should be investigated. ---drugs to be recommended----cam, oral, mg/ times a day or mg/twice a day þ rfp, oral, mg/kg/once a day (maximum: mg/ day, orally administered before meals as a rule) þ eb, oral, mg/kg/once a day (maximum: mg/day) * severe-status patients and those with cavity-type lesions in addition to the above regimen, the intramuscular injection of sm or km should be added. abscessus is sometimes susceptible to lzd, tgc (tigecycline), and ketolides, but it is unclear whether there is a correlation between the drug susceptibility and clinical effects [ , ] . -combination therapy with cam and several intravenous antimicrobial drugs (amk, cfx, and ipm/cs) may control the symptoms and progression of pulmonary infection with m. abscessus [ , ] . however, actually, hospitalization is required to administer these intravenous antimicrobials, and the administration period is limited to e months. subsequently, treatment with oral drugs is performed, but cam is the only reliable oral drug, as described above. on the other hand, monotherapy with cam should be avoided from the perspective of resistance induction. although some studies reported combination therapy with lzd or quinolones, its efficacy has not been established. -based on such a background, a combination of surgical resection of the lesion and combination chemotherapy is the only treatment that is expected to achieve the complete cure of pulmonary infection with m. abscessus in which the lesion is localized [ , , ] . ---drugs to be recommended--- a first choice based on the results of a drug susceptibility test, the following antimicrobial drugs should be combined: -cam, oral, mg/ times a day or mg/twice a day þ amk, intravenous drip, mg/kg/once a day þ ipm/cs, intravenous drip, . g/ times a day or g/ times a day * surgery must be considered. the treatment period should be at least months after culture becomes negative. ---executive summary----for the treatment of childhood tuberculosis, several drugs should be combined, and administered for a specific period (aii). -for the treatment of non-tuberculous mycobacterium infection, several drugs should be combined, and administered for a specific period. however, mycobacterium often resists treatment. if there is no treatment response, surgery must be considered (ciii). ---explanation--- [characteristics and classification of the disease] in japan, tuberculosis is still an important infectious disease. when encountering patients with chronic infection who do not respond to general antimicrobial drugs, tuberculosis should be considered for differential diagnosis. mycobacteria that can be cultured are classified into two types: m. tuberculosis complex and non-tuberculous mycobacteria (ntm) [ ] . m. tuberculosis is a major type of m. tuberculosis complex, and has a strong infectivity from humans to humans. childhood tuberculosis is classified into two types based on age [ ] . briefly, primary tuberculosis represented by hilar lymph node tuberculosis and meningitis, which develop following primary infection, is characteristic of infants and children. the interval from infection until onset is short, and the morbidity rate is high. in addition, this disease may lead to a severe condition. pulmonary/hilar lymph node tuberculosis in infants and children is asymptomatic, or the general condition is favorable even in the presence of fever or cough in many cases. when primary tuberculosis is detected based on dyspnea or an unfavorable general condition in addition to fever or cough, many infants/children have military tuberculosis or meningitis. on the other hand, secondary tuberculosis with a cavity lesion or nodular shadow in the lung field is frequent in junior high school students or older. symptoms such as cough, sputum, fever, and thoracic pain are often observed. in most children, the source of infection can be clarified through detailed peripheral contact screening at the time of onset. usually, the source of infection is clarified in / to / of children. it is often their fathers/mothers or grandfathers/grandmothers [ e ]. as childhood tuberculosis does not form a cavity in the lung field, the m. tuberculosis level in the focus is lower than in adults. in many cases, it is difficult to bacteriologically or histologically make a definitive diagnosis in comparison with adult tuberculosis. usually, it is possible to make a definitive diagnosis by comprehensively evaluating epidemiological/clinical information such as opportunities for the source of infection to contact with tuberculosis patients, tuberculin reaction-or quantiferon tb (qft)-based verification of infection, imaging findings suggestive of tuberculosis, such as chest x-ray findings, verification of m. tuberculosis from sputum or gastric juice, and individuals' resistance including the grade of bcg vaccination-acquired immunity and age, as well as by considering treatment responses in some cases. qft is a very useful testing method to quantitatively measure ifn-g and diagnose tuberculosis infection without being influenced by bcg. however, assessment in infants/children should be further examined in the future [ ] . when a definitive diagnosis of pulmonary tuberculosis cannot be made based on chest x-ray findings alone, thoracic ct, which facilitates the detailed evaluation of the presence or absence and extent of tuberculous lesions, is useful for diagnosis. furthermore, imaging findings of tuberculosis do not change in a short period in many cases. non-tuberculous mycobacterium belongs to mycobacterium, the same category as reported for m. tuberculosis. therefore, it is often detected as a mycobacterium-positive smear of sputum, that is, gaffky's positive reaction. initially, some patients are regarded as having infectious tuberculosis, and admitted to a tuberculosis ward. symptoms and imaging findings are also similar between nontuberculous mycobacterium-and m. tuberculosis-infected patients. unless detected bacteria are identified, or unless either gene is detected using the nucleic acid amplification method, it is difficult to differentiate the two types of bacteria. however, it is important to recognize that tuberculosis and non-tuberculous mycobacterium infection are different diseases [ , ] . the most important point is that non-tuberculous mycobacterium infection does not transmit from humans to humans, differing from tuberculosis, an infectious disease in humans. therefore, it is not necessary to isolate the patient, and, as a rule, patients requiring admission should be managed in a general ward. as there are no public hygieneassociated problems, it is not necessary to submit a report to a health center. [type and frequency of causative microorganisms] in japan, the number of patients with childhood tuberculosis has markedly decreased. the number of newly registered patients with tuberculosis decreased from , ( ) to ( ) in children aged e years [ e ]. however, a decrease in the incidence of smear-positive pulmonary tuberculosis, which is important as the source of infection, is not marked in great urban areas. we cannot conclude that the opportunity of infection in children is favorably decreasing; caution is needed. the number of patients who newly develop non-tuberculous mycobacterium infection in japan is estimated to be approximately . in the adult field, it accounts for about / of that of patients who newly develop tuberculosis. however, it is relatively low in children. approximately % of patients with non-tuberculous mycobacterium infection are infected with m. avium complex (m. avium and mycoboterium intracellulare, pulmonary mac infection), and approximately % are infected with m. cansasii. [rules of antimicrobial drug therapy] -the characteristics of antitubercular chemotherapy in children are that children are tolerable to a relatively high dose per body weight in comparison with adults with respect to pharmacokinetics, and that the incidence of side effects is low [ ] . in the pediatric field, -month treatment with inh, rfp, and pza for childhood pulmonary tuberculosis is internationally selected as standard chemotherapy: three drugs, inh, rfp, and pza, are administered every day for the first months, and inh and rfp every day for the subsequent months. when drug resistance is suspected, these drugs should be combined with sm or eb in the initial phase until the results of a resistance test are clarified. in patients with secondary tuberculosis, -drug combination therapy with inh, rfp, pza, and sm (or eb) should be initially performed. in addition, as a rule, follow-up must be continued for years after the completion of treatment [ e ]. on the other hand, drug resistance, referral to another hospital, and discontinued treatment are present among patients who drop out of treatment, although the number of such patients is small. it is necessary to support the resistance and continuation of treatment. in particular, recently, the number of patients in whom it is difficult to continue treatment has increased. potent compliance support must be considered in connection with direct observed therapy (dot) by health centers and welfare activities [ ] . side effects during treatment include liver dysfunction. however, if the maximum ast or alt levels are approximately , administration should be carefully continued without discontinuing treatment. if these levels exceed , treatment should be transiently discontinued, and additional administration at a low dose should be conducted after confirming the normalization of the liver function. the dose should be gradually increased. liver dysfunction requiring a change of treatment is not frequent. furthermore, there is an increase in the serum uric acid level, but continuous treatment leads to normalization. there have been few patients with arthralgia. -prevention of tuberculosis: to prevent the onset of tuberculosis in uninfected persons, bcg vaccination should be performed. concerning its efficacy, a consensus regarding its potent preventive effects on severe disseminated tuberculosis, such as tuberculous meningitis and military tuberculosis, has been reached. considering the importance of tuberculous meningitis prevention, bcg vaccination in the early phase of infancy ( e months after birth, or earlier in accordance with the state of peripheral tuberculosis prevalence) is still necessary in japan [ ] . -treatment for latent tuberculosis: to prevent the onset of tuberculosis in persons with a history of tuberculosis, treatment for latent tuberculosis (conventional chemoprevention) should be conducted. a large-scale controlled study reported that inh therapy decreased the incidence of tuberculosis by approximately e %. for drug administration, the risk of tuberculosis onset should be concretely and flexibly evaluated based on the tuberculin reaction, opportunity of infection, age, and state of bcg vaccination in individual patients [ ] . -treatment for non-tuberculous mycobacterium infection: nontuberculous mycobacterium infection is refractory despite combination therapy with antitubercular drugs. in particular, there is no evidence regarding treatment in children [ ] . the effects of monotherapy are weak, and monotherapy with cam may lead to the appearance of cam-resistant bacteria within a few months [ ] ; therefore, this therapy should be avoided. the responses of m. kansasii to antitubercular drugs are relatively favorable, and cure may be achieved. however, pulmonary mac infection is often resistant to treatment. if there is no response, surgery must be considered. recurrence after the completion of treatment is also often observed. ---drugs to be recommended--- * as the administration period is longer than that for tuberculosis patients, the development of vision disorder should be considered even at these doses. * if there is no response, surgery must be considered. ---explanation---acute bronchitis is characterized by cough that persists for days or more. in most cases, cough persists for e weeks, but spontaneously subsides [ , ] . sputum is present in some cases, but is absent in others. sputum may be purulent even when viral infection is etiologically involved. neither chest x-ray nor ct shows the appearance of a new abnormal shadow, differing from pneumonia. viruses . there is no evidence that infection with other bacteria directly causes acute bronchitis in adults without an underlying disease [ ] . however, in a study using the transtracheal aspiration method in japan, h. influenzae, s. pneumoniae, and m. catarrhalis were primarily isolated in patients diagnosed with bacterial acute bronchitis in the absence of a chronic lower respiratory infectious disease as an underlying disease [ ] . in cases of pertussis, cough persists particularly over a long period, and paroxysmal coughing, inspiratory whooping, and vomiting after coughing may occur [ ] . acute bronchitis caused by m. pneumoniae also induces severe, persistent cough. in cases of influenza, fever, headache, general malaise, and arthralgia are observed. furthermore, acute viral bronchitis may lead to acute bacterial exacerbation in patients with chronic respiratory lesions as underlying diseases; fever and an increase in the amount of purulent sputum are observed. as a rule, when an underlying disease or complication is absent, the routine administration of antimicrobial drugs for acute bronchitis is not recommended [ , ] . to control symptoms such as cough, symptomatic therapy should be performed if necessary. on the other hand, antimicrobial drug treatment with macrolides is indicated for patients with pertussis. treatment after the catarrhal period does not reduce the degree or duration of cough, but antimicrobial drugs are necessary to prevent infection to peripheral persons [ , ] . when performing antimicrobial drug treatment for acute bronchitis caused by m. pneumoniae or c. pneumoniae, macrolides should be selected as first-choice drugs. however, an increase in macrolide-resistant m. pneumoniae must be considered [ ] . in cases of influenza, anti-influenza therapy should be conducted within h after onset [ ] . in patients with underlying diseases or elderly persons with complications, bacterial (e.g., s. pneumoniae) infection may occur following viral infection, although this is not frequent in healthy adults. when acute bronchitis related to bacterial infection, including secondary infection, is strongly suspected based on cough/sputum, fever, leukocytosis, or findings suggestive of the presence of causative microorganisms on gram staining of sputum despite the absence of a new infiltrative shadow on chest x-ray, antimicrobial drug treatment is considered in accordance with treatment for community-acquired bacterial pneumonia [ , ] . ---drugs to be recommended--- when there are no complications such as chronic respiratory diseases, the administration of antimicrobial drugs for acute bronchitis are not recommended as a rule (with respect to the selection of antimicrobial drugs for acute bronchitis complicated by chronic respiratory diseases with secondary bacterial infection, refer to the section " . miller & jones classification of purulent sputum. m : saliva, complete mucous sputum m : mucous sputum containing a small volume of purulent sputum p : sputum in which the purulent area comprises / or smaller p : sputum in which the purulent area comprises / to / p : sputum in which the purulent area comprises / or greater fever and shortness of breath, in addition to bacterial infection-related respiratory symptoms, such as increases in the frequency of cough, volume of purulent sputum, and degree of purulence, from the chronic, stable conditions of underlying diseases, such as copd, bronchiectasis, and old pulmonary tuberculosis. concerning laboratory data, inflammatory responses involving the leukocyte count and crp level are enhanced, and pao is often reduced on blood gas analysis. [imaging findings] imaging findings are necessary to differentiate chronic respiratory disease-related airway infection from pneumonia. the absence of a shadow must be confirmed. ct should also be performed to evaluate underlying diseases such as pulmonary emphysema and bronchiectasis. [estimation of causative microorganisms and gram staining] it is possible to collect sputum in many patients. gram staining is useful for predicting causative microorganisms or differentiating respiratory tract infection in the presence of chronic respiratory disease from persistent infection. according to a study, the tone of sputum suggests the presence of pathogenic microorganisms rather than the degree of purulence; macroscopic examination is also necessary [ ] . sputum involves much information, and is the most important sample. samples should be collected before the administration of antimicrobial drugs. those collected on waking-up early in the morning are ideal. to evaluate the degree of sputum purulence, the miller & jones classification [ ] (table ) is used, but, if samples are evaluated as p or higher, causative microorganisms may be predicted using gram staining. on gram staining, an area where the number of inflammatory cells is large should be initially searched at a low magnification, and detailed observation should be conducted at a high magnification. before the administration of antimicrobial drugs, sputum should always be submitted for a susceptibility test. as causative microorganisms, h. influenzae, p. aeruginosa, m. catarrhalis, and s. pneumoniae are frequently detected. persistent infection with p. aeruginosa is often observed, but it must be differentiated from acute exacerbation based on clinical symptoms and laboratory data. in addition, s. aureus and k. pneumoniae should be considered [ ] . the involvement of atypical pathogens such as c. pneumoniae or mixed infection with viruses and bacteria must also be considered. [treatment] the purpose of treatment is to reduce clinical symptoms, prevent recurrence, prolong the interval until subsequent exacerbation, and inhibit lung tissue damage. the administration of appropriate antimicrobial drugs relieves clinical symptoms, and maintains the respiratory function [ ] . on the other hand, inappropriate antimicrobial drugs may deteriorate the prognosis, inducing recurrence. in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced [ , ] . several studies have reported that new quinolones are more useful than b-lactams [ , e ] . respiratory quinolones have potent antimicrobial activities against all types of causative microorganisms, and against resistant bacteria [ e ]. concerning the administration period, a study indicated that the efficacy of administration for days was similar to that for days, and that the former was safer than the latter. the administration period should be shortened [ ] . ---drugs to be recommended--a. empiric therapy internationally, some studies have supported the usefulness of b-lactams [ , ] . however, in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced, and p. aeruginosa is also sometimes isolated. therefore, the use of b-lactams and macrolides is limited to patients without risk factors. an international comparative study reported that the efficacy of azm sustained-release preparations was similar to that of new quinolones [ ] . however, in japan, the long-term administration of macrolides is performed in many patients; therefore, circumstances differ. beta-lactamase-producing strains are detected in approximately e % of h. influenzae strains. beta-lactamasenegative, ampicillin-resistant (blnar) strains account for approximately %. therefore, when drug susceptibility is unclear, new quinolones should be selected as first-choice oral antimicrobial drugs. if drug susceptibility is clarified, they should be switched to effective and narrow-spectrum drugs. as injection, penicillins should be initially selected, followed by b-lactamase inhibitor-containing penicillins, carbapenems, and new quinolones. ➁ m. catarrhalis beta-lactamase-producing strains account for % of m. catarrhalis strains. as oral antimicrobial drugs, macrolides should be initially selected, followed by b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, and new quinolones. as injection, b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, new quinolones, or carbapenems should be selected. ➂ p. aeruginosa as oral drugs, new quinolones should be selected. as injection, anti-p. aeruginosa penicillins, cephems, monobactums, carbapenems, or new quinolones should be selected. as the drug susceptibility of this type of bacteria markedly differs among strains, drugs should be selected based on the results of culture tests. ➃ s. pneumoniae as oral drugs, penicillins should be initially selected, followed by new quinolones. in patients with a risk of resistant bacteria, respiratory quinolones such as lvfx and grnx should be selected. as injection, penicillins or ctrx should be selected, but carbapenems must be considered in severestatus patients. ---explanation---[characteristics/classification of the disease] dpb is a chronic inflammatory disease of the respiratory tract, which is frequently observed in east asians including japanese. there is no gender difference, and this disease frequently develops in persons aged e years. it is often detected in patients with a history of chronic sinusitis or in those with the concomitant development of chronic sinusitis. this disease is classified as the category of sinobronchial syndrome. [symptoms] the most typical symptoms of dpb are persistent cough and purulent sputum. symptoms such as exertional shortness of breath and dyspnea appear in accordance with disease progression. in patients with a complication of chronic sinusitis, purulent nasal discharge and nasal obstruction are observed. chest x-ray shows pulmonary overexpansion or a diffuse scattered nodular shadow. thoracic hrct reveals a diffuse centrilobular nodular shadow. furthermore, obstructive respiratory dysfunction, hypoxemia, and an increase in the cold agglutinin value ( -fold or more on the hemagglutination method) are observed. [type and frequency of causative microorganisms] in patients with dpb, persistent respiratory tract infection with h. influenzae, s. pneumoniae, or m. catarrhalis is often observed. however, the incidence of persistent infection with p. aeruginosa increases with progression. [long-term macrolide therapy] previously, the prognosis of dpb was unfavorable; respiratory failure gradually progressed through repeated acute exacerbation related to respiratory tract infection, leading to a fatal outcome; however, the prognosis of dpb has been markedly improved since long-term macrolide therapy with low-dose administration of em or other -membered ring macrolides was established [ e ]. early diagnosis/treatment have facilitated the complete cure of dpb. therefore, if once a diagnosis of dpb is made, long-term macrolide therapy should be started promptly. ---drugs to be recommended---➀ persistent infection -the oral administration of em at e mg/day should be continued for months to be evaluated its clinical effects [ ] . -in many cases, an improvement in symptoms (such as a decrease in the volume of sputum) will be achieved within e months after the start of administration. -in addition, an improvement in imaging findings or the respiratory function will be achieved after e months of treatment. croup syndrome is characterized by acute laryngeal stenosisassociated respiratory disturbance such as barking cough, hoarseness, and inspiratory stridor. most lesions involve not only the larynx but also the trachea/bronchus. this disease is sometimes called laryngotracheobronchitis [ ] . etiological factors are classified into two types: infectious and non-infectious (allergy-/ foreign body-related) factors [ , ] . the incidence of infectious croup syndrome is high in infants/children aged monthse years [ , ] . [type and frequency of causative microorganisms] croup syndrome is primarily caused by viruses. parainfluenza virus type is the most common virus [ ] . in addition, parainfluenza virus type / , influenza a/b virus, rs virus, human metapneumovirus, coronavirus, adenovirus, and measles virus are relatively frequently isolated [ , ] . [rules of antimicrobial drug therapy] in most cases, croup syndrome is caused by viruses, and antimicrobial drugs are not necessary. therefore, no study has evaluated the efficacy of antimicrobial drugs in patients with croup syndrome. there are no treatment guidelines regarding croup syndrome in which antimicrobial drugs are recommended [ , ] . ---executive summary---bronchiolitis is caused by viruses, and the administration of antimicrobial drugs is not necessary (ai). ---explanation--- bronchiolitis is an acute, inflammatory, obstructive disease involving the bronchiole. narrowing of the bronchiolar lumen related to mucosal epithelial injury, inflammatory-cell infiltration, interstitial edema, or an increase in mucus secretion causes air trapping in the peripheral respiratory tract, leading to obstructive respiratory disorder. this disease frequently develops in children aged years or younger. however, infants aged months or younger account for % or more [ ] . [type and frequency of causative microorganisms] bronchiolitis is primarily caused by viruses. rs virus accounts for e %. in addition, parainfluenza virus, human metapneumovirus, adenovirus, and influenza virus are relatively frequently isolated [ e ]. [rules of antimicrobial drug therapy] in most cases, bronchiolitis is caused by viruses, and antimicrobial drugs are not necessary. basic treatment is symptomatic therapy. in double-blind comparative studies involving abpc and non-treated groups [ ] , azm and non-treated groups [ ] , and abpc intravenous injection/oral em and non-treated groups [ ] , respectively, there were no significant differences in the admission period or symptom improvement. however, a small-scale doubleblind comparative study reported that the interval until recovery in the cam-treated group was shorter than in the non-treated group [ ] . according to another study, the incidence of secondary bacterial infection during the course of rs virus-related bronchiolitis was . %, and there was no difference between antimicrobial drug-treated and non-treated groups [ ] . therefore, it is not necessary to administer antimicrobial drugs to children with bronchiolitis for routine treatment or the prevention of secondary bacterial infection. however, follow-up must be carefully continued during the course of bronchiolitis. when a diagnosis of secondary bacterial infection-related pneumonia or otitis media is made, antimicrobial drug therapy should be started. ---executive summary---bacterial tracheitis is a bacterial disease with the rapid progression of dyspnea. if symptoms are progressive, antimicrobial drugs should be used even when a definitive diagnosis is not made (aiii). ---explanation--- fever and croup syndrome-like cough/stridor initially appear, and respiratory disorder rapidly progresses, but there is no specific posture, salivation, or dysphagia, which are characteristic of acute epiglottitis. a definitive diagnosis can be made based on characteristic clinical features and purulent secretion in the respiratory tract. in some cases, a lateral view of the larynx on x-ray shows stenosis below the larynx [ ] . this disease frequently develops in children aged e years [ ] . [type and frequency of causative microorganisms] s. aureus-related tracheitis accounts for approximately %, followed by that related to m. catarrhalis, h. influenzae, streptococcus pneumoniae, and streptococcus pyogenes [ e ]. mixed infection with viruses and bacteria is frequent, and parainfluenza virus type i [ ] and influenza a virus [ ] are often detected. [rules of antimicrobial drug therapy] antimicrobial drugs should be intravenously administered for the following reasons: this disease rapidly progresses, and oral administration is difficult in many cases. as empiric therapy, combination therapy with vcm, which may be effective for infection with s. aureus (including mrsa), and third-generation cephems (ctrx, ctx), which have potent antimicrobial activities against m. catarrhalis, h. influenzae, s. pneumoniae, and s. pyogenes, should be performed. the administration period is e days [ ] . ---drugs to be recommended---refer to the section " . pneumonia (children)---drugs to be recommended . definitive therapy---" (p. ). ---executive summary---acute bronchitis is primarily caused by viruses, and the necessity of antimicrobial drug administration is low (ai). when acute bronchitis is caused by m. pneumoniae, c. pneumoniae, or b. pertussis, antimicrobial drugs should be administered if necessary (aiii). secondary infection with s. pneumoniae or h. influenzae may occur, although its incidence is unclear. therefore, when there is no improvement, the administration of antimicrobial drugs should be considered (aiii). ---explanation---[characteristics and classification of the disease] bronchitis causes symptoms such as cough, fever, and general malaise. various causative microorganisms induce inflammation of the epithelial tracheobronchial tissue, leading to the onset of bronchitis. clinically, there are no special findings on auscultation, or only rough respiratory sounds (intermittent accessory murmurs) are heard. chest x-ray does not also show any marked infiltrative shadow. usually, patients in whom the interval after onset is less than weeks are regarded as having acute bronchitis [ ] . however, bronchitis diagnosed in japan slightly differs from that in europe and the united states. the latter primarily causes persistent cough. in japan, patients in whom there are no findings on chest x-ray despite clinical signs of pneumonia or those in whom chest x-ray is not performed are often diagnosed with bronchitis; the disease entity must be arranged. [type and frequency of causative microorganisms] viruses, such as rhinovirus, influenza virus, rs virus, adenovirus, parainfluenza virus, human metapneumovirus, and human bocavirus, account for % of causative microorganisms. m. pneumoniae, c. pneumoniae, and b. pertussis also cause bronchitis, although such cases are relatively rare [ , ] . [rules of antimicrobial drug therapy] acute bronchitis is primarily caused by viruses, and the administration of antimicrobial drugs is not necessary. a metaanalysis compared adults to whom antimicrobial drugs were administered for bronchitis treatment with non-treated adults, and indicated that there was no difference in the efficacy [ ] . few reports on clinical studies involving children have been published, and the scale is small; objective data are insufficient, but no study has reported that antimicrobial drugs are effective [ e ]. however, if secondary bacterial infection following viral infection causes fever, purulent sputum, leukocytosis, or an increase in the crp level, antimicrobial drugs should be administered, considering s. pneumoniae and h. influenzae. other indications for antimicrobial drug administration include m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis with protracted cough. as m. pneumoniae-or c. pneumoniae-related bronchitis tends to show spontaneous cure, the administration of antimicrobial drugs is not always necessary, but the necessity of administration should be evaluated, considering the severity of symptoms and course (with respect to indications and administration methods, refer to the section " . pneumonia (children)".). first-choice drugs for m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis are macrolides. in patients with b. pertussis-related bronchitis, antimicrobial drugs relieve symptoms only during the catarrhal period, but, if b. pertussis-related bronchitis is suspected based on clinical symptoms, previous vaccination, lymphocyte-predominant leukocytosis, an anti-pt antibody titer, and lamp findings, antimicrobial drugs should be used. however, -membered ring macrolides are ineffective for b. pertussis-related bronchitis. symptoms are similar to those of pneumonia, but, when chest x-ray does not show any abnormalities, or, when it is impossible to strictly differentiate bronchitis from pneumonia due to difficulty in chest x-ray, treatment should be performed in accordance with pneumonia. ---drugs to be recommended--- secondary bacterial infection after viral infection (cases in which fever, purulent sputum, leukocytosis, or an increase in the crp level is observed) a first choices -ampc, oral, e mg/kg/ times a day -sbtpc, oral, mg/kg/ times a day -cdtr-pi, oral, mg/kg/ times a day -cfpn-pi, oral, mg/kg/ times a day -cftm-pi, oral, mg/kg/ times a day <> second choices -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day . definitive therapy ➀ b. pertussis -em, oral, e mg/kg/ times a day -cam, oral, . mg/kg/twice a day -azm, oral, mg/kg/once a day, days ➁ m. pneumoniae ▪ macrolide-sensitive strains -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day ▪ macrolide-resistant strains -mino, oral or intravenous drip, e mg/kg/twice a day (in children aged years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders.) -tflx, oral, mg/kg/twice a day (administration is limited to children aged years or younger in whom mino cannot be used.) ➂ chlamydia (c. pneumoniae, c. psittaci, c. trachomatis) -em, oral, e mg/kg/ times a day -azm, oral, mg/kg/once a day, days -cam, oral, . mg/kg/twice a day . influenza ---executive summary----both m protein inhibitors and neuraminidase inhibitors (nais) are commercially available as anti-influenza drugs as of july . -influenza viruses a (h n ) and a (h n ) pdm (seasonal influenza) have been reported to be resistant to amantadine, an m protein inhibitor which can be used in japan. the use of this drug as an anti-influenza drug should be avoided for a while [ , ] . -during the influenza outbreak period, anti-influenza therapy should be promptly started based on a clinical diagnosis even when patients with influenza-like symptoms show negative results on a rapid diagnosis kits (because influenza cannot be completely ruled out) [ ] (ai). -nais significantly improve influenza survival, and nai administration within days after onset significantly reduces the rate at which the condition becomes severe [ , ] (ai). -currently, the following nais can be selected in japan. during the outbreak period, an appropriate drug should be selected based on the patient background and latest information on a prevalent influenza strain: -oseltamivir (oral), efficacy: a (h n ) pdm , a (h n ), b, resistance: h y mutant -zanamivir (inhalation), efficacy: type a/b -laninamivir (inhalation), efficacy: type a/b -peramivir (intravenous drip), efficacy: type a/b ---drugs to be recommended---there is no meta-analysis of anti-influenza drugs other than oseltamivir and zanamivir as of july . however, it has been shown that the early introduction of anti-influenza therapy for influenza significantly inhibits not only the mortality and admission rates but also the incidences of influenza-associated pneumonia, otitis media, and ischemic heart disease in comparison with symptomatic therapy. nais such as laninamivir and peramivir have also been confirmed to be as effective as oseltamivir at the time of development [ ] (ai). <>outpatient treatment -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -zanamivir, inhalation, mg/twice a day, days -laninamivir, inhalation, mg/single dose -peramivir, intravenous drip, mg/single dose <>hospital treatment ➀ patients with severe, life-threatening influenza in patients with severe, life-threatening influenza requiring admission, respiratory failure or encephalopathy is present. in either case, the complication must be treated, but, as a rule, nais should be introduced within h after the onset of influenza to obtain their effects. -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -peramivir, intravenous drip, mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➁ non-life-threatening influenza patients with pneumonia -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -peramivir, intravenous drip, mg ( mg for patients in whom the condition may become severe)/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➂ non-life-threatening influenza patients without pneumonia -oseltamivir, oral, mg/twice a day, days (as a rule, administration to children/adolescents aged e years should be avoided.) -zanamivir, inhalation, mg/twice a day, days -laninamivir, inhalation, mg/single dose -peramivir, intravenous drip, mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) * patients with a (h n ) -basic treatment is the early administration of anti-influenza drugs [ ] . -according to an article, there were no significant differences in the viral level or mortality rate days after the start of administration between double-and standard-dose oseltamivir therapies [ ] . however, treatment was not started within h after onset in all patients in the article. ---executive summary---influenza is caused by influenza virus. antimicrobial drugs are not necessary. it is recommended that neuraminidase inhibitors should be administered within h after onset (ai). if pneumonia or otitis media occurs through secondary bacterial infection, the administration of antimicrobial drugs must be considered (biii). ---explanation---[characteristics and classification of the disease] influenza often appears with sudden fever and shivering/ headache/general malaise/muscular pain/dry cough, followed by marked respiratory or digestive symptoms. in underlying diseasefree children, recovery is achieved after e days [ , ] . however, during the outbreak period, even underlying disease-free children are often admitted with serious symptoms such as encephalopathy, myocarditis, and pneumonia requiring artificial respiration. although the outbreak period is from december until march every year, outbreaks are observed in the summer in some areas [ , ] . [type and frequency of causative microorganisms] since influenza a (h n ) pdm prevailed in , the outbreaks of types of influenza, a (h n ) pdm , a (h n ), and b, have been repeated. their incidences differ among years. [rules of antimicrobial drug therapy] no antimicrobial drug is indicated for influenza. it is recommended that neuraminidase inhibitors should be administered within h after onset [ ] . the doses of neuraminidase inhibitors approved in japan are presented in table . during the course of influenza, bacterial infection such as pneumonia and otitis media may occur. previously, the incidence of secondary bacterial infection in children exceeded % [ ] , but it has been % or less since neuraminidase inhibitors were developed [ , ] . however, bacterial infection is observed in e % of severe-status patients requiring intensive care [ , ] . streptococcus pneumoniae, s. aureus, and h. influenzae are frequently detected as causative microorganisms [ , , , ] . no study has demonstrated that the prophylactic administration of antimicrobial drugs at the onset of influenza prevents secondary bacterial infection. among children with influenza, antimicrobial drug therapy should be considered in those in whom signs of pneumonia or otitis media do not subside despite the administration of a neuraminidase inhibitor. . parasitic diseases of the respiratory system ---executive summary----parasitic diseases are widely distributed throughout the world. in addition to domestic infection, japanese travelers may be infected in overseas endemic areas. furthermore, parasitic diseases always considered in foreign patients from endemic areas [ ] (biv). -when peripheral blood eosinophilia is observed in addition to respiratory symptoms and abnormal findings of the chest imaging, examinations should be performed to differentiate parasitic diseases [ ] (biv). -parasitic diseases of the respiratory system are caused by paragonimus spp., ascaris lumbricoides, ancylostoma duodenale, necator americanus, strongyloides stercoralis, toxocara canis, toxocara cati, wuchereria bancrofti, brugia malayi, or dirofilaria immitis. -there are two major diagnostic tests for parasitic diseases: ( ) detection of parasite eggs or larvae in sputum or stools, and ( ) detection of parasite-specific antibodies using serum or pleural effusion samples (immunodiagnosis). -an anti-parasite antibody screening test against species of parasite is commercially available; paragonimus spp., strongyloides sp., t. canis, d. immitis, ascaris suum, anisakis simplex, gnathostoma spp., fasciola hepatica, clonorchis sinensis, spirometra erinaceieuropaei, and cysticercus cellulosae. -diagnosis/treatment consultations regarding parasitic diseases by the japanese society of parasitology are available (as of ). refer to the homepage (http://jsp.tm.nagasaki-u.ac.jp). ---explanation----paragonimus spp. several paragonimus species are known to cause human infection. paragonimus westermani and p. miyazakii are distributed in japan. infection occurs through the consumption of freshwater crabs (intermediate host: eriocheir japonica, e. sinensis, geothelphusa dehaani) or wild boars (paratenic host:sus scrofa) contaminated with metacercaria (infective larvae) as a raw or insufficiently cooked food [ ] . typical symptoms are cough, sputum, thoracic pain, and exertional dyspnea. in patients with such symptoms, the presence of peripheral blood eosinophilia suggests this disease. in many cases, a diagnosis is made based on the peripheral blood eosinophilia, a history taking of food and table standard doses of neuraminidase inhibitors in children. oseltamivir a oral mg/kg, twice a day, days (the use of this drug should be avoided in children/adolescents aged e years.) zanamivir b inhalation mg, twice a day, days laninamivir inhalation years or older: mg years or younger: mg, single dose peramivir intravenous drip mg/kg, once a day a the preventive administration of this drug at mg/kg (once a day) for days was approved, but is not covered by health insurance. b the preventive administration of this drug at mg (once a day) for days was approved, but is not covered by health insurance. immunodiagnosis. some of paragonimiasis patients are asymptomatic, and the presence of lung lesions is detected on a health checkup. extrapulmonary paragonimiasis such as cutaneous and cerebral paragonimiasis are classically known form of the ectopic infection with paragonimus spp. in cases of cutaneous paragonimiasis, a slowly moving nodular lesion in the subcutaneous tissue is a characteristic symptom. the worms may migrate through mediastinal soft tissues to the brain, causing eosinophilic meningitis or cerebral paragonimiasis in some cases [ ] . since paragonimus spp. is widely distributed around the world, japanese travel to endemic area such as china, korea, thailand, and philippines possible to infect with paragonimus spp. as well as foreign patients from endemic areas, this disease always is considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia [ ] . chest x-ray findings vary: not only pulmonary parenchymal lesions, such as nodular (±cavity formation) and infiltrative shadows, but also pleural lesions, such as the retention of pleural effusion and pneumothorax, are sometimes observed [ ] . many patients show peripheral blood eosinophilia and/or elevated serum total ige. immunodiagnosis to detect parasite-specific antibody has been proven as the most useful and reliable tool. not only patient's serum but also pleural effusion could examine by immunological test. commercially available anti-parasite antibody screening test is including paragomimus spp. in japan, egg-detection rate among paragonimiasis patients nowadays is low; for example . % in sputum and . % in balf [ ] , . % in bronchoscopic aspirate [ , ] . after a definitive diagnosis is made, oral treatment should be started. the type of treatment to be selected, outpatient or hospital treatment, depends on the patient's general condition. however, usually, outpatient treatment is possible. in patients having pleural effusion, pleural fluid must be extensively drained off before starting chemotherapy [ ] (biii). in patients with chronically encapsulated pleural effusion, surgery is required [ ] (ciii). ---drugs to be recommended---a first choice (same dose for adults and children) -praziquantel, oral, mg/kg/ times a day, e days [ ] (aiii) -a. lumbricoides, a. duodenale, n. americanus the larvae of these parasites pass through the lung in the human body, causing asthma-or pneumonia-like symptoms such as transient fever, cough, and dyspnea. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or elevated serum total ige. previously, the condition was called loffler syndrome. currently, it is classified as simple pulmonary eosinophilia in the category of pie syndrome [ ] . symptoms appear e weeks after the oral ingestion of a. lumbricoides embryonated eggs. the latency period of percutaneous infection with n. americanus larvae or percutaneous/oral infection with a. duodenale larvae is approximately days. larvae appeared in the small intestine penetrate the intestinal wall, enter the portal vein, migrate through liver to heart/lung. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults [ ] . simple pulmonary eosinophilia related to parasites spontaneously resolved in a few weeks. in many cases, a definitive diagnosis is made based on parasite eggs detected on a stool examination, although larvae are sometimes detected in sputum [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -pyrantel pamoate, oral, mg/kg/single-dose administration * dry syrup for children is available. * this drug is effective for adult worms in the intestinal tract, but not for larvae migrating in the human body. therefore, a stool examination should be performed weeks after oral administration. if parasite eggs are detected, additional administration should be conducted. <> second choices (same dose for adults and children) -albendazol, oral, mg/single-dose administration -mebendazole, oral, mg/twice a day, days, or mg/single-dose administration -ivermectin, oral, e mg/kg/single-dose administration (after fasting) -s. stercoralis in japan, s. stercoralis is distributed in nansei islands, chain of islands extending from southwestern kyushu to northern taiwan. there are few young persons newly infected with s. stercoralis, but the incidence of infection is high in elderly persons [ ] . internationally, s. stercoralis is widely distributed in tropical/subtropical areas. not only domestic infection but also japanese travel to endemic area possible to infect with this parasite. foreign patients from endemic areas, strongyloidiasis always considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia. s. stercoralis percutaneously infects humans. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults. parasite eggs delivered by adults are hatched while descending the intestinal tract, and larvae excreted in stools. some larvae developed to infective form again invade/transfer through the mucosa around the anus, maintaining a life cycle in the human body. such a mode of infection is termed autoinfection. with the transfer of larvae to the lung, asthma-or pneumonia-like symptoms, such as transient fever, cough, and dyspnea, appear, as indicated for simple pulmonary eosinophilia related to a. lumbricoides, a. duodenale, or n. americanus. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or an increase in the total ige level. in immunocompromised patients, suppressing the cellmediated immunity such as atl patients, hiv/aids patients, those receiving immunosuppressive drugs, the number of s. stercoralis increases through acceleration of autoinfection, and larvae are disseminated in various organs, leading to a severe condition (disseminated strongyloidiasis). in such cases, stridor, bloody sputum, tachypnea, protein-losing gastroenteritis, ileus, and mobile exanthema are observed. furthermore, severe pneumonia related to enteric bacteria disseminated with larvae, lung abscess, or bacterial meningitis concomitantly occurs [ ] . a diagnosis is made based on s. stercoralis larvae detected in stools/sputum. immunological diagnosis is also useful [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -ivermectin, oral, mg/kg/single-dose administration (after fasting), additional administration at the same dose after weeks (ai) * repeated administration must be considered in cases of disseminated strongyloidiasis [ ] . * a study reported that transrectal or percutaneous administration was useful in patients in whom oral administration was difficult due to digestive symptoms [ ] . <> second choice (same dose for adults and children) -albendazol, oral, mg/twice a day, days (bi) -t. canis, t. cati infection to humans occurs through the oral ingestion of the embryonated eggs of t. canis or t. cati [ ] . it also occurs through the consumption of beef/chicken liver or meat contaminated with larvae as a raw or insufficiently heated food. this is considered to be a dominant route of infection in japan [ ] . as humans are not definitive host for t. canis and t. cati, they do not become adults in the human body [ ] . larvae invading the human body transfer to various organs with blood flow through the intestinal mucosa. target organs are the lung, liver, eyes, and central nervous system including spinal cord. toxocariasis is a typical larva migrans. lesions are often observed in the lung/liver (visceral larva migrans). there are few symptoms, or nonspecific symptoms are present. in many cases, a diagnosis is made based on peripheral blood eosinophilia and multiple nodular shadows of the lung or liver on ct. ocular or central nervous symptoms appear in some patients. it is impossible to make a diagnosis on a stool examination due to larva migrans. immunological diagnosis is useful. after a definitive diagnosis is made, treatment should be started. when visceral larva migrans is asymptomatic, follow-up may be continued. ---drugs to be recommended---a first choice (same dose for adults and children) -albendazol, oral, mg/kg/ or times a day, e weeks [ ] (biii) * this drug should be taken with meals for days. a -day period of discontinuation should be established and then restarted drug if required. <> second choices (same dose for adults and children) -albendazol, oral, mg/twice a day, days [ ] (biii) -mebendazole, oral, e mg/twice a day, days [ ] (ciii) -w. bancrofti, b. malayi w. bancrofti and b. malayi are called lymphatic filaria. infection occurs when these parasites are transmitted to humans through mosquitoes. the pathogenesis of lymphatic filariasis is relate to structural and functional abnormality of lymphatic channels induced by parasitized adult worms. infection with w. bancrofti is characterized by febrile attacks, lymphedema/elephantiasis, hydrocele, and chyluria. in patients infected with b. malayi, neither hydrocele nor chyluria is observed, and lymphedema of the lower limbs/ elephantiasis are localized in the lower thighs. microfilaria produced by adult worms is not pathogenic, but rarely induces allergic reactions in the lung, contributing to tropical pulmonary eosinophilia (tpe) [ ] . this condition shows a chronic course, differing from simple pulmonary eosinophilia. cough, dyspnea, and stridor with exacerbation at night are observed. fever, malaise, and weight loss are noted in some patients. on chest x-ray, bilateral reticular nodular lesions are detected. peripheral blood eosinophilia and an increase in the anti-filaria antibody titer are observed. in case of tpe, no microfilaria is detected [ ] . if treatment is not performed, the condition may gradually exacerbate. it is important to adequately make a diagnosis for the differentiation of this disease. in the world, , , persons are infected with lymphatic filaria, but tpe occurs in less than . % of these. the risk of this disease in travelers is unclear, and most patients consist of foreign persons from endemic areas [ ] . ---drugs to be recommended---a first choice (same dose for adults and children) -diethylcarbamazine, mg/kg/ times a day, days * in cases of malayan filariasis, marked side effects, such as digestive symptoms, fever, lymphangitis/ lymphadenitis, and orchitis/epididymitis, may appear. therefore, a half dose ( mg/kg) should be administered times a day for days [ ] . -d. immitis d. immitis parasitize in the dog right ventricle and pulmonary artery, where mature female worms produce microfilaria that circulate in peripheral blood. infection to humans is mediated by microfilaria-ingesting mosquitoes. d. immitis larvae in human subcutaneous tissue inserted by mosquito bite, some larvae migrate to the heart and die. dead worms produce infarcts then they lodge in pulmonary vessels. there are few symptoms [ ] . in patients with symptoms, thoracic pain, cough, bloody sputum, stridor, and fever have been reported. typical chest x-ray findings include solitary coin lesions. in many cases, an abnormal shadow of the chest x-ray is indicated on a health checkup, and d. immitis infection is pathologically diagnosed from a tissue specimen extirpated under a tentative diagnosis of lung cancer. at this point, peripheral blood eosinophilia is rarely observed [ ] . ---drugs to be recommended (same dose for adults and children)----as a diagnosis is made using pathological specimens in many patients, oral treatment is not necessary. -if necessary, diethylcarbamazine at mg/kg should be orally administered times a day for days. ---precautions for each drug--- in the japanese package inserts, it is described that this drug at mg/kg/day should be orally administered twice a day for days for the treatment of paragominiasis. however, we recommend administration at mg/kg/day ( times a day) for e days based on reference no. . the incidence of side effects is low, but fever, abdominal discomfort, nausea, diarrhea, and headache are sometimes observed. masahumi seki: speaker's honorarium from shionogi & co., ltd., and taisho toyama pharmaceutical co., ltd dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the committee for the japanese respiratory society guidelines for the management of respiratory infections. the japanese 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paragonimiasis: experimental and clinical experience with praziquantel in korea parasitic infections of the lung:a guide for the respiratory physician current status of strongyloides infection in okinawa pulmonary strongyloidiasis: the varied clinical presentations non-oral treatment with ivermectin for disseminated strongyloidiasis how common is human toxocariasis? towards standardizing our knowledge toxocariasis in japan lessons from eight cases of adult pulmonary toxocariasis :abridged republication thiabendazole vs. albendazole in treatment of toxocariasis: a clinical trial a case of adult hepatic toxocariasis tropical pulmonary eosinophilia: a case series in a setting of nonendemicity study group regarding the establishment of medical management by appropriate treatment with rare-disease drugs for imported tropical diseases/parasitosis. guidelines for drug therapy for parasitosis. study group regarding drugs for tropical diseases drug to be contraindicated for combination therapy: rfp precautions for combination therapy: decrease in the blood concentration: dexamethasone, phenytoin, carbamazepine, chloroquine increase in the blood concentration: cimetidine ➁ pyrantel pamoate patients can take this drug regardless of meals. there are few side effects. ➂ albendazol this drug should be taken with meals for days. a day period of discontinuation should be established and then restarted drug if required.as liver dysfunction is frequently observed, caution is needed during the administration period. bone marrow suppression and stevens-johnson syndrome must also be considered.precautions for combination therapy: decrease in the blood concentration: ritonavir, phenytoin, carbamazepine, phenobarbital increase in the blood concentration: praziquantel ➃ mebendazolealthough this drug has been used in few children, the same dose as established for adults is used. in children weighing kg or less, a half dose should be used. this drug is contraindicated for pregnant women or those who may be pregnant. combination therapy with cimetidine may increase the blood concentration of mebendazole. combination therapy with metronidazole may cause toxic epidermal necrolysis (ten) and stevens-johnson syndrome. after fasting, this drug should be taken with water. the incidence of side effects is low, but nausea/vomiting and mild hepatic disorder are sometimes observed. the safety of this drug in pregnant women or children weighing less than kg has not been established. to these patients, this drug should be administered only when its therapeutic advantage is considered to exceed its risk. ➅ diethylcarbamazine as side effects, fever, lymphangitis/lymphadenitis, and orchitis/epididymitis, which result from anti-parasitic actions, are observed in addition to abdominal discomfort, nausea, and abdominal pain. the safety of this drug in pregnant women has not been established. upper row: dose (mg/kg), lower row: frequency of administration per day key: cord- -ukz hnmy authors: nan title: poster date: - - journal: j frailty aging doi: . /jfa. . sha: doc_id: cord_uid: ukz hnmy nan background: frail older adults are at increased risk of postoperative morbidity compared with robust counterparts. simple methods testing frailty such as grip strength have shown promising results for predicting post-operative outcome, but there is a debate regarding the most appropriate and precise frailty assessment method. objectives: we compared the predictive value of multidimensional frailty score (mfs) with grip strength or conventional risk stratification tool for predicting postoperative complications in older hip fracture patients. methods: from january to december , older hip fracture patients (age >= years) who underwent surgery and comprehensive geriatric assessment (cga) were retrospectively included for analysis. hip-mfs was calculated based on the cga with component of sex, charlson comorbidity index, serum albumin, koval grade, cognitive function, risk of falling, mini-nutritional assessment and mid-arm circumference. grip strength was also measured before surgery. the primary outcome was a composite of postoperative complications (e.g. pneumonia, urinary tract infection, delirium, acute pulmonary thromboembolism, and unplanned intensive care unit admission). results: among patients (mean age . ± . years, . accordingly, grip strength could be used for screening tool to identify high-risk patients who need for further comprehensive geriatric assessment among older hip fracture patients. information and data suspected of post-operative infections. the diagnostic criteria of infection dealt with grade ii or more of clavien-dindo classification. diagnosis of infectious disease was made with reference to vital sign, blood test, imaging and bacterial test results. surgical site infection (ssi) was evaluated based on the infectious control team surveillance. results: elderly patients were registered with necessary data. the average age was . years, males and females were included. in the sarcopenia evaluation, there were cases without sarcopenia and cases with it. cases developed some infectious complications postoperatively. the types of infectious complications (including duplication) were cases of some surgical site infections including suture failures, of pneumonia, of urinary tract infection, of pneumonia and cases of sepsis in patients. infectious complications occurred in cases in the non-sarcopenia group and in the sarcopenia group (p = . ). the average postoperative hospitalization was . days overall, . in the group with postoperative infectious complications, and . in the group without sarcopenia. conclusion: in this study, there was no relation in the incidence of postoperative infections and preoperative sarcopenia. however, the postoperative hospitalization in the group with postoperative infectious complications was almost tripled. background: hypertension is one of the major risk factors for cardiovascular disease. lowering blood pressure is effective for preventing stroke, heart failure (hf), myocardial infarction and possibly dementia. in france, the prevalence of elderly people treated for hypertension rising leading to a possible increase of potentially inappropriate antihypertensive prescribing (piap) that may cause adverse drug events. objectives: to identify associated factors with potentially inappropriate antihypertensive prescribing (piap) in elderly people. methods: we conduct a retrospective observational study based on a cohort from geriatric day hospital for assessment of frailty and prevention of disability in toulouse, between january and april . piap was defined with several explicit criteria: the european list of potentially inappropriate medications, alert and control of iatrogenesis (aci) criteria by the french health authority, the french society of hypertension guidelines, screening tool of older people's potentially inappropriate prescriptions (stopp) version two and summary of product characteristics. the piap has been considered as a binary variable (logistic regression) then as a counting variable by number of nonconformities on antihypertensive drugs (negative binomial regression). results: among the patients, % had piap. frailty, polypharmacy, history of angina and hf are associated with a higher risk of piap. similarly: frailty, polypharmacy and history of angina are associated with an increase in the number of non-conformities antihypertensive drugs. analysis of subgroup of patient hf -piap indicated that % had aci criteria whose % the aci criteria " antihypertensive drugs or more" and % the aci criteria " diuretics or more". analysis of subgroup of patient history of angina -piap indicated that % had stopp criteria, focused on loop diuretics. conclusion: our work suggests that some elderly people characteristics are associated with an increase likelihood of piap. targeting these patients would be beneficial in preventing medicine-related illness. background: social frailty was reported to be associated with age, sex, income, education, marital status, and household status. however, mood status including depression and emotion was relatively less investigated. objectives: the aim of this study is to clarify the association between depression and apathy status and social frailty in community-dwelling japanese elderly. methods: a health promotion project (teng tv project) is designed to distribute health promotion programs including enhancement of nutrition and physical activity via cable tv channel for community-dwelling elders. we ran a cross-sectional analysis using baseline characteristics of all participants (n= ). demographic data, socio-economic status, comorbidities, and nutrition evaluated by mininutritional assessment-short from (mna-sf) were recorded. functional capacity was assessed by the japan science and technology agency index of competence (jst-ic). mood status including depression, and emotion was measured by geriatric depression scale (gds- ) and apathy evaluation scale (aes). social frailty was defined by household status (living alone or not), financial difficulty, social activity, and fulfilment of social needs. we defined total deficit scores of or more as social frailty, as social pre-frailty, and as robustness. we used a linear regression model to analyze the association between mood status and social frailty after adjusting for age, sex, education, marital status, comorbidities, bmi, mna-sf, jst-ic. results: at baseline, mean age of all participants ( . % men) was ± . years. a total of . % and % of all participants were categorized as social prefrailty and social frailty, respectively. the mean scores of gds- and aes were . ± . , . ± . , respectively. in linear regression model after full adjustment, participants with social pre-frailty and social frailty were associated with increased gds- scores (social pre-frailty vs. social robustness: b= . , %ci . - . ; social frailty vs. social robustness: b= . , %ci . - . ) and aes scores (social pre-frailty vs. social robustness: b= . , %ci - . - . ; social frailty vs. social robustness: b= . , %ci . - . ). in addition, jst-ic was also associated with gds- and aes scores. conclusion: social pre-frailty and social frailty were associated with greater level of depression and apathy. future studies are warranted to determine the causal relationship among mood status and social participation. inthira roopsawang , , hilaire thompson , oleg zaslavsky , basia belza (( ) ramathibodi school of nursing, faculty of medicine ramathibodi hospital, mahidol university, bkk, thailand; ( ) biobehavioral nursing and health informatics, school of nursing, university of washington, seatlle, usa) background: frailty is a common geriatric condition with an impact on surgical outcomes. no research has been published on frailty assessment in hospitalized orthopedic patients in thailand. having a valid frailty measure has the potential to improve screening and could enhance quality of care. objectives: to test the ability of the reported edmonton frailty scale-thai version (refs-thai) in predicting hospital outcomes compared with preoperative assessment measures, the american society of anesthesiologists physical status classification (asa) and the elixhauser comorbidity measure (emc) in older thai orthopedic patients. methods: a prospective study was conducted at a university hospital. the hospitalized patients aged years or older scheduled for elective orthopedic surgery were recruited in this study. multiple firth logistic regression modeled the effect of frailty on postoperative complications, postoperative delirium (pod), and discharge disposition, while length of stay (los) was examined by poisson regression. the area under the receiver operating characteristic curve (auc) and mean squared errors (mse) were used to compare predictive ability of the instruments. results: two hundred participants with mean age of (range - years) were mostly female , % were frail, and % underwent knee surgery; of which . % had postoperative complications, . % developed pod, and % were unable to be discharged home. average los was days. adjusting for other variables, frailty was significantly associated with postoperative complications (or = . , p = . ), pod (or = . , p = . ), and prolonged los (relative risk [rr] = . , p = . ). applying the refs-thai alone shows good performance in predicting postoperative complications (auc = . , % ci = . - . ) and pod (auc = . , % ci = . - . ). the combination of refs-thai with asa and emc demonstrates improvement in predicting postoperative complications (auc = . , % ci = . - . and . % ci = . - . , respectively) and pod (auc = . , % ci = . - . and . % ci = . - . , respectively). conclusion: frailty assessment using the refs-thai was useful in predicting adverse outcomes in older adults undergoing orthopedic surgery. integrating the refs-thai for preoperative assessment may be useful for enhancing orthopedic care quality. anthony frioux , matthieu faure , margot de battista , benoit roig (( ) université de nîmes, france; ( ) université de france) background: the attention of the scientific community to frailty has been drawn over the past several years. frailty is defined as a state of increased vulnerability that may lead to functional disability. if this state is managed soon enough it may be reversible. in parallel, the possibilities of monitoring health status through connected objects such as smartphones are increasing. similarly, it is possible to measure the activity of the inhabitants of a house collecting usage data (water and electricity consumption). our project is in the field of smart home and aging monitoring. objectives: therefore, the objective of our work is to develop an integrative model of frailty based on the contributions of existing scientific tools (fried et al., ; mitnitski, mogilner, & rockwood, ) and current sensors to measure a person's activity. eventually, we are aiming for the detection of the frailty trajectory early on. for example, real-time activity monitoring is used to detect a fall and alert rescue. in our case, these sensors will allow us to identify as soon as possible a dimension that would be abnormal in order to intervene and propose an appropriate intervention. methods: our tool will be able to measure the five fried's frailty criteria which are currently used in clinical practice. we compare the data from the sensors with the results of the evaluation of fried's frailty phenotype. results: we expect to obtain a correlation between our data and phenotype results. conclusion: the main contribution of our tool resides in the possibility to observe deviations from an individual's normal aging trajectory. thus, the evaluation we propose would be more ecological as it will enable us to consider the individual's habits and to have a more detailed assessment of his activity evolution. in conclusion, the holistic aspect of our work will allow the practitioners to base their intervention on a wide range of health data. l. van wagenberg, r.m. wösten-van asperen (department of paediatrics, paediatric intensive care unit. wilhelmina children's hospital, utrecht, the netherlands) background: a frail phenotype is recognized in the elderly population. frailty is associated with a higher mortality for adult intensive care (icu) patients. research in oncology suggests biological age is not the key contributor to frailty, since frailty is also found in the younger population. in paediatrics frailty is an unknown concept and as a consequence, the prevalence and meaning of being frail at young age are unknown. objectives: to assess whether a possible frail phenotype can be found in a critically ill paediatric oncological population. methods: a retrospective cohort study in a paediatric oncological icu population between january and september . demographic data and need for icu resources (mechanical ventilation, inotropic support and s continuous renal replacement therapy (crrt)) were collected. since specific paediatric frailty scores are not available, we addressed patients as having a frail phenotype by textmining their electronic health records on the words "fatigue", "cachexia" and "diminished physical activities" before, during, and after paediatric icu admission. risk factors for a possible frail phenotype (cachexia, use of corticosteroids and lowest serum albumin levels) were collected. primary endpoint was mortality during icu treatment or course of illness. results: admissions were included, of which admissions had a possible frail phenotype. these admissions included unique patients. % of patients was male and the median age was years (iqr - ). patients were predominantly treated for a haemato-oncological malignancy ( %). mortality during icu-admission was %, and % died subsequently during the course of disease after picu discharge. patients were severely ill, with a mean icu length of stay of . days (± ), % on ventilator support, % receiving vasopressor or inotropic support, and % on crrt. loss of muscle function or fatigue was present in % before icu admission and in % acquired atrophy or cachexia was documented during icu treatment. % were treated with corticosteroids during picu stay. in % a serum albumin ≤ gram/dl was measured. conclusion: a possible frail phenotype is present in the oncological patient population of a paediatric icu. more research on the contributing factor of frailty on outcome of these patients is needed in the near future. john muscedere , , amanda lorbergs , jayna holroyd-leduc , anik giguere , leah gramlich , heather keller , ada tang , danielle bouchard , donna fitzpatrick-lewis , , diana sherifali , (( ) canadian frailty network, kingston, on, canada; ( ) queen's university, kingston, on, canada; ( ) university of calgary, calgary, ab, canada; ( ) laval university, quebec city, qc, canada; ( ) background: despite research evidence related to nutritional and physical activity interventions, there is a gap in provision of evidence-based care focused on preventing and managing frailty among older adults. objectives: to systematically generate evidence-based nutrition and physical activity (pa) clinical practice guidelines to improve health and functioning in older adults with or at risk of frailty. methods: we are using the agree ii guideline development protocol to generate guidelines to improve health and functioning in older adults. for each guideline, systematic review of meta-analyses was conducted by searching three databases for english language citations published since that included adults aged y and older with frailty and/or pre-frailty. nutrition or pa interventions with a comparison group were considered eligible. acceptable study designs included rcts, quasi-experimental trials, and observational cohorts with a comparison group. in a face-to-face meeting with multidisciplinary content experts, healthcare professionals, and end-users we will further appraise the quality and strength of the evidence using the grade approach. this group will use this evidence to form recommendations related to nutrition and pa in this population. results: the nutrition and pa searches resulted in and citations, with and eligible for full-text review, respectively. the results will inform guideline recommendations. knowledge translation strategies will be developed to support guideline dissemination and implementation. conclusion: the guidelines will inform health professionals by providing evidence-based nutrition and pa interventions for adults with frailty. ( background: physical and psychosocial factors play important roles in the severity and progression of frailty. frailty screening tools include measures of the more common risk factors, including advanced age, comorbidities, poor diet, weight loss, lower socioeconomic status, and physical inactivity. however, there has been limited standardization in the us on specific frailty screening measures to include in national health surveys or frailty tools/protocols for community health settings. this makes it difficult to monitor frailty incidence/prevalence in the older adult population and to best identify and treat individuals at risk. results: we reviewed the most recent versions of us national health surveys that include older adults, to identify whether frailty screening measures were included in. no national surveys had a battery of measures that would allow for frailty risk screening. most commonly, questions on weight, disability, mental health, physical functioning were included. however, physical functioning measurements such as grip strength or gait speed, measured height and weight, unintentional weight loss, dietary intake or appetite changes were not. further, we used the world health organization criteria for effective community screening programs to review published evidence of the validity, reliability, and feasibility of data-driven screening tools for frailty risk among community-dwelling older adults. of the frailty screening tools reviewed, the frail scale was identified as the most promising, based on test characteristics and cost/ease of use. more community-level s research is recommended, particularly on predictive validity of favorable outcomes following physical activity/nutritional interventions. finally, because nutrition plays a significant role in frailty risk, we surveyed registered dietitian nutritionists who work with older adult populations (n= ) to identify their awareness/use of frailty screening protocols/tools and dietitians' potential role in frailty screening. dietitians practicing in the community recognized a potential role, but few dietitians were aware of (< %) or using (< %) specific frailty screening tools. conclusion: future opportunities to better support healthy aging include: addition of frailty screening measures to national health surveys to help prioritize high-risk populations, conduct additional research to validate/recommend a common community-level screening tool, and promote engagement by dietitians and other health professionals who can establish protocols for community-based frailty screening. ming-yueh chou , , ying-hsin hsu , yu-chun wang , chih-kuang liang , , li-ning peng , , liang-kung chen , , yu-te lin (( ) center for geriatrics and gerontology, kaohsiung veterans general hospital, kaohsiung, taiwan; ( ) aging and health research center, national yang ming university, taipei, taiwan; ( ) department of geriatric medicine, national yang ming university school of medicine, taipei, taiwan; ( ) center for geriatrics and gerontology, taipei veterans general hospital, taipei, taiwan) background: older people with frailty are at risk of adverse outcomes, such as falls, functional decline and mortality, and multi-domain intervention program may prevent those. objectives: the purpose of this study is to evaluate the effectiveness of multi-domain intervention program among those community-dwelling frail older people in southern taiwan. methods: a week multi-domain intervention program were provided for all participants, including physical activity, high protein diet education, medical knowledge education and cognitive simulation activity for hours per week. comprehensive geriatric assessments were performed before and after the intervention program, including basic demographic data, risk for malnutrition (by mna-sf), mood condition (by gds- ), cognitive condition (by mmse) and frailty status according to the definition by the cardiovascular health study (chs) . results: during jan and may , totally participants were invited for study ( . % female, mean age . ± . years). among them, ( . %) were clarified as frailty status and ( . %) as prefrailty status. after the multi-domain intervention program, their mood condition ( . ± . to . ± . , p< . ) and cognitive condition ( . ± . to . ± . , p< . ) improved significantly. in addition, the walking speed ( . ± . to . ± . m/s, p< . ) and physical activity ( . ± . to . ± . mets/week, p< . ) improved, but not handgrip strength (p= . ). for the frailty status, those clarified as frailty status decreased from . % to . % and prefrailty status from . % to . % (p< . ). conclusion: our results showed that through the week multi-domain intervention program, those frail older people could improve their mood condition, cognitive condition, usual gait speed and frailty status. sarah b. lieber , stephen a. paget , , jessica r. berman , , medha barbhaiya , , lisa sammaritano , , kyriakos a. kirou , , john a. carrino , dina sheira , mangala rajan , yingtong lyu , lisa a. mandl , (( ) division of rheumatology, hospital for special surgery, new york, ny, usa; ( ) department of medicine, weill cornell medicine, new york, ny, usa; ( ) department of radiology and imaging, hospital for special surgery, new york, ny, usa) background: frailty is a clinical phenotype that increases with age, but can occur in younger patients with chronic disease. based on few studies, frailty has been found in up to . % of patients with systemic lupus erythematosus (sle) and is associated with increased mortality. whether frailty is prevalent in other sle cohorts and associated with objective and subjective factors is unknown. objectives: we aimed to determine the prevalence of frailty in a prospective cohort of women with sle and whether inflammatory biomarkers, body composition, and patient-centered domains differed between frail and non-frail women. methods: adult women < years old who fulfilled american college of rheumatology sle criteria were recruited from one center. exclusions included pregnancy, dialysis, active malignancy, overlap autoimmune syndromes, and severe sle disease activity. frailty was measured according to fried criteria. patient-reported outcomes (pros) were measured using pro measurement information system (promis) computerized adaptive tests; lupusqol; and disability based on valued life activities. physicianreported sle disease activity and damage indices were collected. inflammatory biomarkers and sarcopenia according to dual-energy x-ray absorptiometry were assessed. differences between frail and non-frail women were evaluated using chisquare tests and kruskal-wallis tests; the association between frailty and disability was determined using logistic regression. results: women enrolled from / - / . despite age under years old, % were frail. frail women had greater disease damage (p= . ) and were more often smokers (p= . ). high-sensitivity c-reactive protein (p= . ) and interleukin- (p= . ) were higher and sarcopenia trended toward greater prevalence (p= . ) in frail women. significant differences in promis mobility, physical function, pain interference and behavior, and fatigue and lupusqol physical health and pain (all p< . ) were observed between frail and non-frail women, with frail women reporting consistently worse scores. frail women were . x more likely to be disabled than non-frail women, including after adjustment for age, comorbid conditions, and disease activity/damage. conclusion: the prevalence of frailty was high in this cohort of mid-aged women with sle. frail women had poorer health-related s quality of life than non-frail women, including substantially higher disability. if frailty is associated with worse health outcomes, it could be a potential therapeutic target. chariya sumcharoen, supreeda monkong, nuchanad sutti (ramathibodi school of nursing, faculty of medicine ramathibodi hospital, mahidol university, bangkok, thailand) background: bed bound older adults need caring of physical activities, mental, mood, and social from family caregivers. family caregivers usually gets the role strain from caregiving. there are many factors associate with the caregiver role strain but have been rarely reported in bed bound older adults at home. objectives: the study examined age, adequacy of incomes, mutuality, health status, preparedness, and social support influencing caregiver role strain from caregiving activities for bed bound older adults at home. methods: caregiver role strain concept by archbold and colleagues with literature review were used to guide this study. the sample was recruited by purposive sampling consisted of caregivers aged years or older, who have cared for bed bound older adults at home in thailand. data were collected by structured interview using the questionnaires including demographic data, preparedness, health perception, mutuality, social support, and caregiver role strain from the care activities. data was analyzed using descriptive statistics, pearson's product moment coefficients, and multiple regression analysis. results: the most of participants were women ( . %), age ranging from to years (m= . , sd= . ) . the result showed that age, adequacy of incomes, mutuality, health status, preparedness, and social support jointly significantly explained . % of the variation in caregiver role strain from caregiving activities. the regression effects were strongest for health status (beta=-. , p=. ), followed by preparedness (beta=-. , p=. ), age (beta=. , p=. ), and adequacy of incomes (beta=-. , p=. ) respectively. conclusion: this finding suggests that healthcare providers should find strategies for promoting health status and preparedness of family caregivers for decrease caregiver role strain from caregiving activities. of life, and hospital admissions. objectives: we estimated the prevalence and describe the characteristics of the population with recurrent falls and fear of falling and their association with frailty, physical performance and cognitive fragility. methods: data came from the "salud, bienestar y envejecimiento" (sabe) colombia study, a cross-sectional study conducted in at the urban and rural research sites ( municipalities) in colombia. sociodemographic, health, cognitive and anthropometric measures were collected from community-dwelling adults aged years and older, representative form the total population. frailty was defined using the frailty phenotype proposed by fried. cognitive frailty was defined using the inaa/iagg consensus definition. low performance was evaluated with sppb (short physical performance battery). logistic regression analyses were used to identify factors associated with recurrent falls and fear of falls. results: our study identified elderly who had recurrent falls and fear of falling ( . % and . % respectively). young elders (≤ years) had more falls and greater probability for fear of falling compared to older ages. sex had no significant differences. the factor associated with an increased risk of recurrent falls and fear of falling in the elderly were low physical performance, fragility and polypharmacy. chronic illness such as osteoarticular disease, mental disease, diabetes and chronic pulmonary disease were significantly associated with recurrent falls and fear of falling. finally, when adjusted for age, sex, sociodemographic factors and comorbidities in a logistic regression model, frailty was associated with fear of falling and recurrent falls, while cognitive frailty and low physical performance only were associated with fear of falling. conclusion: recurrent falls have a significantly association with frailty. there are cognitive, physical performance and clinical factors associated with fear of falling that could be preventable and treatable. rubbieri gaia , ceccofiglio alice , mazzeo nicla , pupo simone , cartei alessandro , rostagno carlo , mossello enrico (( ) department of perioperative medicine, careggi hospital and university of florence, italy; ( ) department of geriatric medicine, careggi hospital and university of florence, italy) background: the prevalence of frailty in patients with hip fracture is high, but little is known about the choice of the best frailty tool in terms of prediction of functional recovery. objectives: the aim of this preliminary study was to determine the most predictive validated frailty tool in older people with hip fracture and to determine whether frailty can predict functional recovery during the hospital acute phase. methods: this study was observational prospective cohort study. participants aged + admitted to hip fracture units in florence, were assessed pre surgery (t ), and post surgery. each participants underwent a comprensive geriatric assessment and frailty was defined using: clinical frailty scale (csf), frail scale (fs), reported edmonton frail scale (refs), postal frailty screening (pfs). the outcome was functional recovery, evaluated by a score of postoperative performance on the cumuleted ambulation score (cas). data recorded included pre-recovery barthel index (bi), charlson comorbidity index (caci), handgrip strenght test (hg), asa score, mini nutritional assessment short-form (mna-sf), delirium. results: sample included patients (mean age ± years, female . %). cfs was the most predictive frailty tool, with a % sensitivity and a % specificity (auc = . , cut off > ). dividing the sample according to premorbid bi, while bi itself had the highest predictive value when premorbid level was < %, cfs was the best predictor of functional outcome in the %+ subsample (auc= . ). conclusion: frailty defined by cfs can predict short-term functional recovery during acute phase following hip fracture. this appears particularly relevant for subjects with a higher pre-morbid functional independence. s % were women. individuals had data for all five frailty measures. nine percent of participants were non-frail by all instruments, % were frail by all measures and thus % had discordant frailty measurements. % were frail by at least one measure method. the prevalence of frailty ranged from % to % for the different measures. those classified as frail by cfs and non-frail by bp were more likely to be men, be co-living, have lower cognitive function and a higher dependency in iadl compared to those classified as frail by bp and non-frail by cfs. conclusion: frailty measures cannot be used interchangeably. specifically the cfs might not identify physical frail women, with high cognitive ability who lives alone. factors contributing to the heterogeneity of groups classified as frail by different measures need to be further explored. background: polypharmacy is increasingly common amongst older, multimorbid adults. in these individuals, studies have shown a high prevalence of frailty. identification of frailty can be performed using comprehensive assessments registering accumulation of deficits like in the frailty index, or using single-trait markers of frailty like gait speed and handgrip strength. polypharmacy is recognized as an independent risk factor for the development of frailty, and the subgroup of psychotropic drugs may be particularly important in the development of this syndrome. objectives: our objectives were to study the relationship between the total burden of polypharmacy on frailty status using three different measurements of frailty, and specifically the influence of psychotropic drug use on frailty status. our overall aim was to explore whether either of these could be used as independent predictors of frailty. methods: we used data from a -year follow-up study of older people living in the community and receiving home care nursing, i.e. the cascade-study. data collection was completed in june . all participants were aged > years (mean years). a item frailty index was calculated based on results from a comprehensive geriatric assessment performed in the patients' own home. a fourmeter gait speed test was performed, as well as measurement of handgrip strength. information on regular medications was collected from the patients if they administered own medications, or from the home care nursing service if they were responsible for administering the patients' medications. psychotropic drugs were selected based on beers criteria. results: we found a significant association between the use of psychotropic drugs and frailty index, and frailty index increased by . for each psychotropic drug added (p< . ). one additional psychotropic drug decreased gait speed by , m/s (p< , ). there was no statistically significant association between psychotropic drug use and handgrip strength. conclusion: our study showed that psychotropic drug use was a significant predictor of increased frailty index and reduced gait speed. this was not the case for handgrip strength in our material. laetitia beernaert , frédéric schuind , sandra de breucker (( )department of geriatrics, hôpital erasme -université libre de bruxelles, belgium; ( ) department of orthopedics, hôpital erasme -université libre de bruxelles, belgium) background: anemia is a condition whose prevalence might reach % in the geriatric population. anemia and frailty are two prognostic factors for patients admitted for a hip fracture. objectives: we analyzed retrospectively if preoperative frailty and anemia were independently predictive of postoperative complications and mortality in old patients admitted for hip fracture. methods: ninety-seven patients above years old have been admitted for urgent surgery for a hip fracture during and . we excluded patients with a pathological fracture or fractures due to high energy trauma. preoperative anemia was defined as an hemoglobin level under g/dl for women and g/dl for men. frailty was assessed with the isar (identification of seniors at risk) score. results: seventy-five percents of patients were considered as frail (isar score> ). the prevalence of preoperative anemia was %. we found no statistically significant correlation between anemia and frailty (r = - . -p = . ). in multiple regression logistic analysis, the only independent parameter associated with anemia was the presence of comorbidities (or . ( . - . )-p = . ), and the only parameter associated with frailty was the presence of malnutrition (or . ( . - . )-p = . ). neither anemia nor frailty was associated with postoperative complications and mortality. conclusion: preoperative anemia and frailty are not interrelated in patients admitted for hip fracture. anemia is associated with comorbidities, but not postoperative mortality. frailty is associated with preoperative malnutrition. the isar score may not be ideal to screen for frailty in old patients admitted for hip fracture, an item being attributed to the current loss of autonomy. settings. m martinez , maria montoya , , davide angioni , lizeth canchucaja , natalia ronquillo , maria luz gallego , claudia bejar , emmanuel gonzalez , olga vazquez , anna renom (( ) institute de viellisement toulouse, france; ( ) hospital del mar, barcelona, spain; ( ) hospital de terrasa, barcelona, spain; ( ) parc tauli, barcelona, spain) background: frailty is a common critical geriatric syndrome which has been associated with poor health outcomes.a wide variety of frailty indices (fis) have been developed. frail-vig («vig» is the spanish/catalan abbreviation for comprehensive geriatric assessment).it contains simple questions that assess different deficits. it has been inspired by the rapid geriatric assessment. objectives: the aim is to compare the prediction capacity of clinical rockwood index frailty (rif) and frail-vig index (vif) for poor health outcomes (pho) defined as: emergency department visits and/or hospital admission and/or mortalityamong elderly patients. methods: a retrospectiveobservational study was conducted with a followup up to months or pho occurred. patients were admitted in acute geriatric unit care and geriatric day hospital at hospital del mar; barcelona; spain during august and march . the inclusion criteria were the admission ones. frailty was measured at admission. survival analysis was conducted; cox proportional hazards regression was used to build a pho predictive model based on both indexes. best model according to contrast of hypothesis log-rank ,aic; bic and c harrel was selected.diagnoses of the chosen model was done. results: a total of patients were included, mean age was and . % female. the mean of follow-up was . , % patients presented a pho. . % died, % were admitted at emergency department, . % were hospitalized and % presented more than one event.survival curves for frail and non-frail according to pho showed statistically significance for vif (x = . p= . )but not for rif (x = . p= . ). cox proportional hazards regression showed vif hazard ratio . (p= . ) and rif hazard ratio . (p= . ). predictive capability resulted in a model for vif containing cognition and sex, with harrel c of . . as for rif the most parsimonious model rif would be absent and harrel c . . the diagnoses of the model showed time covariate variable test with p= . , p= . , p= . for each predictive variable; squared linear predictor with p= . of and outliners. conclusion: the vig frailty index performed better; compared to rockwood clinical index; in predicting a composite outcome composed by mortality, hospitalization and visits to emergency departments in patients admitted in acute and outpatient settings. after hospital discharge. methods: this study was conducted in the departments of internal medicine and neurology of the university hospital of araba (basque country, spain). participants were >= years, scoring >= on the mmse test and able to stand and walk independently for at least -meter. participants performed twice-weekly moderate intensity group sessions of multicomponent exercise at the hospital during -week, followed by a home-based intervention ( week) . both were focused on balance, aerobic capacity and strength. taking together both interventions, participants completed -week of physical exercise. at the beginning and the end of the program, frailty was measured though fried´s index and sarcopenia with different criteria : muscle strength ( -chair stand), muscle quality (dxa) and physical performance (sppb). we compared the results before and after the intervention by mcnemar test. results: patients ( females, %) were enrolled, were lost to follow-up at the -week time point and people finished the intervention. the intervention decreased significantly the percentage of frail individuals (p< . ) according to fried´s index, and the percentage of people who met sarcopenia criteria for sitto-stand (p= . ) and sppb (p= . ). however, there were no differences in the percentage of people with low appendicular muscle mass. conclusion: our study showed that a multicomponent exercise program is effective for posthospitalization patients because after -week intervention there were significant reductions in frailty and improving results in muscle strength and physical performance. we did not find changes related to muscle mass. references: . background: alcohol addiction can impact every part of the body, including bones. research shows that chronic heavy alcohol use, especially during adolescence and young adult years, can dramatically affect bone health and increase the risk of osteoporosis and bone fracture later in life. objectives: the purpose of this study is to compare data from international scientific literature with data from the study of patients admitted for alcohol dependence, to assess whether there are significant connections between alcohol dependence and unrecognized fractures. methods: we analyzed meta-analysis's studies from the pubmed search engine to evaluate the association between bone fractures with alcohol use disorders. only humans studies from the last years have been analyzed. subsequently, data related to patients admitted for an alcohol rehabilitation cycle were analyzed. results: scientific literature show that there is a close correlation between alcohol abuse and greater frequency of bone fractures. this is partly due to association between alcohol consumption and both osteoporotic fracture and bone density, and partly to the fact that there is an increased risk of falls in alcohol intoxicated patients compared to the general population. patients were considered: % male and . % female. the average age was years. of these , . %, patients, had unrecognized fractures. conclusion: intoxicated patients admitted in alcoholic rehabilitation with recurrent falls anamnesis often did not perform any diagnostic assessment. this is due to the lack of pain perception in the patients or due to family members or emergency physicians who placed the state of drunkenness before any consequences caused by repeated falls. there is an increased risk of unacknowledged fracture in the patients admitted in alcohol rehabilitation this is partly due to the fact that alcohol intoxicated patients often do not perceive the pain and therefore do not investigate any falls that occurred in a state of drunkenness, in part it is due to the damages that alcohol causes on the bone. our data show that alcohol dependence and unrecognized fractures can often be associated. studies in the literature confirms that there is an increased risk of non-cone fractures in patients with alcohol dependence. zamudio-rodríguez, hélène amieva, luc letenneur, karine pérès (centre de recherche inserm u université de bordeaux -isped, bordeaux, france) background: although conceptually distinct, frailty and disability are very common among older adults. both are multifactorial conditions and share some risk factors and pathophysiological mechanisms, such as inflammation or sympathetic-parasympathetic balance alteration. furthermore, each individual component of the frailty phenotype defined by the cardiovascular health study (chs) has been associated with disability in basic and instrumental activities of daily living. objectives: the present study aimed to determine whether pre-frail and frailty are part of the natural history of the disability process. methods: a sample of people aged of the three cities ( c) study in bordeaux were followed for four years. pre-frailty and frailty were defined according to the original phenotype proposed in the chs. disability was defined using the basic (adl) and instrumental (iadl) activity of daily living scales. seven mutually exclusive hierarchical groups were distinguished at inclusion: ) robustness (no frailty or disability); ) pre-frail (without disability); ) frailty (without disability); ) iadl (without pre or frailty or adl) ) pre-frail with iadl (no adl); ) frailty with iadl (no adl); ) frailty with iadl and adl. results: deaths ( . %) occurred during the four years follow-up. compared to the robust group, all other hierarchical subgroups had an increased risk of death, with an increasing gradient: pre-frailty (hr= . ; ic %= . - . ); frailty (hr= . ; ic %= , ) , iadl disability (hr = . ; ic %= . - , ); pre-frailty with iadl disability (no adl) (hr= , ; ic %= , - . ); frailty with iadl disability (no adl) (hr= , ; ic %= . - . ); frailty with iadl and adl disability (hr= , ; ic %= . - . ) were significant after adjustment by age and sex. conclusion: there is a gradual risk of mortality across the different groups ( i.e., ) robust; ) pre-frail; ) frail; ) iadl disability without pre or frailty; ) pre-frail with iadl disability; ) frail with iadl disability; ) frail with iadl and adl disability) thus suggesting a hierarchical relationship. this study could have important clinical implications since pre-frailty and frailty are assumed more effectively reversible conditions in order to interrupt the continuum at the early phase of the disability processes. background: joint replacement provides significant improvement in pain, physical function, and quality of life in patients with osteoarthritis. with a growing body of evidence indicating that frailty can be treated, it is important to determine whether targeting frailty in joint replacement patients is feasible and improves post-operative outcomes. objectives: to examine the feasibility of a preoperative multi-modal frailty intervention (mmfi) compared to usual care in pre-frail/ frail older adults undergoing elective unilateral hip or knee replacements. methods: in this pilot randomized controlled trial (rct), participants who are )>= years old; ) pre-frail (score of - ; (fried frailty phenotype (ffp)) or frail (score of - ; ffp); ) having elective unilateral hip or knee replacement with surgery wait times between - months were recruited from the regional orthopaedic clinic mcmaster university, ontario canada. the mmfi included tailored exercise, protein ( - gm/day), vitamin d ( iu/day) supplementation, and medication review with recommendations sent to family physicians. frailty and mobility were assessed at baseline and -weeks post-operative using ffp, short performance physical battery (sppb) and oxford hip/knee score (ohs/ oks) respectively. results: we recruited and randomized participants between september and may . of those, . % were referred for total hip replacement and . % for knee replacement. the included participants' mean age (standard deviation (sd)) was . ( . ) years; . % were women; . % lived alone, body mass index was . kg/ m ( . ) and . % were former smokers. at the baseline assessment, on the ffp, % were prefrail, % were frail and the sppb was . ( . ). for participants with hip osteoarthritis, ohs mean (sd) was . ( . ) and for participants with knee osteoarthritis, oks mean (sd) was . ( . ). the study recruitment rate was . %, and the retention rate was %. eighty three percent of participants of the intervention group completed the intervention. self-reported adherence to the intervention components was as follow: ) exercise sessions: . %, ) protein supplement: . %, ) vitamin d supplement: . % and ) medication review completion: %. conclusion: this is the first study to examine the feasibility of a multi-modal frailty intervention in pre-frail/frail older adults undergoing joint replacement. this study showed that frailty screening, assessment and management is feasible for older adults undergoing joint replacement in orthopaedic surgery clinics. results have informed the current multi-centre rct to determine effectiveness. christine tocchi , sathya amarasekara , michael cary (( ) school of nursing, duke university durham, nc usa; ( ) school of nursing, duke university durham, nc usa; ( ) school of nursing, duke university durham, nc usa) background: inpatient rehabilitation facilities (irfs) provide intensive rehabilitation therapy to patients to reduce functional impairment, enhance independence and return patients to the community. determination of eligibility for irf is currently based on preadmission screening. subpopulations of older adults may require special consideration in determination of irf admission due to greater risk for poor functional recovery such as those with pre-existing functional limitations and those who are frail. frailty, a pervasive characteristic in older adults with hip fractures has not been examined as a clinical factor influencing discharge destination outcomes in irfs. objectives: ) determine the prevalence of frailty among older adult with hip fracture receiving inpatient rehabilitation; and ) determine the association between frailty and discharge destination among hip fracture patients receiving inpatient rehabilitation. methods: a retrospective cohort study design using cms inpatient rehabilitation facility-patient assessment instrument file. multivariate regression models were performed to examine the association between frailty and discharge destination. frailty status was measured using a frailty index of items with the following cut-off points: - . robust/non-frail; . - . pre-frail; and . or greater as frail. the final sample included , hip fracture patients. results: frailty, pre-frailty, and nonfrail were present in . % (n= ), . % (n= ), and % (n= ) of hip fracture patients, respectively. the majority ( %) of the frail hip fracture patients were discharged home. there were significantly greater proportion of females than males discharged home and those of white race, to years of age, and with higher functional status. regression analysis showed significantly lower functional status at discharge (p < . ) for patients with these characteristics: males, non-white race, and older age. additional factors that influenced discharge destination included: marital status, living in the community prior hospitalization, and length of stay. conclusion: frailty was the most common frailty status on admission to irf. home is the most common discharge destination for all frailty status groups. frailty status could be used to identify hip fracture patients at high risk for adverse outcomes. future studies should be used to explore the potential of frailty to provide valueadded utility to clinical settings such as irfs. background: front-line care providers are seeking direction on how frailty measures may be integrated into existing or future care pathways to enhance the experience of individuals who live with it. multidimensional frailty measures such as the edmonton frail scale offer the potential for case-finding, estimation of severity, and definition of frailty components. objectives: test the feasibility of the implementation of a multidimensional frailty order set into acute care. methods: in , we conducted a literature search to identify existing frailty guidelines and systematic reviews related to frailty in acute care. an expert panel graded the quality the evidence, then generated recommendations, graded by strength to inform the generation of a clinical knowledge and content management (ckcm) topic for dissemination throughout alberta health services (ahs). ahs is the largest province-wide, fullyintegrated health system in canada. this ckcm would include graded statements and recommendations, clinical decision support, electronic alerts, and a frailty order set. results: four guidelines, systematic reviews, and one scoping review informed the development of the frailty ckcm. from this, we developed eight recommendations, covering topics such as prevention, case-finding, estimation of severity, definition of components, triggers for expert assessment, and linkage to care processes. the recommendations also addressed safeguards to avoid labelling and other unintended consequences. an order set employs the clinical frailty scale, electronic frailty index, and edmonton frail scale to support a clinician to develop a personalized care plan. the order set empowers front-line clinicians to administer these frailty measures, based on cut points that prompt personalized recommendations on diet, activity, fall prevention, bladder management, and infusions. depending on the frailty component of concern, clinicians are also prompted with specific options to address cognitive impairment, functional dependence, falls and immobility, social isolation, nutritional risk, polypharmacy, urinary incontinence, chronic pain, and constipation. in preparation for the conversion to a province-wide electronic medical record (emr) in november , the ckcm was released in may and the frailty order set was built into the emr by september . conclusion: development and implementation of a multidimensional frailty order set in the setting of acute care is feasible. masayo kojima , toshihisa kojima , yuko nagaya , yasumoto matsui (( ) national center for geriatrics and gerontology, obu, aichi, japan; ( ) nagoya university, nagoya, aichi, japan; ( ) nagoya city university, nagoya, aichi, japan) background: prevention programs for frailty at community usually target healthy older people. to further prolong healthy life expectancy, we need to approach those who already have got chronic diseases such as rheumatoid arthritis (ra). objectives: the aim of this study is to assess the prevalence and factors associated with frailty in japanese ra patients. methods: ra patients aged - -yearold who visited two university hospitals between march and july were consecutively invited to join the study. those who agreed to participate the study provided written consent forms. frailty was assessed by the total score of the kihon checklist >= . self-report questionnaires were used to evaluate patients' demographic characteristics, perceived degree of pain, depression (the beck depression inventory-ii) and physical function (the health assessment questionnaire, haq). rheumatologists' global assessment of disease severity, swelling and/or tender joint counts, years of ra duration, frequency of arthritis surgery and crp level were also measured. results: total of ra patients were included in the study ( women, average age: . ± . years, average disease duration: . ± . years), and the prevalence of frailty was . %. the higher the age and the longer the duration of the disease, the higher percentage of ra patients with frailty was observed. . % among ra patients of working age ( - years), were frail, whereas . % and . % were frail among those aged - years and >= years, respectively. stepwise logistic regression analysis revealed that age, haq, depression severity and trust in neighbors were independently associated with frailty in ra. no significant gender difference was observed. conclusion: frailty is common even among working age in ra patients. physical function, depression and social capital were suggested to be independently associated with frailty. on-going followup study will disclose the influence of frailty on fracture, dependency, and mortality among ra patients. background: frailty is an important modulator of ageing and might impact on clinical presentation and progression of parkinson's disease. objectives: to evaluate the prevalence of frailty and correlation with motor and non motor symptoms as well as mri atrophy and white matter hyperintensities in parkinson's disease. methods: consecutive parkinson's disease patients underwent a comprehensive motor and non motor evaluation and geriatric assessment using multidimensional prognostic index (mpi). a subset of patients underwent mri with assessment of atrophy and white matter hyoperintensities by visual rating. results: pd outpatients (mean age . y, mean disease duration . years) entered the study. pre-frailty assessed by mpi was presented by % of patients and correlated with age and disease duration. when adjusting for these ariables, mpi correlated with updrs-iii, non motor symptoms assessed by umsar, prevalence of prevalence of orthostatic hypotension, rbd and depression. the mri assessment showed a correlation between global atrophy and frailty indipendently from mmse and educational levels. no association between frailty and wm hyperintensities was found. conclusion: frailty is a possible important modulator of pathology and brain vulnerability in parkinson's disease and could explain different severity in motor and non motor symptoms. longitudinal studies are warrented to evaluate the impact of frailty in disease progression. background: accidental falls in older adults have been associated with worse health-related outcomes especially in the frailest individuals, such as nursing home (nh) residents. in this special population of older adults, falls have been related to greater morbidity and mortality, but their impact on nutritional status is still unclear. moreover, so far there are no data on the potential role of unmodifiable (e.g. cognitive impairment [ci] ) and modifiable factors (e.g. assistance from informal caregivers) in influencing the impact of falls on nutritional status in older residents. objectives: we aimed to evaluate the changes in body weight during the six months after the occurrence of a fall in nh residents, and the possible influence of severe cognitive impairment, depressive symptoms and of the assistance from informal caregivers on such variations over time. methods: the sample included older residents who experienced at least one fall since nh admission. for each participant, we collected data on sociodemographic information, mean frequency of visits from informal caregivers, medical history, and cognitive and functional status at nh admission. severe ci was defined as the presence of a physician-based diagnosis of ci or a mini-mental state examination < points. the frequency of the visits from informal caregivers was categorized as none or (low) vs > (high) per week. falls' date and characteristics were obtained from structured forms completed by physicians. monthly body weight in the six months before and after the fall were derived from the nh medical records based on nurses' assessments. linear mixed models were used to evaluate the body weight changes after a fall, as a function of the presence of severe ci and low visits' frequency from informal caregivers, alone or in combination. results: the mean age of our sample was . ± . years and % were women. more than half ( . %) of residents involved had severe ci and . % had low visits' frequency from informal caregivers. after adjusting for potential confounders, the presence of severe ci (b=- . , se= . , p< . ) and the report of low visits' frequency from informal caregivers (b=- . , se= . , p= . ) were associated with steeper decline in body weight during the six months after the fall. when combining these variables, we found an additive effect of severe ci and low visits' frequency from informal caregivers in influencing weight loss (b=- . , se= . for residents with severe ci and high visits' frequency, and b=- . , se= . for those with severe ci and low visits' frequency; p< . for all). conclusion: our results suggest that cognitive impairment may worsen the impact of falls on nutritional status in nh residents, and that this effect may be exacerbated by scarce assistance from informal caregivers. ( ) tokyo women medical university, tokyo, japan, japan; ( ) department of geriatic medicine, kyorin university medical hospital, tokyo, japan; ( ) tokyo metropolitan institute of gerontology, tokyo, japan) background: in consideration of the future rapid aging of the society, to achieve healthy and active aging is indispensable. because especially the major issue is to prevent "multi-faceted frailty", it is necessary to reconsider regarding nutrition, physical activity and sociality/sociability in the elderly. sarcopenia is associated with adverse health outcomes, such as frailty, limited physical function, falls, disability and loss of independence. objectives: our aim to notice evidencebased new information, leading to frailty prevention, and let the community-based activity by elderly citizen only promote as a voluntary motion in each community. methods: we have already established many new evidences from our on-going japanese large-scale longitudinal study 'kashiwa study'. these evidences include the impact of overlapping of slight oral dysfunction, namely "oral frailty", as well as unbalanced diet and inadequate physical activity in early-stage sarcopenia. furthermore, we found the negative impact of several social disengagements including eating alone, so-called "social frailty", leading to subsequent sarcopenia. we developed a simple screening tool, ''frailty check-up activity'', which elderly citizen supporters only can operate in each small gathering place (e.g. community salon) via support by its local government. results: based on the concept of all-including three pillars, ) nutrition (i.e. dietary food intake including diversity and adequate protein intake, and treatment/maintenance against oral frailty), ) physical activity (not only exercises but also social daily activity) and ) social participation, the newly citizen activity ''frailty check-up'' has developed. after elderly citizen supporters received training fully, they could implement this activity completely and repeatedly in each local municipality. elderly participants could learn how to improve/conquer by themselves with raising their self-awareness for the importance of early frailty/sarcopenia prevention and could change their behavior modification. in addition, using big data combined with preexisting database of new-onset regarding care needs and/or all-cause mortality, we found the new cut-off point in our frailty check-up activity. conclusion: we could confirm that our interdisciplinary "action-research" can raise the citizen's early awareness and affect their behavior modification via elderly citizen supporter system for frailty prevention, consequently leading to extend healthy life expectancy. saguez, carlos márquez, bárbara angel, mario moya, lydia lera (inta, universidad de chile, santiago, chile) background: physical phenotype of frailty has been associated with quality of life deterioration and some studies have calculated cost-effectiveness of interventions on frailty in quality-adjusted life years (qalys), however studies on the direct burden of frailty expressed in qualys lost in community dwelling older adults are scarce. objectives: to forecast qalys lost caused by frailty in older chileans and describe health profiles as determined by euroqol (eq- d) in community-dwelling older chileans with and without frailty. methods: cross sectional study in ( , % women, mean age y± . ) community dwelling people >= years participants in alexandros cohorts. the frailty phenotype was defined as having >= from the following criteria: weak handgrip dynamometry, unintentional weight loss, fatigue/ exhaustion, five chair-stands/slow walking speed and low physical activity. qol was evaluated trough euroqol (eq- d) five dimensions: mobility, self-care, usual activities, pain/ discomfort and anxiety/depression and self-rated health trough eq -visual analogue scale (eq- d-vas). qualys were calculated by the eq -d time trade-off (tto) method. to estimate life expectancies (le), multistate methods based on the follow-up of alexandros cohorts, were employed. results: frailty was identified in , % of the sample. selfrated health according to eq- d-vas was lower in frail than non-frail people ( . ± . vs . ± . , p< , ). after adjusted multinomial logistic regression, the eq- d dimensions of anxiety/depression (very depressed rrr= . ; %ci: . moderate rrr= . ; %ci: , ) and pain (much pain rrr= . ; moderate pain rrr= . ; had the highest association with frailty. the valorisation of years in qualys was lower in frail than in non-frail people ( . ± . vs. . ± . qalys per year, p< , ) and among those frail, much lower in people >= y than in the group - y ( . ± . vs. . ± . , p< , ). the qualys remaining years were lower in frail people than in non-frail:total le at - y was , y corresponding to , qalys in frail and , qalys in the non-frail; in the group >= y tle was , y corresponding to , qalys in frail people and , in the non-frail. conclusion: the high burden of frailty on qalys, mostly related to pain and anxiety/depression makes compulsory its early detection and treatment. its knowledge allows calculating cost-effectiveness of interventions. background: + agil barcelona is a real-life a multicomponent intervention against frailty implemented in a primary care center, which promotes a comprehensive and coordinated approach between primary care, geriatrics teams and community resources, to detect and reverse frailty in the older adults. objectives: we aimed to assess the -months impact on physical function of +agil barcelona in community-dwelling frail older adults with cognitive impairment. methods: the study population was driven from the +agil barcelona program population. we included participants with cognitive impairment or dementia past history and those who performed a minicog test < points. after frailty screening by the primary care team, a geriatric team performed the comprehensive geriatric assessment. according to cga results, a tailored and specific multidisciplinary intervention for each person was designed. the intervention could include a) multi-modal physical activity (pa) sessions, b) promotion of adherence to a mediterranean diet c) health education and d) medication review. the physical performance was assessed at baseline and at -omths follow-up by the short physical performance battery (sppb) and gait speed. the pre/post intervention analysis was done by a paired sample t-test for repeated samples for continuous variables and chi-square for categorical variables. results: we included participants (mean age= . ± . , . % woman and . % lived alone). despite being independent in daily life, . % had fallen the past year, . % were vulnerable or frail according to the csf. physical performance was impaired: sppb= . ± . and gait sped= . ± . m/sec and . % had balance impairments. after months, . % of participants completed >= . physical activity sessions. the mean improvements were + . ± . points (p< . ) for sppb, + . ± . m/ sec (p< . ) for gait speed, - . ± . sec (p< . ) for chair stand test, and . % (p . ) improved their balance. additionally, psychoactive treatment was withdrawn in . %. conclusion: according to our results, a multidisciplinary and comprehensive geriatric intervention for frail elderly people with cognitive impairment of the community improves physical function and could reverse fragility at months. clarence mwelwa patrick chikusu, amritha narayanan, joel james (ashford and st peter's nhs foundation trust, chertsey, uk) background: frailty and muscle strength are a critical component of walking ability and presence of these can result in high prevalence of falls. it also results in increased morbidity and mortality among the elderly. despite sarcopenia being very common and a reversible condition in its early stage it is a frequently overlooked and undertreated geriatric syndrome a greater understanding of sarcopenia and frailty among healthcare professionals could have a dramatic impact on outcome and quality of life of the elderly. objectives: this study aimed to assess the current knowledge about the concept of sarcopenia and frailty among the healthcare professionals working in an nhs district general hospital in surrey. methods: this longitudinal study included nhs healthcare professionals (n = ) who were asked to complete a questionnaire regarding awareness of concept, risk, diagnostic strategy and management of frailty and sarcopenia. results: . % of healthcare professionals stated to know the concept of sarcopenia, % indicated to know how to diagnose sarcopenia and % had seen patients with suspected sarcopenia in the last one month. only % knew the risk associated with sarcopenia. . % used sarc f questionnaire as diagnostic method for sarcopenia. percent of the cohort experienced bottle necks during the implementation of diagnostic strategy. lack of awareness and time ( . %) was the main reason for this . . percent heard the term frailty and . % knew that sarcopenia and frailty is not the same . . percent was aware of the scoring methods for the frailty and . % used clinical frailty score as the method. . % was aware of the frailty pathway but only . % knew whom to contact regarding managing frailty. . % heard the term comprehensive geriatric assessment. only . % was aware of key recommendations of managing frailty in the acute settings. conclusion: although concept of sarcopenia and frailty is familiar to most nhs healthcare professionals, the practical and clinical application is limited due to a lack of awareness regarding the diagnostic methodology, risks as well as time constrains. as such the benefits and potential treatment options may be overlooked and we aim to improve awareness so that these measures can improve outcomes for patients. mahtab alizadeh-khoei , fatemeh sadat mirzadeh , reyhaneh aminalroaya , fati nourhashemi (( ) gerontology & geriatric department, medical school, tehran university of medical sciences, ziaeian hospital, tehran, iran; ( ) department of internal medicine and clinical gerontology, toulouse, france) background: frailty is a potentially reversible geriatric syndrome associated with geriatric risk factors. detecting risk factors is a useful purpose to predict frailty levels incidence to plan for institutional or home care services. objectives: the aims were finding frail and prefrailty frequency in iranian geriatric outpatients' and determining demographics related factors and geriatric syndrome predictors on frailty levels, based on frailty fried index. methods: in this cross-sectional study elderly >= years old, selected by convenience sampling from geriatric day clinics in the area of tehran university of medical sciences. the effect of risk factors (adl and iadl dependency, obesity, and polypharmacy) and geriatric syndromes (falling, chronic pain, sleep problems, vertigo, vision and hearing impairments, incontinence, dementia, and depression) were evaluated on frailty fried index. predictor factors by logistic regression model were analyzed, according to demographic risk factors and geriatric syndromes. results: the mean age was / ± / years old, majority were male ( %). prefrailty was . % in men and . % in women based on fi. the significant risk factors in elderly prefrail women were depression ( . %), polypharmacy ( . %), visual impairment ( . %), and chronic pain ( . %); although, in prefrail men were vertigo ( . %), falling ( %), sleep disorder ( . %), and incontinence ( . %). in prefrail older adults>= years, only sleep disorder was significant. in logistic regression model, six significant predicted factors were included depression, iadl dependency, falling, chronic pain, vertigo, and age. depression increased the risk of prefrailty by . times, dependency in iadl increased . times; moreover, chronic pain and vertigo increased prefrailty risk about times. dependency on iadl increased the risk of frailty . times, and chronic pain and falling increased the risk of frailty about . times. by logistic regression model, % of prefrail outpatients elderly could be diagnosed. conclusion: geriatric syndromes in outpatients' elderly could predict prefrail more than frail elderly. in the iranian community dwellers prevalence of prefrailty was high, so the on-time screening and outpatients' interventions can help to prevent frailty. background: frailty is a key condition to be screened among elderly oncological patients. nevertheless, the use of the frailty index (fi) in onco-geriatrics is still limited. objectives: aim of our work is to measure the functional and prognostic value for -year mortality of the frailty index (fi) in a cohort of older women with gynecological cancer. methods: the prognostic value of fi was tested in older women with gynecological cancer (mean age = . years). fi was retrospectively calculated following the rockwood model[ ]. spearman's rho test was used for correlations with other oncological scales: eastern cooperative oncology group performance status (ecog); karnofsky performance status (kps); vulnerable elders scale- (ves- ). cox proportional hazard models and roc curve were performed to estimate prognostic role of -year mortality. sensitivity and specificity were also calculated. results: fi is normally distributed and descriptive statistics define our population as frail (mean = . ± . , range . - . ). . is confirmed as an upper limit compatible with life. fi doesn't significantly correlates with age, ecog and kps while it positively correlates with ves- (r= . , p < . ). fi is the strongest predictor for -year mortality confirmed after all adjustments for confounders (or . ; % ci . - . , p < . ) and by roc curve analyses ( . , % ci . - . , p=. ). conclusion: frailty index is a useful tool to detect vulnerability in onco-geriatrics and it predicts -year mortality. it predicts negative health-related outcomes (mortality) better than other traditional scales. its adoption may support a more efficient identification of patients in the need of adapted and personalized care. further studies are needed to confirm and extend these findings. background: frailty has been studied in the old population due to its association with negative outcomes but more information is needed about frailty in very old samples. the fried frailty phenotype (ffp) has been widely used and includes a set of objective indicators: weakness, slowness, unintentional weight loss, exhaustion and low physical activity. objectives: to determine which sociodemographic, functional and health-related variables predict ffp in a sample of community-dwelling individuals aged +yrs. methods: data from individuals living in the metropolitan area of porto were considered: sociodemographic information (age, sex, education level, living status), ffp ( - ), functionality (basic and instrumental activities of daily living), health information (nr. medicines, nr diseases, nr. falls, cognitive impairment, and self-perception of health). descriptive and correlational analysis were conducted and followed by a linear regression analysis (stepwise method) of variables significantly associated with ffp. results: participants' mean age was . years (sd= . ), they were mainly women ( . %), with - years of education ( . %) and living with a relative ( . %). high disability levels were found both for basic and instrumental activities of daily living. the mean of medicines intake was . (sd= . ) and of diseases . (sd= . ); . % of the participants rated their health as poor. the median number of falls in the last year was (iqr= ). participants scored on average . points (sd= . ) in mmse. gender or age were not associated with ffp. basic and instrumental activities of daily living, selfperception of health and cognitive performance significantly predicted ffp. in the adjusted model (r = . ), the stronger predictor was the higher dependency for basic activities of daily living, followed by worst self-perception of health and lower scores of cognitive performance. the dependency for instrumental activities of daily living lost its significance in the adjusted model. conclusion: our results identify three main predictors of ffp (basic activities of daily living, selfperception of health, and cognitive performance) in participants with advanced age. these results provide relevant information for further understanding of frailty and the ffp among the oldest old. background: unplanned hospital readmissions are associated with poorer prognosis and increased risk of functional decline and dependence in older people. identifying major risk factors and assessing clinical risk scores can help to distinguish patients at risk of worse outcomes and rehospitalization, allowing the proposal of preventive measures. the aim of this study was to compare the accuracy of different instruments and risk factors in predicting readmission, functional decline and death in hospitalized older patients in a brazilian geriatric unit. methods: in a cohort study performed at a geriatric unit, patients, years old or over were included. demographic data, functional status, prisma scale, geriatric depression scale, mini mental state examination, timed get up and go test, gait speed, mini nutritional assessment, palmar prehension strength, charlson comorbities index, frailty score of the cardiovascular health study and the senior index risk for rehospitalization were assessed at study admission. all patients received a follow-up telephone call at days after discharge to assess potential readmissions, deaths and functional status. results: mean age was . years (sd +- . ) and the mean barthel adl score was . (sd +- . ). altered barthel ( . ; ci % . - . ; p< . ), chs score ( . ; ci % . - . ; p< . ), isar-hp ( . ; ci % . - . ; p= . ), tgug ( . ; ci % . - . ; p< . ), palmar prehension ( . ; ci % . - . ; p= . ) and gait speed ( . ; ci % . - . ; p= . ) were associated with higher mortality days after discharge. the risk of functional decline at -month follow up evaluation was higher in patients with altered barthel ( . ; ci % . - . ; p< . ), lawton ( . ; ic % . - . ; p= . ), chs score ( . ; ci % . - . ; p< . ), isar-hp ( . ; ci % . - . ; p< . ), prisma ( . ; ci % . - . ; p= . ), tgug ( . ; ci % . - . ; p< . ), palmar prehension ( . ; ci % . - . ; p< . ) and gait speed ( . ; ci % . . ; p= . ). conclusion: altered iadl, frailty chs score, isar, tgug, palmar prehension strength and gait speed are predictive of functional decline and mortality days after hospital discharge. these tools can be useful to pinpoint frailty in older patients, allowing the implementation of preventive interventions to avoid functional decline. more research is needed to evaluate the role of these tools in predicting rehospitalization. to limit the strain on available resources and prevent an unnecessary increase in patient burden. objectives: this study aimed to improve patient selection for multi-disciplinary care by identifying risk factors for disability after cardiac surgery in elderly patients. methods: two-centre prospective cohort study in patients aged >= years undergoing elective cardiac surgery. before surgery frailty characteristics were investigated. outcome was disability at three months defined as world health organisation disability assessment schedule . >= %. multivariable modelling using logistic regression, concordance statistic (c-statistic), and net reclassification index were used to identify factors contributing patient selection. results: disability occurred in ( %) patients. ten out of frailty characteristics were associated with disability. a multivariable model including euroscore ii and preoperative haemoglobin yielded a c-statistic of . ( % ci . - . ). after adding prespecified frailty characteristics (polypharmacy, gait speed, physical disability, preoperative health related quality of life, and living alone) to this model the c-statistic improved to . ( % ci . - . ). net reclassification index was . (p< . ) showing improved discrimination for patients at risk for disability at three months. conclusion: using preoperative frailty characteristics improves discrimination between elderly patients with and without disability at three months after cardiac surgery and can be used to guide patient selection for preoperative multi-disciplinary team care. fabiola valero , , henry tapia , , enrique valencia , , tania tello , , (( ) facultad de medicina, universidad peruana cayetano heredia, lima, peru; ( ) instituto de gerontología, universidad peruana cayetano heredia, lima, peru; ( ) hospital cayetano heredia, lima, peru) background: frailty is increasingly recognized as a risk assessment to detect vulnerability and complexity. currently, there are limited tools to predict adverse perioperative outcomes for the geriatric population with hip fracture. objectives: to determine frailty and functional dependence as predictors of intrahospital adverse events in hospitalized older adults with hip fractures in the orthogeriatric unit of a general hospital in lima, peru. methods: we conducted a prospective cohort involving patients aged years or older who were admitted to the orthogeriatric unit with hip fracture from june to june . data were obtained at the time of admission to our unit: frailty was assessed with the frail scale, function ability with the barthel scale, cognition with the short portable mental state questionnaire (spmsq) scale of pfeiffer, comorbidities, socio-family assessment and geriatric syndromes. patients were followed up to discharge, and adverse events were evaluated during this period. univariate models were performed, and logistic regression was done subsequently. results: patients with hip fractures were evaluated, the mean age was . ( . ) years, . % ( ) were women and . % ( ) came from nursing homes. hypertension was the most frequent comorbidity in . % ( ). % ( ) had a history of functional dependence on basic activities of daily living (abvd), % ( ) had some degree of cognitive impairment, . % ( ) had social problems, polypharmacy in . % ( ) and . % ( ) history of falls in the last year. according to frail scale, . % (n = ) were robust, . % (n = ) were pre-frail and . % were frail (n = ). . % ( ) had an adverse event while hospitalized (pneumonia, uti, delirium, acute renal injury, pet), of whom % ( ) were robust, . % ( ) pre-frail and % ( ) frail (p = . ). . % of patients with functional dependence on abvd presented adverse events. in the multivariate analysis, the factors associated with in-hospital adverse events were functional dependence in abvd, or: . , (ci: . - . ); frailty with an or: . ic ( . - . ) and social problem, or: . ic ( . - . ). conclusion: older adult patients hospitalized for hip fracture who had frailty, functional dependence, and social problems had significant adverse events at a general hospital in lima, peru. aiko inoue , chi hsien huang , , chiharu uno , kosuke fujita , , tomoharu kitada , , joji onishi , hiroyuki umegaki , masafumi kuzuya , (( ) institutes of innovation for future society, nagoya university, japan; ( ) department of community health and geriatrics, nagoya university graduate school of medicine, nagoya, japan; ( ) department of business administration, seijoh university, aichi, japan) background: social frailty was associated with age, sex, income, education, marital status, and household status. however, the risk factors of social frailty relatively less investigated. objectives: the aim of this study is to clarify the risk factors of social frailty in community-dwelling japanese elderly. methods: a health promotion project (nagoya-teng project) is designed to distribute health promotion programs including enhancement of nutrition and physical activity via cable tv channel for community-dwelling elders. of all participants (n= ), participants with complete baseline information (mean age . ± . years, men ( . %)) were included in our cross-sectional analysis. at baseline, demographic data, socio-economic status, geriatric depression scale (gds- ), japanese version of european health literacy survey questionnaire (j-hls-eu-q ) were obtained. social frailty was defined by household status (living alone or not), financial difficulty, social activity, and fulfilment of social needs. total deficit scores of or more were defined as social frailty, as social pre-frailty, and as robustness. results: a total of ( . %), ( . %), and ( . %) of all participants were categorized as social non-frailty, pre-frailty and social frailty, respectively. in multivariable logistic regression model after adjusting for age, sex, bmi, and education level, living without a spouse is a significant risk factor (p< . ) for social pre-frailty (or . , % ci . - . ) and social frailty (or . , ). low gds- scores were associated with high risk of social prefrailty (or . , % ci . - . ) and social frailty (or . , % ci . - . ). in addition, health literacy was inversely associated with social frailty (or . , % ci . - . ). age, sex, and education level were not associated with social frailty. conclusion: regardless of age and sex, living with a spouse and depression which is associated with activity of daily living and quality of life are associated with social frailty. low health literacy is also a risk factor of social frailty. in literature, loneliness and social frailty were associated with functional decline and mortality in the elderly. future approaches incorporating health literacy interventions are warranted to prevent social frailty in the aged society with increasing number of physical frail older adults. background: frailty increases the risk for morbidity and mortality after cardiac surgery. the influence of frailty on postsurgical functional outcomes is largely unknown. objectives: the aim of this research was to study the association of preoperative frailty characteristics on adverse functional outcomes and to investigate the trajectory of functional recovery among frail and non-frail elderly patients up to one year after elective cardiac surgery. methods: a prospective two-centre observational cohort study in elective cardiac surgery patients aged >= years. preanaesthesia assessment was supplemented with frailty tests covering the physical, mental, and social domain. functional outcomes were assessed at one year and included change in health related quality of life (hrql) measured by the short form and disability measured by the world health organisation disability assessment schedule . . adverse functional outcome was considered when worse physical or mental hrql or disability was present after surgery. results: frailty characteristics were present in ( %) patients of whom ( %), ( %) and ( %) showed frailty in the physical, mental or social domain respectively. adverse functional outcome at one year after surgery occurred in ( %) patients. patients with an adverse functional outcome were more often frail ( ( %)) than patients without an adverse functional outcome ( ( %) p< . ). worse physical or mental hrql occurred in ( %) and ( %) patients respectively. the most important frailty characteristic associated with worse physical hrql was high preoperative physical hrql (β - . per point ( % ci - . to - . ). preoperative mental hrql showed the strongest associations for worse mental hrql (β - . per point ( % ci - . to - . )). disability was reported by ( %) patients and associated with preoperative polypharmacy, gait speed, health related quality of life, living alone or dependent living. gait speed had the strongest association (β . per second ( % ci . to . )). conclusion: preoperative frailty characteristics were common and predictive for adverse functional outcome one year after cardiac surgery. frailty screening can be used to improve risk stratification and decision making in older cardiac surgery patients. background: frailty frailty has many elements and these can be characterised as physical, nutritive (including body composition), cognitive and sensory (including hearing and seeing). the relative prevalence and importance of these elements are not known. objectives: to estimate the prevalence of frailty and relative contribution of physical/ balance, nutritive, cognitive and sensory frailty to important adverse health states (falls, physical activity levels, outdoor mobility, problems in self-care or usual activities, and lack of energy or accomplishment) in an english cohort. methods: analysis of community-dwelling older people. the sample was drawn from a random selection of all people aged or more registered with general practices across england. data were collected by postal questionnaire. frailty was measured with the strawbridge questionnaire. we used cross sectional, multivariate logistic regression to estimate the association between frailty domains and adverse health outcomes. some models were stratified by sex and age. results: mean age of participants was years (sd . ), range to and . % ( / ) were men. the prevalence of overall frailty was . % ( / ) and there was no difference in prevalence by sex (odds ratio . ; % confidence interval . to . ). sensory frailty was the most common and this was reported by more men ( / ) than women ( / ; odds ratio for sensory frailty . , % confidence interval . to . ). men were less likely than women to have physical or nutritive frailty. physical frailty had the strongest independent associations with adverse health states. however, sensory frailty was independently associated with falls, less frequent walking, problems in selfcare and usual activities, lack of energy and accomplishment. conclusion: physical frailty was more strongly associated with adverse health states, but sensory frailty was much more common. the health gain from intervention for sensory frailty in england is likely to be substantial, particularly for older men. sensory frailty should be explored further as an important target of intervention to improve health outcomes for older people both at clinical and population level. background: it live independently. our goal is to encourage independent living, wellbeing and to relieve health and care services budget pressure. longevity is one of the biggest achievements of modern societies. by , a quarter of europeans will be over years of age. combined with low birth rates, this will bring about significant changes to the structure of european society, which will impact on our economy, social security and health care systems. the most problematic expression of population ageing is the clinical condition of frailty. frailty develops because of age-related decline in multiple physiological systems. it is estimated that a quarter to a half of people over years are frail , and this is set to reach epidemic proportions over the next few decades. while frailty increases, the average amount of health spending increases as well with the frailty level in a range from , to , €/person year, depending upon the frailty status and the setting of care. frailty usually comes along associated with another risk facto; loneliness. then, ageing, frailty and loneliness constitute overlapping conditions submitted to multiple health and care interventions. ecare project aims to deliver disruptive digital solutions for the prevention and comprehensive management of frailty to encourage independent living, wellbeing and to relieve health and care services budget pressure, throughout the implementation of a pre-commercial procurement scheme. pre-commercial procurement is an ideal framework for the delivery of innovative solutions. the ecare network of procurers and the service providers are often on the frontline as new needs emerge. this pcp will allow the procurers to voice out their unmet needs, create a new demand to access sustainable products of higher quality, and develop new applications with lower life cycle costs. the demand and the supply side will work together to co-create and co-design the solutions and validate their functionalities against the specific challenges outlined in the pcp call for tender. this will clearly maximize the engagement of innovation in health and care services. solutions should improve outcomes for frailty in old adults entailing the physical and the psychosocial factors. the target group are the pre-frail/frail old adults with emphasis on those that feel lonely and/or isolated. the project will procure the development, testing and implementation of digital tools/services and communication concepts to facilitate the transition to integrated care models across health and social services and country-specific cross-institutional set-ups, including decentralised procurement environments and collaboration across institutions. objectives: the project objectives are: • newly development easy-to-use and reliable solutions that facilitate early detection of frailty based on the most efficient standards and methods. • improve the understanding of the factors affecting frailty and the feelings of loneliness and isolation, and how they do correlate (e.g.: gender dimension, social context, etc.). • deliver personalised intervention plans taking into account the end-user societal context. • innovative and meaningful means to tackle the feelings of loneliness and isolation. • new approaches to engage patients as active self-managers of their own health. • new technology developments designed and oriented to the target end-user. • and among all, investigate to deliver cost-efficient solutions, affordable to the payers involved. methods: ecare procurers will proactively organize the requirements of the demand for care solutions in a coherent way. the procurers (buyers' group) will assess the solution adequacy to the targets. the preferred partners will contribute with solid knowledge of innovative procurement paths to the innovation procurement tender. the project partners will do this by: • providing a solid and informed base for dialogue between stakeholders by determining a coherent picture of the market state of the art of the sector based on practical experience of customers and suppliers. • enabling a genuine and credible dialogue between the supply-chain and customers to determine the practical policy and procurement actions required to deliver the ecare solutions. • defining the common unmet needs, communicating these to stakeholders and initiating a mobilization plan for a pcp addressing ecare needs. the pcp may be summarized in a series of actions: • convey the relevance of innovation procurement to public procurers: encouraging suppliers to offer novel solutions to address ecare challenges rather than the lowest price solutions. • analyze the state of the art of the market with all potential suppliers, as well as the main problematic and barriers faced in the sector and that need to be overcome a set of actions involving both the supply and demand sides will be carried out: a coordinated first analysis of the state of the art conducted by all project members followed by a coordinated market sounding through all dissemination channels managed by the consortium will be undertaken to spread project results aiming to receive feedback from all key market players. for this, the role of procurers is vital to replicate and stretch the impact of the project. • providing public procurers with procurement know-how to improve public sector procurement efficiency and increase public sector market power by giving support to apply the methodologies of innovation procurement. market sounding will provide an opportunity for engagement and two-way dialogue with innovative companies that can offer solutions and guidance on how to overcome the procurement barriers. • launching an agreed, realistic and validated joint pcp tender. results: the ecare consortium is immerse in a deep process of unmet needs detection. our goal is to be extraordinarily concrete when defining what the end users and the healthcare professionals are willing for. those unmet needs will be critical for the definition of the requirements and uses cases that the it suppliers will have to follow to design the ict solutions. then… what a better way to know their needs that asking them personally? the vision of providing tailored fit solutions and tools to the end users led to the consensus in creating and facilitating focus group sessions across the procurers regions -campania (italy), barcelona (spain), santander (spain) and wroclaw (poland)-. these sessions will be involving end users, health and social care professionals, and it internal departments of the procurers' organisations. -the focus group script for the end users sessions integrates as main topics the specific condition and related symptoms; experiences of services and care provided; experiences of managing condition when progressing rapidly ; needs for symptom management and how these can be met ; integration of it supportive tools in the management of frailty and loneliness. -the professionals are invited to reflect and discuss the topics of common symptoms and actual care model; experiences of monitoring elderly when condition is progressing rapidly; views about the supportive care needs of elderly and caregivers; early integration of the new care in the management of frailty and loneliness; integration of it supportive tools in the management of frailty and loneliness. -the identified and proposed topics for the it staff would be the state of the art of the relation in between it and social/healthcare; state of the art of interventions on frailty and loneliness. all the four procurers were challenged to organize, at least, focus sessions, one with each specific target group. so far, all the procurers already organized and scheduled the sessions that will occur until the end of january. in terms of impact, participants are expected to be involved ( end users, healthcare professionals and it people). all the representative of the procurers reported so far that the participants have been considering the sessions so interesting and useful. in fact, new topics have been put in the table for discussion in all the different sessions, adding more important information for the definition of the unmet needs. the journey of the project so far has been providing very powerful insights and evidences that people and professionals appreciate to be involved and e(motionally) cared. conclusion: ecare will progress beyond the state of the art by approaching older people not just in terms of their diseases but also in terms of physical, cognitive and psychosocial care and support to prevent functional decline, frailty and disability. the project key components to address frailty are those that define also integrated care, with the addition of targeting high risk frail individuals, an enablement attitude and a focus on outcomes most relevant to frail individuals and their caregivers. for these, a multimodal comprehensive system able to provide the most effective care will need to be provided. background: maintaining autonomy as life progresses has become a challenge for the health systems. this objective can only be achieved by moving the axis of health policies and health care practice from the disease to the preservation of functional capacity. objectives: the aim of this study is to design and pilot a model for the assessment and support of functionality for community dwelling older people. methods: a space in which nurse and social worker jointly assess the functional capacity of older people and identify and provide responses to the detected deficits was proposed. this study was performed in osi donostialdea (gipuzkoa, spain). three main tasks were carried out: . definition of the joint assessment procedure of functionality. . identification of the existing resources and community assets to give answer to the identified needs. . piloting the model in a sample of older people. the identified needs and the availability of resources to respond to them were obtained from the pilot phase. results: in the initial version of this integral assessment were included, functional capacity, physical activity, cognitive capacity, sense organs, nutritional status, social assessment and housing and environmental conditions. a total of individuals ( % women; mean age years, sd= . ; barthel index, mean . , sd= . ; % living alone; % without cognitive impairment) were recruited during the pilot. the following needs were identified: personalized workout routines, fine motor skill exercises, visual and efficient diets adjusted to each patient, make sure resources reach the community, promote the use and design of gadgets to assist the needs of basic and instrumental activities of daily living, improve strategies to prevent cognitive function impairment, ease loneliness and avoid or minimize physical and environmental barriers to access home, to walk the streets and, particularly, to use public transport. there were no resources available for all the identified needs. conclusion: this study will allow the development of a model for the integral assessment of functionality for the aged population, based in a multidisciplinary team, a space and a new way of working in primary care. mónica machón - , maider mateo-abad , , mercedes clerencia-sierra , , , carolina güell , , beatriz poblador-pou , , kalliopi vrotsou - , antonio gimeno-miguel , , alexandra prados-torres , , itziar vergara - ( ( ) background: multimorbidity and frailty are often present in older people and are found to be associated to increased risk of adverse health events. it is necessary to improve the knowledge of the characteristics of such populations to design adequate clinical guidelines seeking to avoid or delay the onset of dependence. objectives: the aim of this study was to identify clusters of chronic diseases in robust and frail individuals and compare sociodemographic and health characteristics between these clusters. methods: this was a cross-sectional study based on data from two longitudinal studies. the sample was composed of functionally independent community-dwelling older people with multimorbidity living in gipuzkoa (basque country, spain). information from electronic health records (diagnose diseases and medication) and a baseline assessment (sociodemographic characteristics, functional status, self-perceived health, cognitive status, sight and hearing impairments, history of falls and nutritional status) was used in the analysis. the timed up and go test of physical performance was included as a measure of frailty. multiple correspondence and cluster analyses were performed to identify groups. results: the study population consisted of individuals ( . % women; mean age . years, sd= . ). frail individuals (n= ) were older, had a lower educational level and a poorer health status than robust individuals (n= ). three clusters were obtained in robust (rc , n= ; rc , n= and rc , n= ) and four among the frail individuals (fc , n= ; fc , n= ; fc , n= and fc , n= ). in rc and fc , none of the chronic diseases had a higher prevalence than in rc -rc and fc -fc -fc , respectively. individuals pertaining to rc and fc presented more frequently diseases related to mobility limitation or limb pain compare to the other clusters. higher rates of cardiovascular diseases and risk factors were seen in rc and fc . in frail individuals a new cluster emerged, fc , containing individuals with higher rates of cognitive and eye problems and a clearly poorer health status. conclusion: the findings obtained in this exploratory study may provide insight for the designing of more specific health interventions for older patients with multimorbidity, even though the chronic diseases cluster identified were similar in robust and frail individuals. background: older african americans (oaa) are at high risk for becoming frail in later life. interventions can reverse or delay frailty, yet oaa have largely been excluded from frailty intervention research. many interventions are also time and resource intensive, making them inaccessible to socially disadvantaged oaa. objectives: we present results of a feasibility trial of a low dose frailty prevention intervention among community-dwelling, pre-frail oaa aged + recruited from a primary care clinic between june st and october st . methods: using a -arm rct, participants were assigned to the intervention, which was delivered by an occupational therapist (ot) and comprised of four sessions over four months (an ot evaluation, and sessions on healthy dietary practices, increasing physical activity, and maintaining a healthy lifestyle), or enhanced usual care (publicly available information about healthy lifestyle, home safety, and local elder services). feasibility criteria were set a priori at % for participant retention (including attrition due to death/ hospitalization), % for session engagement, participants/ week for mean participant accrual, and % for program satisfaction. results: participants were % female with an average age of . years, . % of which lived alone and . % lived off of less than k per year. feasibility metrics were met. the study recruited . participants per week and retained % of participants who attended % of scheduled sessions. mean satisfaction scores were %. the treatment also resulted in positive trends in the expected direction in the treatment group for the following outcomes (d = effect size): global health (d = . ), mental health (d = . ), qol (d = . ), social functioning (d = . ), depression (d = . ), and pain reduction (d = . ). descriptively, treatment group participants were also less likely to experience a progression (deterioration) in three frailty status indicators at -months compared to controls: weight lost, walking speed slowness, and grip strength weakness. conclusion: the intervention was feasible to deliver. qualitative findings from exit interviews suggested changes to the program dose, structure, and content that could improve it for future use. background: it is well known that frail patients are potentially most at risk of functional decline following a hospital admission. objectives: to measure the effects of an augmented prescribed exercise programme versus usual care, on physical performance, quality of life and healthcare utilisation for frail older medical patients in the acute setting. methods: this was a parallel single-blinded randomised controlled trial. within two days of admission, older medical inpatients with an anticipated length of stay >= days, needing assistance/aid to walk, were blindly randomly allocated to the intervention or control group. until discharge, both groups received twice daily, monday-to-friday half-hour assisted exercises, assisted by a staff physiotherapist. the intervention group completed tailored strengthening and balance exercises; the control group performed stretching and relaxation exercises. length of stay was the primary outcome measure. blindly assessed secondary measures included readmissions within three months, and physical performance (short physical performance battery) and quality of life (euroqol- d- l) at discharge and at three months. time-to-event analysis was used to measure differences in length of stay, and regression models were used to measure differences in physical performance, quality of life, adverse events (falls, deaths) and negative events (prolonged hospitalisation, institutionalisation). results: of the patients allocated, patients' (aged ± . years) data were analysed. groups were comparable at baseline. in intention-to-treat analysis, length of stay did not differ between groups (hr . ( % ci, . - . ) p= . ). physical performance was better in the intervention group at discharge (difference . ( % ci, . - . ) p= . ), but lost at follow-up (difference . ( % ci, - . - . ) p= . ). an improvement in quality of life was detected at follow-up in the intervention group (difference . ( % ci, . - . ) p= . ). overall, fewer negative events occurred in the intervention group (or . ( % ci . - . ) p= . ). conclusion: improvements in physical performance, quality of life and fewer negative events suggest that this intervention is of value to frail medical inpatients. its effect on length of stay remains unclear. background: to propose a simple frailty screening tool able to highlight frailty profiles, already since the initial screening phase. methods: a -item questionnaire (lorraine frailty profiling screening scale, lofpross), constructed by an experts' working group, was administered by health professionals to participants > years old (n= ) and living at home, in different clinical settings: a primary care outpatient clinic (rural population, n= ), a geriatric day clinic (day-clinic population, n= ) and healthy volunteers (urban population, n= ). a multiple correspondence analysis (mca) followed by a hierarchical clustering of the results of the mca performed in each population was conducted to identify participant profiles based on their answers to lofpross. a response pattern algorithm was resultantly identified in the rural (main) population and subsequently applied to the urban and day-clinic populations and, in these populations, the two classification methods were compared. finally, clinically-relevant profiles were generated and compared for their ability to similarly classify subjects. results: the response pattern differed between the subpopulations for all items, revealing significant intergroup differences ( . ± . positive responses for urban vs. . ± . for rural vs. . ± . for day-clinic, all p< . ). five clusters were highlighted in the main rural population: "non-frail", "hospitalizations", "physical problems", "social isolation" and "behavioral", with similar clusters highlighted in the remaining two populations. identification of the response pattern algorithm in the rural population yielded a second classification approach, with % of tested participants classified in the same cluster using the different approaches. three clinically-relevant profiles ("non-frail" profile, "physical frailty and diseases" profile and "cognitive-psychological frailty" profile) were subsequently generated from the clusters. a similar double classification approach as above was applied to these profiles revealing a very high percentage ( . %) of similar profile classifications using both methods. conclusion: the present results demonstrate the ability of lofpross to highlight frailty-related profiles, in a consistent manner, among different older populations living at home. such scale could represent an added value as a simple frailty screening tool for accelerated and better-targeted investigations and interventions. ( ) homburg/saar/germany, saarland university medical center, neurology, homburg/germany) background: frailty is the most important short and long term predictor of disability in the elderly. no study to date evaluate the impact of frailty on short and long term independently from neurological outcome measures. objectives: the aim of the study was to evaluate whether diagnosis frailty predicts short and long-term mortality and neurological recovery in old patients who underwent reperfusion acute treatment in stroke unit. methods: consecutive patients were older than years who underwent thrombectomy or thrombolysis in a single stroke unit from to . predictors of stroke outcomes were assessed including demographics, baseline nihss, time to needle, treatment and medical complications. premorbid frailty was assessed with a comprehensive geriatric assessment (cga) including functional, nutritional, cognitive, social and comorbidities status. at and months, all-cause of death and clinical recovery (using mrs) were evaluated. results: patients, of whom underwent mechanical thrombectomy and venous thrombolysis (mean age . , - years) entered the study. frailty was diagnosed in out of patients and associated with older age (p= . ) but no differences in baseline nihss score or treatment strategies. at follow-up, frail patients showed higher incidence of death at ( % vs %, p= . ) and ( % vs %, p= . ) months. frailty was associated with worse neurological recovery at month (mrs . + . vs . + . , p= . ) and one year followup (mrs . + . vs . + . ) for free survival patients. conclusion: frailty is an important predictor of efficacy of acute treatment of stroke beyond classical predictors of stroke outcomes. larger prospective studies are warranted in order to confirm our findings. background: frailty becomes increasingly common as adults age and has known associations with activity limitations and injurious falls among older adults. while it is believed that frailer older adults are less socially connected than their more functional counterparts, less is known about the relationship between frailty and social isolation among community-dwelling older adults. objectives: the purpose of this study was to examine associations of frailty indicators on self-reported social isolation risk among community-dwelling adults age years and older. methods: the upstream social isolation risk screener (u-sirs) was developed to assess social isolation risk among older adults within clinical and community settings. comprised of items (cronbach's alpha= . ), the u-sirs assesses physical, emotional, and social support aspects of social isolation. using an internet-delivered survey, data were collected from a national sample of , adults age years and older. participants completed the u-sirs and additional items on sociodemographics and other health risks. theta scores for the u-sirs serve as the dependent variable, which were generated using item response theory. an ordinary least squares regression model was fitted to identify frailty indicators associated with social isolation risk. results: participants' average age was . (± . ) years. the majority of participants was female ( . %) and lived with a partner/spouse ( . %). twenty eight percent of participants reported difficulty walking or climbing stairs, . % reported difficulty dressing or bathing, and . % reported a fall in the past year. higher u-sirs theta scores were reported among males (b= . , p< . ) and those with more chronic conditions (b= . , p< . ). participants who reported difficulty walking or climbing stairs (b= . , p< . ), difficulty dressing or bathing (b= . , p= . ), or a fall in the past year (b= . , p< . ) also reported higher u-sirs theta scores. further, higher u-sirs theta scores were reported among participants who had not left their home in the past three days (b= . , p< . ). conclusion: findings suggest frailer older adults and those with functional limitations may have greater risk for social isolation. this highlights the critical demand for easy-to-administer and practical assessments for frail older adults that identify their social isolation risk and link them to needed resources and services. background: peak expiratory flow (pef) has been linked to several negative health-related outcomes in older people, but its association with frailty is still unclear. objectives: this study investigates the association between pef and prevalent and incident frailty in older adults. methods: data come from community-dwelling participants of the swedish national study on aging and care in kundgsholmen (snac-k), aged >= years. baseline pef was expressed as standardized residual (sr) percentiles. frailty was assessed at baseline and over six years, according to the fried criteria. associations between pef and frailty were estimated crosssectionally through logistic regressions, and longitudinally by multinomial logistic regression, considering death as alternative outcome. obstructive respiratory diseases and smoking habits were treated as potential effect modifiers. results: our crosssectional results showed that the th- th and < th pef sr-percentile categories were associated with three-and fivefold higher likelihood of being frail, than the th- th one. similar estimates were confirmed longitudinally, i.e. adjusted or= . ( %ci: . - . ) for pef sr-percentiles< th, compared with th- th. associations were enounced in participants without physical deficits, and tended to be stronger among those with baseline obstructive respiratory diseases, and, longitudinally, also among former/current smokers. conclusion: these findings suggest that pef is a marker of general robustness in older adults and its reduction, exceeding that expected by age, is associated with frailty development. background: as consistently reported in the literature, muscle strength (ms) decreases at a higher rate than muscle mass (mm) during aging resulting in a decreased muscle quality (mq). loss of mq has been associated with loss of mobility, falls, frailty and an increased risk of mortality. however, the degree of muscle declines is varying throughout the population leading to states: successful, normal or pathological. it has been proposed that healthy life habits such as be physically active, having a healthy diet etc. could reduce the muscle aging decline. thus, identifying if life habits could counteract or maintain muscle quality during successful aging is important to better characterize aging and to intervene more specifically. objectives: the aim of the present study was to identify whether a physically active lifestyle could attenuate the effects of aging on mq. methods: active young were compared to active older men. to be considered active, young and older men need to practice voluntary physical activity at least min/week since yrs. body composition (dxa; mri) and maximum knee extension strength were measured. mq was calculated as the ratio of ms to mm. aerobic capacity (vo max; moxus©) and muscle contractility (emg) were also measured. muscle biopsies were performed to determine fiber typing, size, intermuscular adipose tissue (imat) and intramyocellular lipid content (imcl). results: absolute mm (p< . ) and ms (p= . ) was greater in young participants compared to their older counterparts while mq was similar between them. even if total (p= . ) and type iia (p= . ) fiber size were greater in ya than in oa, muscle fiber proportion, muscle contractility and lower limb fat mass (imat, imcl) were similar between both groups (p> . ). conclusion: mq was similar between younger and older physically active men suggesting that being physical activity may have mitigated the loss of mq with aging and delayed some physiological age-related changes (muscle composition, contractility). i r a t x e e g a ñ a , itxaso mugica , , nagore arizaga , maider ugartemendia , nagore zinkunegi , janire virgala , maider kortajarena ( ( ) and sppb test (p< , ). similar results have been found in other researches. the parameters that have higher influence in cognition are handgrip test (p< , ) and frailty (p< , ). in other investigations, they got the same results; better cognition is related to better physical capacity and less fragility. in regards with functionality, the values of tug test (p< , ) and gait speed (p< , ) are the ones that show stronger relation. in other investigations, they observed that physical state and functionality were related. conclusion: the quality of life, the functionality and moca test are interconnected and the parameters that have the strongest statistical relationship are fragility and physical state. the greater the physical capacity of the older person is, the greater the functional capacity is too and the fragility decreases. in conclusion, the quality of life is better. kazuki kaji , jun kitagawa , takahiro tachiki , naonobu takahira , masayuki iki , junko tamaki , etsuko kajita , yuho sato , jpos study group (( ) national center for geriatrics and gerontology, obu, aichi, japan; ( ) nagoya university, nagoya, aichi, japan; ( ) nagoya city university, nagoya, aichi, japan) background: the skeletal muscle mass index (smi), which is the appendicular skeletal muscle mass (asm) adjusted for height squared (kg/m ), is used to assess skeletal muscle mass. we reported at this conference last year that smi was overestimated by height loss due to aging in elderly women. furthermore, age-related changes in smi were inconsistent with changes in physical function such as grip strength and walking speed. objectives: the purpose of this cross-sectional study was to investigate the effects of height loss on agerelated changes in smi and physical function in japanese women aged or older. methods: this study was part of the / -year follow up survey of the japanese population-based osteoporosis (jpos) cohort study conducted in / . the jpos study was started in . the subjects of the / year follow-up were women (mean . ± . years). we divided the subjects into quartiles based on years of height loss (q : the lowest, q , q and q : the highest). asm was measured by dual x-ray absorptiometry (qdr a, hologic, usa). grip strength, maximum walking speed, and timed up and go (tug) were also measured. results: the mean change in height during the / -year follow-up was - . ± . cm. mean changes in height in q (n= ), q (n= ), q (n= ) and q (n= ) were - . ± . cm, - . ± . cm, - . ± . cm and - . ± . cm, respectively. the trend test demonstrated significant increases in the mean age and smi from q to q . on the other hand, there was a significant decrease in asm from q to q . the mean grip strength and maximum walking speed significantly decreased from q to q . tug results were similar, suggesting that greater height loss led to longer times. conclusion: in japanese elderly women with height loss, asm and physical function decreased with age, but the smi adjusted for height increased. it may be necessary to establish a muscle mass parameter other than smi to investigate the relationship between muscle mass and physical function. kota tsutsumimoto , takehiko doi , sho nakakubo , satoshi kurita , hideaki ishii , hiroyuki shimada (( ) section for health promotion, department of preventive gerontology, center for gerontology and social science, national center for geriatrics and gerontology, aichi, japan; ( ) center for gerontology and social science, national center for geriatrics and gerontology, aichi, japan) background: sarcopenia was defined as decline in skeletal muscle mass and muscle function, leading to serious health problems including disability. the modifiable risk factors of sarcopenia should be elucidated to contribute to develop intervention from sarcopenia. objectives: to examine the association between anorexia of aging and sarcopenia among community-dwelling elderly japanese individuals. methods: population-based, cross-sectional cohort study in japanese older adults was conducted and participants were identified from the database of the national center for geriatrics and gerontology-study of geriatric syndromes. anorexia of aging was assessed via a simplified nutritional appetite questionnaire. handgrip strength and walking speed were tested, and skeletal muscle mass was assessed using a bio-impedance analysis device. subjects with sarcopenia were defined as those who met the criteria of the asian working group for sarcopenia. the association between anorexia of aging and sarcopenia was then analyzed via multiple regression analysis. results: in total, , elderly japanese individuals were evaluated. the prevalence of sarcopenia and anorexia of aging was . % and . %, respectively. in multivariable logistic regression model adjusted for the covariates except for nutritional status such as albumin, anorexia of aging was independently associated with sarcopenia (or: . , % ci: . to . ; p = . ). this significant association remained even after adjusting for all covariates including nutritional status (or: . , % ci: . to . , p = . ). conclusion: anorexia of aging is associated with sarcopenia among japanese older adults. further studies are needed to determine whether a causal association exists between anorexia and sarcopenia. background: low grip strength is consistently associated with higher rates of mortality, disability and other age-related health outcomes, and is a key characteristic of sarcopenia. grip strength has thus been proposed as a general biomarker of ageing. life expectancy in russia is substantially lower than in norway but whether this is reflected in differences in grip strength across adulthood, as observed in previous comparisons of older adults from russia, denmark and england, needs to be established and explained. objectives: we aimed to compare grip strength in norwegian and russian populations by age and gender, and investigate whether any observed differences were explained by contrasts in height, weight, smoking or education. methods: we used harmonised cross-sectional data on grip strength for , men and women aged - years. this comprised participants from the russian know your heart study (n= , ) conducted in the cities arkhangelsk and novosibirsk in - , and from wave of the norwegian tromsø study (n= , ) conducted in - . grip strength was assessed using the jamar+ digital dynamometer in both studies, and the maximum of six measurements (three in each hand) was used. the association between grip strength and covariates was assessed using linear regression. results: norwegian males had stronger grip than russian males at all ages, for example they were an average of . kg ( % confidence interval (ci) . , . ) stronger at age years and . kg ( % ci . , . ) stronger at age years. among women, corresponding numbers were . kg ( % ci . , . ) at age and . kg ( % ci . , . ) at age . adjustment for weight, education and smoking did not affect the results, but height attenuated the between country differences, especially at older ages. among women aged +, differences in height between countries fully explained the differences observed in grip strength. conclusion: norwegian -year-olds had the grip strength of -year-old russians suggesting that russians are ageing more rapidly in terms of muscular strength than their norwegian counterparts. the important role of height in explaining these differences, especially at older ages, suggest contrasts in early life circumstances may be of key importance. eleanor lunt , , paul greenhaff , , adam l gordon , , , john rf gladman , ( ( ) background: frailty is a state of vulnerability to stressors resulting in adverse clinical outcomes including falls and fragility fractures. identifying biomarkers associated with these outcomes may help target interventions. objectives: to compare parameters of body composition, muscle thickness and muscle strength between patients and healthy older and young volunteers. methods: six young ( - years) and older (>= years) healthy female volunteers were recruited by advert from community groups. female patients (>= years) with an acute fragility fracture were recruited from hospital wards and measured during first week of admission (median th day (iqr - )). frailty was determined by the -item frail scale. height, weight, handgrip (jamar dynamometer) and knee extension (lafayette manual muscle tester) were assessed. body composition was estimated using whole body bioelectrical impedance (bodystat quadscan ®). midpoint vastus lateralis (vl) muscle thickness and mid-thigh subcutaneous fat thickness were assessed using ultrasound (mylab gold, esaote biomedica, italy) with a hz linear-array probe. oneway anova and post hoc tukey's test were used to compare end-point measures between groups. results: frailty was significantly more prevalent in the patient group ( % frail, % pre-frail, % robust) than the healthy older group ( % robust, p< . ). the patient group was older ( ± years vs ± years, p< . ) and had more co-morbidities (p< . ). there were no significant differences between the patient and healthy older group in weight, height, bmi, percentage body fat or subcutaneous fat thickness of lateral thigh. vl muscle thickness was lower in the patient group compared to healthy older and young volunteers ( . ± . cm, . ± . cm and . ± . cm respectively, p< . ). the patient group also had lower handgrip strength ( . ± . kg, . ± . kg, . ± . kg respectively, p< . ) and lower knee extension strength ( . ± . kg, . ± . kg, . ± . kg respectively, p< . ). vl muscle thickness associated with muscle strength (knee extension r= . , p< . and handgrip r= . , p< . ) and was significantly lower in the frail compared to pre-frail or robust participants ( . ± . cm, . ± . cm, . ± . cm respectively p< . ). conclusion: female patients presenting to hospital with a fall and fragility fracture have lower muscle thickness in the thigh compared to non-frail older women, despite no difference in other body composition variables. register, health technology assessment, nhs economic evaluation database) were searched from inception to april , . cross-sectional and cohort studies that reported adjusted risk ratios with % confidence intervals (ci) for frailty with serum level of total testosterone, free testosterone, sex hormone-binding globulin (shbg) were selected. a metaanalysis was carried out by using fixed effects and random effects models to calculate the or of relationship between low level of testosterone and risk of frailty. results: the crosssectional study concluded articles, there was statistically significant association between lower level of total testosterone and risk of frailty (or= . ; %ci, . - . , i = %), as well as free testosterone (or= . ; %ci, . - . ,i = % ), the highest level of shbg was no significant associated with the risk of frailty(or= . ; %ci, . , . ; i = %). the prospective cohort studies obtain articles, no significant were found between frailty and low total testosterone and frailty (pool or= . ; %ci, . - . , i = %). conclusion: the meta-analysis indicates that low level of serum testosterone is significantly associated with the risk of frailty in the crosssection studies. however, we found no significant relationship between low total testosterone and frailty in the cohort studies. more research is needed to address the underlying mechanisms to explain this relationship and to determine whether testosterone supplementation is effective for preventing frailty syndrome. background: although frailty and abdominal obesity are known risk factors for disability in older persons, few studies have investigated the interaction between both factors on the association with disability. objectives: to investigate the association of frailty and abdominal obesity with disability in older persons. methods: we used data from , participants ( % men) in the prospective, population-based singapore chinese health study cohort, who were interviewed and examined for frailty, abdominal obesity and disability at mean age of (range to ) years from - . we defined frailty as having three or more features of weak handgrip strength, slow timed-up-and-go test, low energy level, multiple comorbidities, and difficulty carrying out usual activities. we defined abdominal obesity by waist circumference using sexspecific cut-offs, and assessed disability using the lawton instrumental activities of daily living (iadl) scale. we used multivariable logistic regression models to compute the odds ratio (or) and % confidence interval (ci) for the association between frailty/abdominal obesity and disability. results: about . % of participants were frail and . % had abdominal obesity. frailty was associated with increased or ( % ci) of . ( . - . ) for disability. conversely, the or ( % ci) for the association between abdominal obesity and frailty was only . ( . - . ). compared to participants who were neither frail nor abdominally obese, the or ( % ci) for disability was . ( . - . ) in those who only had frailty, and . ( . - . ) in those who only had abdominal obesity. however, participants who were both frail and abdominally obese had markedly increased or ( % ci) of . ( . - . ) for disability; p-value for interaction between frailty and abdominal obesity was . . furthermore, while men who were both frail and abdominally obese had increased or ( % ci) of . ( . - . ) for disability compared to their counterparts who were neither frail nor obese, the corresponding or ( % ci) was much higher at . ( . - . ) in women; p value for heterogeneity by sex < . . conclusion: frailty and abdominal obesity interacted synergistically to increase the risk of disability in older persons, and the combined effect of both factors on disability was much stronger in women than in men. background: as the world's population ages, the prevalence of cognitive impairment associated with age increases exponentially. objectives: objective of this study was to investigate the longitudinal association of physical activity and cognitive function in two deferentl populations; older adults from mexico representing latin america and south korea representing asia. based on two large population-based longitudinal studies. methods: this is a secondary analysis of two surveys, mhas and klosa, designed to study the aging process of adults living in mexico and south korea. participants> were selected from rural and urban areas. here we investigate the longitudinal association of exercise and cognition using the two waves of each study. cross cultural cognitive examination and mini-mental state examinarion were used to analyze the association between physical activity and cognition in mexican and korean older adults. multivariate logistic regression models were used to evaluate the said association. results: in mexico, the prevalence of physical activity was . %, physical active older adults obtained a higher score in ccce ( . ± . ) p-value < . . they also had more years of education ( . ± . vs. . ± . ) p-value < . , had depression ( . % vs. . %) . and consumed less alcohol ( . vs. . ) p-value < . . in korea, the prevalence of physical activity was . %. the physical active group performed better in mmse (- . ± . vs. . ± . ) p-value < . . the no physical active group had a higher proportion of women, less alcohol consumption ( . vs. . %) p-value < . , fewer years of education p-value < . and a higher prevalence of depression ( . % vs . %) p-value . . in the multivariate analysis an independent association was found in the korean population between physical activity and mmse score even after adjusting for confounders ( . ( . ; . ) p value . ). conclusion: physical activity could have a protective effect on the cognitive decline associated with ageing. background: aging is related to the increase of several chronic diseases, such as, osteoarthritis, osteoporosis, diabetes, hypertension and sarcopenia. sarcopenia (progressive loss of muscle mass and physical performance) is related to difficulties in treating other comorbidities, whether pharmacologically or non-pharmacologically. it's important to understand the relations between muscular strength (w), muscular mass and the phase angle (pa) of bioimpedance, in sarcopenic subjects to prescribe more accurate treatments. objectives: to study the relations of skeletal muscle index (smi) with w, pa and the presents of comorbidities (nc) in elderly subjects. methods: a prospective, observational secondary analysis of data from the "the sarcopenia screening and health related issues in the region of algarve", was performed. community independent living elderly subjects were recruited. body composition was measured by bioimpedance (seca analytics ), knee flexion and extension isokinetic strength ( º/sec) (humac norm). a screening questionnaire was used to determine the presence of comorbidities. smi levels were assessed using european working group on sarcopenia in older people cut-off points. results: a total of female and males, were included, mean age , (± , sd). subject were divided into groups according to smi: normal (n= ), moderated impairment (n= ) and severe impairment (n= ). pearson correlation were calculated within each group for w; pa and comorbidities. normal smi level, were correlated to knee extensors w in both legs (right: r= , , p< , and left r= , , p< , ) . no significant correlations were found with pa. moderate smi level: were correlated to knee extensors w in both legs (right: r= , , p< , and left r= , , p≤ , ), and also with knee flexors w (right: r= , , p< , ; left: r= , , p< , ). a moderate correlation was also found in this group with pa (r= , , p< , ). severe smi level: no correlations were found, in this group, with w. a moderate correlation was found with pa (r= , , p< , ). comorbidities did not have any correlations with smi levels. conclusion: our results seem to indicate that isokinetic strength (work) may have in the future a role in understanding sarcopenia, once it is related to smi. also, pa may indicate moderate and severe smi impairment. background: body characteristics as low muscle mass and high fat mass (fm) affect the physical function of older people. physical function is a fundamental component for the performance of daily activities and for the maintenance of the independence of older adults. however, the relationship between body composition and physical performance varies in different studies and still demands further research. objectives: this study aimed to investigate the association of fat mass index (fmi) determined by dual-energy x-ray absorptiometry (dxa) with physical performance in brazilian communitydwelling older adults. methods: a cross-sectional study with a sample of participants aged years and older, living in ribeirão preto, brazil, including both men and women, was conducted. fm was measured by dxa and fmi was calculated as fat mass/height (kg/m²). the physical performance was assessed by the -minute walk test, and walking distance was recorded as the main parameter, considering the distance predicted by sex. the kolmogorov-smirnov test was used to verify the normality of data distribution. the association of physical performance and fmi was analyzed using the pearson's correlation test and statistical significance was set at p ≤ . (two-sided). results: the participants were aged . ± . years, fmi was . ± . kg/m and distance walked was . ± . m. there was a significant negative association (r = - , p = . ) between fmi and distance walked, showing that higher fat mass index is associated with worse performance in the -minute walk test. conclusion: high fat mass index is associated with worse physical performance in brazilian older adults. background: sarcopenia and physical frailty have been shown to be risk factors for mortality and major morbidity in older adults suffering from various forms of cardiovascular disease. ultrasound measurement of quadriceps muscle thickness (qmt) is an emerging biomarker for sarcopenia, which we hypothesized could be conveniently acquired during the routine echocardiographic exam. objectives: to demonstrate the feasibility of measuring qmt at the time of echocardiography, and determine the association between qmt and clinical indictors of frailty. methods: adult inpatients and outpatients undergoing a clinically-indicated echocardiogram for known or suspected cardiovascular disease were recruited for this cross-sectional study at the jewish general hospital. prior to the echocardiogram, trained research assistants measured height, weight, and three clinical indicators of frailty: rockwood's clinical frailty scale, handgrip strength (jamar dynamometer), and bioimpedance phase angle (inbody ). at the conclusion of the echocardiogram, cardiac sonographers blinded to the preceding assessments acquired a biplane image of the anterior thigh midway between the anterior superior iliac spine and knee, and measured qmt as the combined thickness of the rectus femoris and vastus intermedius muscles. a cardiac ultrasound machine and probe were used (ge vivid e /e , . - . mhz probe). results: the cohort consisted of patients, of which had an available measure of qmt. the acquisition and measurement of qmt added - minutes to the echocardiographic exam. the mean age was +/- years with % females. the mean qmt was +/- mm, similar in men and women, with the lowest quintile being < . mm. higher age and lower body mass index were associated with lower qmt. after adjustment for age, sex, and body mass index, qmt was found to be associated with the multivariate composite of frailty indicators (p< . ), particularly with the clinical frailty scale (beta - . per mm; ci - . , - . ) and bioimpedance phase angle (beta . per mm; ci . , . ). additional adjustment for heart failure and inpatient status did not alter results. conclusion: qmt can be efficiently measured during a routine echocardiographic exam and can add incremental insights about frailty in a diverse group of patients with cardiovascular disease. background: frailty is a clinical syndrome whose signs and symptoms are predictors of health complications, making this a major public health problem. objectives: this study aims to evaluate the prevalence of frailty, in communitydwelling older adults enrolled in a physical exercise program in the north region of portugal, based on fried's phenotype, its association with other variables. methods: in this crosssectional analysis, we used data from individuals who were enrolled in physical exercise programs. gender and age standardized prevalence and the association between frailty and sociodemographic (age, gender, marital status, education, shortage of money) physical (self-perceived health, polypharmacy, physical fitness, vision, hearing), cognitive (memory), social (emptiness, loneliness and abandonment) and psychological (depression and anxiety) variables were evaluated. results: of the participants, the mean age was . ± . years old, and . % were female. prevalence of pre-frailty and frailty were of . % and . %, respectively. from the fried's phenotype criteria, exhaustion is the most common reported by . % of the pre-frail and . % of the frail participants. age, marital status, self-perception of health, physical fitness, memory and depression were found to be independently associated with pre-frailty, while age, education, self-perception of health, physical fitness and anxiety were independently associated with frailty. conclusion: we reported lower prevalence of pre-frailty and frailty compared with other studies, showing that physical exercise may delay the progression of frailty. interventions aimed to prevent frailty must address the diversity of the associated variables. background: frailty is related with ethnicity and impaired physical capacity which is also affected by diabetes. however, little is known about how physical health indicators of frailty are associated with each other in older hispanics with diabetes. objectives: the goal of this study was to investigate the relationship between physical health indicators of frailty in older hispanics with diabetes. methods: thirty-eight older hispanics with diabetes ( women, men, age = ± years) participated in the study. the variables included age, weight, body mass index, body composition (% of muscle mass and body fat -bio-impedance), fear of falls (falls efficacy scale international -fes-i), chair stands in sec, grip strength (jamar® dynamometer), balance with eyes open and closed (force plate), preferred walking speed, gait velocity during regular and reduce time street crossing simulations (gaitrite®). results: characteristics: body mass = ± kg, % of muscle mass = ± %, % of body fat = ± %, fes-i score = ± points, chair stands = ± repetitions, grip strength = ± kg, center of pressure area with eyes open = ± cm and with eyes closed = ± cm , preferred walking speed = ± cm/s, gait velocity during regular = ± cm/s and during reduced time street crossing = ± cm/s. there were significant correlations (*p< . , **p< . ) between age and gait velocity during regular street crossing (r = - . *); grip strength and % of body fat (r = - . **) and % of muscle mass (r = . **); chair stands and preferred walking speed (r = . **), gait speed during regular (r = . **) and during reduced time street crossing (r = . **) and center of pressure area with eyes closed (r = - . *), and between fear of falls and center of pressure area with eyes closed (r = . **). conclusion: gait speed during street crossing simulations decreased with age. greater grip strength was associated with lower % of body fat and higher % of muscle mass. people who completed less chair stands in s also walked slower and had worse balance, and those with poor balance had increased fear of falls. britta c arends, lisa verwijmeren, peter g noordzij, douwe h biesma, leon timmerman, eric pa van dongen, heleen j blussévan oud-alblas (st. antonius hospital -nieuwegein, netherlands) background: chronic pain after cardiac surgery is common and has a negative impact on quality of life. frailty is an important risk factor for adverse surgical outcomes. the influence of frailty on chronic pain after cardiac surgery is unknown. objectives: this study aimed to address whether frailty characteristics were associated with chronic pain after cardiac surgery in an older population. methods: this study was based on the anesthesia geriatric evaluation (age) and quality of life after cardiac surgery study, which included patients >= years undergoing elective cardiac surgery. preoperatively, frailty was tested in physical, mental and social domains. pain was evaluated with the short form questionnaire (sf- ) preoperatively and one year after surgery. multivariate logistic regression was used to investigate the association between frailty and chronic pain. change in health related quality of life (hrql) was analyzed to evaluate the impact of chronic pain. results: ( %) patients were included in the analysis. / patients ( %) reported new or increased pain one year after surgery. in patients ( %) at least one frailty characteristic was present and patients ( %) were frail in two or more domains. after adjustment for possible confounders in multivariate analysis, patients with single status and polypharmacy were at increased risk for new or increased chronic pain (aors . ( % ci . - . ) and . ( % ci . - . ). new or increased chronic pain was associated with a worse hrql (aor . ; % ci of . - . ). conclusion: frail patients are at risk for chronic pain and worse hrql after cardiac surgery. future research should focus on perioperative interventions to reduce chronic pain in elderly patients. background: frailty is a vulnerability state that is associated with negative outcomes such us falls, in-hospital admissions and mortality. many factors can contribute to the pathogenesis of frailty and nutritional status is playing and important role. that´s why undernutrition and frailty must be overview in older adults before surgical procedures in order to treat them earlier. objectives: identify the relationship between physical frailty and undernutrition in older adults undergoing elective abdominopelvic surgery in a general hospital in lima-perú. methods: this is a secondary database study from the original "physical frailty and adverse events in older adults undergoing elective pelvic abdominal surgery in a general hospital, lima-perú", it was realized between august and march , using validated face to face questionnaires. physical frailty was determined with fried criteria, undernutrition by mini nutritional assesment (mna). in adition, they also evaluated functional status and cognition. univariate models were performed, and logistic regression was done subsequently. results: older adult met inclusion´s criteria, the mean age was . (+ . ) years old, , % ( ) were female, , % ( ) had hypertension, , % ( ) were diabetic, the mean number of comorbidities were . (+ . ), , % ( ) had functional impairment, , %( ) had cognitive impairment. the mean bmi was . ± . . , % ( ) were underweight, . %( ) normal , . %( ) overweigth and . % ( ) obese. by mna % ( ) had risk or undernutrition, . % ( ) of them had functional impairment in contrast with , %( ) who weren´t at risk or undernutrition; p= . . also, . %( ) who had risk or undernutrition had cognitive impairment in contrast with . %( ) who weren´t at risk or undernutrition; p= . . by fried criteria, % ( ) were frailty, % ( ) prefrailty and , % ( ) robust. the frailty patients % ( ) had risk or undernutrition vs , %( ) in prefrailty and . %( ) in robusts; p= . . conclusion: there is an increased risk of undernutrition in frail older adults undergoing abdominopelvic surgery at a general hospital in lima, peru. background: cognitive frailty increases the risk of dementia, dependency and mortality in older people. moderatevigorous physical activity (mvpa) improves frailty syndrome and cognitive functions in older people, but being physically inactive is still prevalent. walking is the most common and inexpensive form of physical activity in older people and brisk walking is a form of mvpa. m-health has been successful in changing health behaviours in many populations. however, its effect in treating cognitive frailty through promoting mvpa in older people is not known. objectives: the aims of this study were to examine the effects and feasibility of an m-health intervention. methods: a pilot randomized controlled trial was employed. eligibility criteria include ) age > years, ) living in community, ) having cognitive frailty, and ) mobility at "outdoor walker" level. the study was conducted in community settings. subjects were recruited in the elderly community centres. subjects were randomized into either intervention or control at a : ratio. in the intervention groups, the subject received a smartphone pre-installed with physical activity tracking and social media applications. they received a course of brisk-walking in daily living training, health education, and a -week behavioural change intervention on the smartphone platform. in the control group, participants received a course of brisk-walking in daily living training, health education, and telephone follow-up. the outcomes were frailty (ffi), cognitive function (moca) and mvpa (actigraph). we targeted at recruiting totally subjects. nonparametric tests were used to compare the effects within and between groups. missing values were replaced by last observed values. results: this study recruited subjects (intervention: n= , control: n= ). significant improvements in frailty (p< . ), cognitive function (p< . ), and mvpa (p< . ) were observed in the intervention group after the completion of the intervention. only cognitive function was also observed to be improved in the control group (p< . ). the compliance of wearing devices (i.e., smartphones and actigraphs) and the usage of the smartphone applications were highly satisfactory. three subjects withdrew from the study (intervention: n= , control: n= ). conclusion: m-health intervention is feasible to treat cognitive frailty in older people. it is more effective to ameliorate frailty and increase mvpa in older people with cognitive frailty when compared to conventional training. background: the prevalence of dementia and associated healthcare cost increases with aging population. population health management and proactive screening with increased emphasis on primary risk reduction may reduce the overall prevalence of dementia. motoric cognitive risk syndrome (mcr) has been increasingly studied as a pre-dementia stage to identify older adults at risk of transiting to dementia while few studies explored the association between mcr and functional capabilities. objectives: the aims are to investigate the prevalence of mcr and its associated factors among community-dwelling older adult and also to examine possible impact of mcr on functional capabilities. methods: data for older adults aged above years old staying in northwest region of singapore was used. mcr was defined as slow gait speed over m ( sd below population mean) with subjective memory complaints in the absence of dementia. functional capability was determined by administering the lawton instrumental activities of daily living (iadls). differences in demographics, socioeconomic and lifestyle factors between mcr positive and mcr negative groups were found using independent t-test and chi-square test. risk factors of mcr and impact of mcr on functional capability were examined using logistic regression. results: the prevalence of mcr in the studied population was . %. after adjusting for demographics and socio-economic factors, indians (adjusted or = . , % ci = . - . , p = . ), increasing age (adjusted or = . , % ci = . - . , p < . ), higher bmi (adjusted or = . , % ci = . - . , p < . ) increased likelihood of mcr while increased years of education decreased likelihood (adjusted or = . , % ci = . - . , p = . ). the odds of having at least one impairment in iadl after adjusting for demographics, socio-economic and health factors amongst those with mcr were . (adjusted or = . , % ci = . - . , p = . ). conclusion: our study found in to have mcr, the pre-dementia stage. indian ethnicity, those with increased age and higher bmi are at greater risk of having mcr. as mcr is also associated with functional impairment, it can serve as a useful screening tool to identify those at risk of progressing to dementia. background: sleep disturbance has been found in older persons with dementia, which impact on the quality of life of older persons and on the caregiving burden of the family. little is known about the sleep patterns and sleep problems of older persons with dementia. exploring these data would provide basic information to develop interventions for this population. objectives: to explore sleep patterns and sleep problems in community-dwelling older persons with dementia. methods: the sample recruited by purposive sampling consisted of community-dwelling older persons with any stage of dementia who used healthcare services at outpatient departments of a university hospital, thailand. data were collected using a demographic data questionnaire, a sleep diary recorded by caregivers, and an electronic wrist activity tracker to assess sleep data for consecutive nights. the data had been collected for three months and were analyzed using descriptive statistics. results: the sample had an age range from to years (m= . , sd = . ). the total sleep data of the older persons with dementia consisted of episodes. almost all of the sleep data showed the polyphasic sleep pattern (sleeps for several periods of time a day), but a few had monophasic and biphasic sleep patterns. the total sleep time per night ranged from to hours with a mean of hours. the mean sleep latency was minutes, by which two-thirds of them had sleep latency less than minutes. three-quarters of the data woke up at night. the mean duration of waking up at night was minutes. two-thirds of the data had sleep problems, including insomnia, waking after sleep onset, and excessive daytime sleeping. also, most of them had snoring ( %), followed by sleep talking ( %). conclusion: the polyphasic sleep pattern was found mostly in older persons with dementia. also, they had sleep problems of insomnia at night and excessive sleeping during the daytime. healthcare providers may use the results from this study to understand the sleep patterns and then find strategies to promote the sleep quality of older persons with dementia. yumi umeda-kameyama , masashi kameyama , taro kojima , masaki ishii , shinya ishii , mitsutaka yakabe , kiwami kidana , tomohiko urano , , sumito ogawa , masahiro akishita (( ) department of geriatric medicine, the university of tokyo school of medicine, tokyo, japan; ( ) department of diagnostic radiology, tokyo metropolitan geriatric hospital and institute of gerontology, tokyo, japan; ( ) department of geriatric medicine, international university of health and welfare, narita, chiba, japan) background: «perceived age» of facial appearance in elderlies was shown to be a robust biomarker of aging that predicts survival, telomere length, and dna methylation. it is also reported to correlate with carotid atherosclerosis and bone status. objectives: this study aims to determine whether perceived age is a better biomarker than chronological age for a variety of aspects in dementia assessment, which includes general cognition, vitality, depressive state, and selfsupportability. methods: one hundred twenty-six patients admitted to the department of geriatric medicine, the university of tokyo hospital with suspect of cognitive decline were enrolled. mmse, vitality index, gds , iadl, and barthel index were performed. ten geriatricians and clinical psychologists determined the perceived age of subjects based on their photographs. results: the average values of rates showed excellent reliability (icc( , )= . ). perceived age showed significantly better correlation with mmse (female), vitality index (total, female), and iadl (total) than chronological age by steiger's test, but not with gds and barthel index. conclusion: perceived age was demonstrated to be a better biomarker for cognitive assessment than chronological age. l a u r a t a y , h u d a m u k h l i s , jolene ho , aisyah latib , eeling tay , shimin mah , candy chan , yeesien ng (( ) department of general medicine, sengkang general hospital, singapore; ( ) office of regional health system, singhealth, singapore; ( ) department of physiotherapy, sengkang general hospital, singapore; ( ) dietetics, sengkang general hospital, singapore) background: cognitive frailty is characterized by co-existence of physical frailty and cognitive impairment. earlier studies reported aggravated health outcomes attributable to cognitive frailty over physical frailty alone. objectives: we examine risk factors for cognitive frailty, and its impact on physical performance and health outcomes, compared with isolated occurrence of cognitive impairment or physical frailty. methods: cross-sectional analysis of communitydwelling older adults who completed multi-domain geriatric screen assessing for social vulnerability, mood, cognition, functional performance, nutrition, physical frailty (frail) and sarcopenia (sarc-f). cognitive impairment was defined using locally validated education-adjusted cut-offs on modified-chinese mini-mental state examination. participants underwent physical fitness tests comprising grip strength, gait speed, lower limb strength and power, flexibility, balance, and endurance. health outcomes included hospitalization, emergency department visits, falls and self-rating of health. each participant was categorized as robust-cognitive intact (pf--/ ci-), pre-frail/ frail only (pf+/ ci-), cognitive impaired only (pf-/ ci+), and cognitive frailty (pf+/ ci+). results: mean age of study cohort was . ( . )years. ( . %) were pf-/ci-, ( . %) pf+/ci-, ( . %) pf-/ci+, and ( . %) pf+/ ci+. in multi-nomial logistic regression referenced to pf-/ci-, older age significantly increased risk for pf-/ci+ and pf+/ci+. cognitive frailty contributes to worse physical performance and poorer health outcomes compared to physical frailty and cognitive impairment in isolation. while social vulnerability and depression were differentially associated with isolated frailty status, malnutrition and sarcopenia should be targets for preventing frailty and cognitive impairment. osamu katayama, sangyoon lee, seongryu bae, keitaro makino, ippei chiba, kenji harada, yohei shinkai, hiroyuki shimada (department of preventive gerontology, center for gerontology and social science, national center for geriatrics and gerontology, japan) background: cognitive frailty is a condition recently defined by operationalized criteria describing coexisting physical frailty and mild cognitive impairment (mci). however, there is no consensus on the definition of cognitive frailty for use in clinical and community settings. objectives: this study aimed to use latent class analysis (lca) to discover potential subtypes of cognitive frail older people. in addition, we explored the relationship between the identified cognitive frailty subtypes, and their demographical, neuropsychological, body composition, and lifestyle activity characteristics. methods: a total of community-dwelling older adults aged >= years participated in the study. we characterized physical frailty as >= of the following criteria: slow walking speed, muscle weakness, exhaustion, low physical activity, and weight loss. we used tests of word list memory, attention, and executive function, and processing speed to screen for cognitive impairment. the presence of >= cognitive impairments were defined as mci. we defined the condition where physical frailty and mci coexist as cognitive frailty. lca was applied to characterize classes or subgroups with different cognitive frailty phenotypes. subsequently, we performed multinomial logistic regression analysis with cluster membership as dependent variable and dichotomized demographics and lifestyle activity characteristics as independent variables. results: lca identified eight distinct subgroups included three different cognitive frailty phenotypes: cognitive frailty composed of physical frailty and amnestic mci (acf), cognitive frailty composed of physical frailty and non-amnestic mci (nacf) and, cognitive frailty in which physical frailty and global cognitive impairment (gcf). cognitive frailty subtypes were associated with distinct demographical, neuropsychological, and lifestyle activity characteristics. in particular, the acf cluster was associated with younger age and also related to the inactivity of productive and cognitive activities (p< . ). the nacf cluster was related to the inactivity of social and cognitive activities (p< . ). finally, the gcf cluster was associated with older age (p< . ). conclusion: using lca, we identified eight distinct subgroups included three different cognitive frailty phenotypes in a large sample of community-dwelling older adults. cognitive frailty subtypes were associated with distinct demographical, neuropsychological, and lifestyle activity characteristics. sara g aguilar navarro, alberto j mimenza alvarado, itzel aparicio gonzález, clarita cabrera juárez, alejandra samudio cruz, monsal alexa, ja avila funes, teresa juarez-cedillo (instituto nacional de ciencias médicas y nutrición salvador zubiran, ciudad de méxico, mexico) background: the prevalence of mild cognitive impairment (mci) ranges between - % and is times more frequent than dementia. the dcl has been associated with cardiovascular risk factors, mainly changes at the executive level. the apoe genotype, on the other hand, is a gene that confers susceptibility to alzheimer's disease in addition to participating in lipid metabolism, giving greater risk of atherosclerosis and cardiovascular risk. however, given the genetic heterogeneity of the mexican population, this association is not clear. objectives: to establish the strength of association between the different types of dcl (amnesic and non-amnesic) in mexican mestizo older adults according to their carrier status of the apoe allele and cardiovascular risk factors. methods: patients in a memory clinic were evaluated from to , older than years, without sensory deficit, psychiatric diseases or uncontrolled metabolic pathology, separating them into mutually exclusive groups: healthy controls, group with amnesic mci, group with nonamnesic mci, performing geriatric and neuropsychological evaluation. parametric and nonparametric statistics (x , anova, multivariate linear regression analyzes) were used to find statistical differences between groups. results: multivariate linear regression analyzes were performed to examine the relationship between vascular risk factors, the presence of the apoe ε allele, and cognitive change. apoe genotype significantly modified the associations between both hypertension and cardiovascular disease and a decline in language abilities as well as diabetes and decline in verbal memory, attention, and visuospatial abilities in non-amnestic mci. associations between increased vascular risk burden and greater cognitive decline were observed among apoe ε carriers but not non-carriers with mci. conclusion: the present study revealed an increase in the association between non-amnestic mci (apoe ε carriers with vascular risk factors) and suggests that the treatment of vascular risk factors could contribute to reducing the risk of progression of cognitive impairment, particularly among patients with apoe ε mexicans. background: a number of cross-sectional and longitudinal studies have demonstrated an association between physical frailty and cognitive impairment ( ). many mechanisms have been suggested to explain the presence of cognitive impairment in frail subjects, such as cardiovascular risk, hormonal disturbances, chronic inflammation or nutrition ( , ). another hypothesis is that cognitive impairment in frail patient may be due to alzheimer's disease (ad) ( , , ). however, the link between frailty and amyloid deposition has to date never been studied in vivo. objectives: ( ) to examine the prevalence of cerebral amyloid pathology as measured with amyloid positron emission tomography (pet) or amyloid-β- - level in cerebrospinal fluid, among frail and pre-frail individuals presenting an objective cognitive impairment ( ) to characterize the cognitive and clinical progression of frail cognitively impaired patients according to the amyloid status. methods: cogfrail is a monocentric observational prospective study of cognitive frail and prefrail older participants (according to fried criteria), aged >= years, with an objective cognitive decline (defined by a clinical dementia rating (cdr) scale scoreat . or ). the participants will be followed up every months, during years. in addition to cerebral amyloid pathology (measured by amyloid positron emission tomography (pet) or amyloid-β- - level in cerebrospinal fluid), measurements include cognitive performance, physical function, nutritional status, depressive symptoms biology, nutrition, magnetic resonance imaging (mri), and body composition to better understand the mechanisms and progression of cognitive frailty. results: the study is currently being recruited. to date, patients were included. mri pet scan and lumbar puncture have been performed. subjects completed the study. conclusion: this study will allow us to determine, for the first time, the prevalence of amyloid pathology, a marker of ad, among frail and pre-frail patients presenting objective memory impairment. the results will help characterize the cognitive decline in frail and pre-frail patients, with important implications for the detection, management and ultimately prevention of neurocognitive disorders among frail old individuals references: ) kojima g, taniguchi background: cognitive impairment is a well-known risk factor for falls in older adults. the risk of falls is increased in those with diminished executive function and reduced processing speed. while participants with cognitive deficits are more prone to falling, it is unknown whether risk of falling on cognitively intact individuals placing them at higher risk for future cognitive decline. objectives: to ascertain the incident development of cognitive decline in those at higher risk for falls using the center for disease control's fall risk assessment tool, steadi (stop elderly accidents, deaths, and injuries) in community dwelling individuals > years of age. methods: we identified individuals >= years old using the longitudinal national health and aging trends study (nhats) that consists of eight years of follow-up. these individuals did not have cognitive impairment at baseline. fall risk was defined using the algorithm from the center for disease control's steadi initiative. participants were classified at baseline in three categories of fall risk (low, moderate, severe). impaired global cognition was defined as nhats-defined impairment in either the alzheimer's disease- score, immediate/delayed recall, orientation, clock-drawing test, or date/person recall. the primary outcome was the risk of incident cognitive impairment over time. cox-proportional hazard models and linear mixed-effects modeling ascertained the incidence of cognitive impairment, adjusting for age, sex, smoking status, education, co-morbidities and an ability to walk. our referent variable was individuals at low steadi fall risk. results: of the , participants ( . % female), median age category was - years. prevalence of baseline fall risk using the steadi measure in participants was low ( . %), medium ( . %) and high ( . %). the rate of cognitive impairment in our sample was . %. in our fully adjusted model, the risk of developing cognitive impairment was hr . [ %ci: . - . ] in the intermediate risk group, and hr . [ %ci: . - . ] in the high risk group. using linear mixed-effects modeling yielded similar results. conclusion: steadi fall risk at baseline was predictive of higher rates of cognitive decline in those with normal cognition. elevated fall risk by steadi may suggest need for more thorough cognitive assessment. background: the concept of cognitive reserve (cr) has been developed as a potential factor able to describe individual differences in vulnerability to cognitive, functional, or clinical decline along aging. the progressive reduction of cognitive and functional performances represents an outcome commonly associated with aging. objectives: the aim of this crosssectional study is to investigate the association of cr with cognitive and functional outcomes in a sample of elderly outpatients. methods: subjects aged >= were consecutively recruited. patients who were unable to undergo the execution of required tasks due to severe cognitive, functional or sensory impairment were excluded. mini mental examination (mmse), brief intelligence test (tib) and cognitive reserve index questionnaire (criq) were administered. handgrip strenght, gait speed and daily life autonomy were measured; a frailty index (fi) was eventually calculated. results: data from patients were analyzed. criq was significantly correlated with mmse (r = . , p < . ), handgrip (r = . , p < . ) and gait speed (r = . , p= , ). furthermore, criq was correlated with badl (r = , , p= , ), iadl (r= , , p= , ) and inversely with fi (r= - . , p < . ). significant correlations were found between tib and mmse (r = . , p < . ), between tib and criq (r = . , p < . ), and between tib and iadl (r = , , p= , ). conclusion: this preliminary report highlighted that patients with higher cr showed not only better overall cognitive functioning, but also better functional status and a lower degree of frailty. in the light of a multidimensional geriatric assessment, the integrative evaluation of cr in elderly might offer the opportunity to track possible trajectories of aging, since it appeared related either to cognitive status, either to functional oucomes and to frailty. background: the clinical syndrome of "physical" frailty has been conceived without regard for cognitive decline. nevertheless, it has been suggested that frail elders exhibit frailty-specific cognitive impairments, and that the cognitive correlates of frailty may be dementing in their own right. meanwhile, we have used confirmatory factor analysis (cfa) in a structural equation model (sem) framework to construct a latent dementia phenotype, "δ". our approach is modular and can be redirected to other clinical targets. objectives: in this analysis, we create a δ ortholog representing the "cognitive correlates of frailty" (df). methods: first, we constructed a frailty index (if) from wave- data collected as part of the hispanic established population for epidemiological studies in the elderly (h-epese). a δ ortholog targeting if was then constructed from a cognitive battery that included the mini-mental status exam (mmse) and clox: an executive clockdrawing task (clox). results: the model fit the data well and df exhibited factor determinance. dfrailty was strongly indicated (r = . , p< . ) by if and explained % of the index's variance. it was also significantly indicated by mmse and clox scores. df was strongly correlated (r = . , p< . ) with instrumental activities of daily living (iadl), independently of age, gender and education. the remaining % of if's variance had no significant association with iadl. the orthogonal latent variable "g'", df's residual in spearman's general intelligence factor "g", was strongly indicated by all three cognitive performance measures. nevertheless, it was weakly associated with iadl. measure specific cognitive performance, residual to both df and g', had no independent: association with iadl. conclusion: these results suggest that the frailty syndrome does indeed have specific cognitive correlates. these are strongly associated with iadl and therefore potentially "dementing". like δ, the cognitive correlates of frailty are extractable from spearman's g, which may constrain the biology and psychometric properties of frailty-specific cognitive changes. independently of df, cognition has little association with iadl. this suggests that frailty may be a major determinant of iadl performance in elderly ma, and possibly a major etiology of "all cause" dementia in that population. background: cognitive-frailty has been proposed as a distinctive entity which preludes dementia. objectives: we aimed to examine the relationship between physical frailty, cognitive status, and gait performance as predictors of cognitive decline and incident dementia. methods: cohort study of community older adults free of dementia at baseline with a year follow-up. inclusion criteria: > years, english speaking, able to ambulate one city block. exclusion criteria: hip/knee joint arthroplasty in past months, parkinsonism, major depression, and diagnosis of dementia (dsm-iv criteria). cognition was assessed using the moca, the mmse, and the clinical dementia rating (cdr) scale was performed. physical frailty was defined using the phenotypic criteria described by fried and walston. cognitive-frailty was defined as the simultaneous presence of physical frailty with objective cognitive impairment, and absence of concurrent dementia. the main outcome measure was all-cause dementia (dsm-iv criteria). cox proportional hazards models were used to estimate the risk of cognitive decline and incident dementia. results: over a -year follow-up, participants experienced cognitive decline and participants progressed to dementia (global incidence rate (ir): per -person/y). participants with frailty had a higher prevalence of cognitive impairment ( %) compared to those without ( %, p= . ) but the risk of progression to dementia was not significant. adding cognitive impairment to the frailty phenotype (cognitive-frailty) predicted further cognitive impairment and progression to dementia. dementia ir for frailty was per person/y and for cognitive-frailty, per person/y. however, when slow gait was combined with baseline cognitive impairment, it showed the highest risk of progression to dementia (hr: . , %ci: . - . ; p = . ) with an ir of per person/y. conclusion: frailty and cognitive impairment are common and often coexist in the same individuals. however, slowing gait seems to be the frailty component driving the association with future dementia. background: assisted bathing requires the most hours of home care. for the frail elderly and their caretakers, the bathroom presents the most risk factors for falls and injury. bathroom adaptation is the primary reason for consultation in community occupational therapy and available resources cannot meet the increasing demand. the hygiene . (h . ) website (https://algo.grismoir.com/) addresses this need by offering a structured questioning to identify bathing assistive technology for the frail elderly living at home. objectives: our actionresearch protocol aims to establish a partnership between actors in the home care social economy enterprises (eÉsad), the home care programs offered through the healthcare system and the private sector (e.g., assistive technology providers). this implies: ) adapting h . to the home care service workers' needs; ) designing an implementation model for h . in order to formalize a partnership in the community; ) conducting pilot testing in two eÉsad. methods: ) user-centered design and a multiple case study where a case represents a home care worker (n= ) from a eÉsad (québec, canada) offering bathing assistance for the elderly. during testing, the home care worker will explore the h . prototype with an elderly in his or her home, sharing their thoughts out loud. the unit of analysis is the usability of h . , allowing improving to the prototype after every three participants. ) all collaborators will participate in the iterative modification of a preliminary logic model for the implementation of h . . modifications suggested will be integrated to the model throughout three meetings, or until a consensus is reached. ) the adapted version of h . (obj. ) will be tested according to the implementation model developed (obj. ). a pilot project using mixed methods in collaboration with two eÉsad will be conducted with older adults having difficulty bathing. results: anticipated results: responsive h . website adapted to the users' needs, an implementation model and pilot data allowing scaling-up technology meeting needs of frail elderly and their caretakers issues during bathing. li-ning peng , , , fei-yuan hsiao , , , wei-ju lee , , , shih-tsung huang , liang-kung chen , , ( ( ) background: the theory of cumulative deficits using big data to develop the multimorbidity frailty index (mfi) has become a widely accepted approach in public health and healthcare services. however, constructing the mfi using the most critical determinants and stratifying different risk groups with dose-response relationships remain major challenges in clinical practice. objectives: this study aimed to develop the mfi by using machine-learning methods that select variables based on the optimal fitness of the model and to further establish four entities of risk using a machine-learning approach as well as to ensure the dose-response relationship and the best distinction between groups. methods: in this study, we used taiwan's national health insurance research database to develop a machine-learning multimorbidity frailty index (ml-mfi) using the theory of cumulative diseases/deficits of an individual older person. compared to the conventional mfi, in which the selection of diseases/deficits is based on expert opinion, we adopted the random forest method to select the most influential diseases/deficits that predict adverse outcomes for older people. to ensure that the survival curves showed a dose-response relationship with overlap during the follow-up, we developed the distance index and coverage index at any time point to classify the ml-mfi of all subjects into the categories of fit, mild frailty, moderate frailty and severe frailty. survival analysis was conducted to evaluate the ability of the ml-mfi to predict adverse outcomes, such as unplanned hospitalizations, intensive care unit (icu) admissions and mortality. results: the final ml-mfi model contained diseases/deficits in this study. compared with conventional mfi, both indices had similar distribution patterns by age and sex; however, among people aged - , the mean mfi and ml-mfi were . (standard deviation (sd) . ) and . (sd . ), respectively. the difference may result from discrepancies in the diseases/deficits selected in the mfi and the ml-mfi. a total of , subjects aged to years were included in this study and were categorized into groups according to the level of the ml-mfi. both the kaplan-meier survival curves and cox models showed that the ml-mfi significantly predicted all outcomes of interest, including all-cause mortality, unplanned hospitalizations and all-cause icu admissions, at , and years of follow-up (p< . ). in particular, a doseresponse relationship was revealed between the four ml-mfi groups and adverse outcomes. conclusion: the ml-mfi consists of diseases/deficits that can successfully stratify risk groups associated with all-cause mortality, unplanned hospitalizations and all-cause icu admissions in older people, which indicates that precise, patient-centered medical care can be a reality in an aging society. to return home. understanding the home environment prior to discharge is crucial. occupational therapists (ots) often depend on client's verbal descriptions, pictures and sketches when planning rehabilitation exercises and suggesting adaptations. the information obtained is therefore partial. mapit is a new mobile application which scans a room producing a d representation with virtual measurements of environmental elements. this could provide a more complete representation of the home needed by inpatient rehabilitation ots. objectives: to target mapit's clinical applications for inpatient rehabilitation of the frail elderly. methods: multiple case study where mapit was introduced in three inpatient geriatric rehabilitation units over days. five ots maintained a logbook and participated in four individual semi-structured interviews. a deductive thematic analysis of the logbooks and interview transcripts was corroborated by two additional ots. results: mapit is useful for ots in rehabilitation settings by allowing them to ) see it: see the home environment, ) measure it: take measurements of desired environmental elements, ) document it: have a copy of the environment on hand, ) communicate it : facilitate exchanges with the client and with colleagues. with mapit, ots gain a better understanding of the environment, which informs the rehabilitation intervention. better communication could also improve the client's implementation of the therapeutic strategies. conclusion: mapit is a useful resource to optimise intensive rehabilitation for the frail elderly. sonia jiménez-mola , javier idoate-gil , david idoate , maría plaza carmona (( ) geriatric department, complejo asistencial universitario, león, spain; ( ) university of salamanca, salamanca, spain; ( ) urgency department, complejo asistencial universitario, león, spain) background: as the age of the population increases, the incidence of osteoporosis and its direct consequence, fragility fractures, are also increasing. hip fractures are associated with the greatest number of complications, functional deterioration, and mortality of up to % one year after the fracture. objectives: the aim of this study is to determine the prevalence of previous diagnosis of osteoporosis in elderly patients who suffer hip fracture and its relationship with age distribution ( - , - and > years old), gender, type of fracture and funtionality. methods: we enrolled patients with hip fracture, aged years or older in an orthogeriatric unit between december and november . underwent comprehensive geriatric assessment that evaluates comorbidities, medication use, ability to perform basic activities of daily living, place of residence, anesthesia risk as measured by the asa score, type of fracture, type of surgery and anesthesia and in-hospital mortality. spss®, v. . . results: the mean age was . ± . years ( - years). . % female. % pertrochanteric fractures. ( %) underwent surgery. only . % received general anesthesia. % walked independently, % had barthel > , ( %) had a previous diagnosis of dementia, and % live in nursing home prior to fracture. we found a previous diagnosis of osteoporosis in patients ( . %). in these patients, statistically significant differences were shown for sex p< . ( . % female vs . % male), age distribution p< . ( . %( - ) vs . % ( - ) vs . % (> ) and the presence of anti-osteoporotic treatments p< . . all other measurements (barthel index, cognitive degree, type of fracture, asa score and type of surgery, did not show statistically significant differences (p>. ). conclusion: patients in very advanced age showed neither significantly higher percentage of diagnosed osteoporosis, not significantly higher amount of preexisting osteoporosis-related medication. although the prevalence of osteoporosis increases with age, the diagnosis and treatment prevalence decreased in higher age groups. background: aging is associated with a decrease in bone density, muscle mass and a gain in fat mass which increase physical disabilities and falls. nevertheless, the impact of obesity on bone density and architecture is still controversial. furthermore, protein intake appears to be associated with maintenance of muscle and physical function, but also with bone density and architecture. however, the role of initial protein intake in osteopenic-obese older adults is still unclear. objectives: to examine the influence of initial protein intake on muscle and bone function in osteopenic-obese older adults. methods: cross-sectional a-posteriori matched study design. fourteen obese (total fat (%): men > ; women: > ) osteopenic (bmd t-score <- . ) older adults (age > years old) were divided in groups according to their initial protein intake (prot-(n= ): < g/kgbw/d or prot+ (n= ): > . g/ kgbw/d) and were matched for age (± years) and gender. body composition (fat, fat-free and bone masses, dxa), muscle composition and bone architecture (qpct), muscle function (grip strength, knee extension strength, muscle power), physical performance (walking speed ( m), tug ( m), unipodal balance, stair and chair tests), cardiorespiratory function ( min walking test) and lifestyle habits (physical activity level: -axial accelerometer and nutritional status: food record) were assessed. results: our groups (prot-vs. prot+) were similar (p> . ) in terms of age ( . ± . vs. . ± . years), bmi ( . ± . vs. . ± . kg/m ), body fat (total(%): . ± . vs. . ± . ), muscle quantity (fat-free mass or limb muscle area) and quality (intra & submuscular adipose tissues), bone density (total hip or spine) and architecture (marrow, cortical or total area, and compressive or torsion strength), physical performance (walking speed(m/s): . ± . vs. . ± . ), cardiorespiratory function, lifestyle habits (steps: ± vs. ± ), except (by design) for the initial amount of protein intake ( . ± . vs. . ± . g/kgbw/d) respectively. conclusion: the initial protein intake does not seem to influence bone architecture, muscle function, or physical performance in elderly osteopenicobese. obesity but also the level of protein intake above the official recommendation (> . g/kgbw/d) could explain these conclusions. thus, future studies are needed to confirm our preliminary results. background: the glim definition of malnutrition is the first intended to be used globally. glim uses five criteria (two phenotypic and three etiologic) for the diagnosis of malnutrition, which is made when at least one etiologic and one phenotypic criterion are present. mna-sf is a validated widespread screening tool used in geriatric settings. glim and mna have not been compared in acute geriatric care. objectives: to measure the prevalence of malnutrition in older patients admitted to an acute geriatric unit using glim criteria and to assess the accuracy of the mna-sf in predicting glim defined malnutrition. methods: a prospective study was conducted among all patients older than years old admitted to an acute geriatric unit. end-of-life situations and wearers of pacemakers were excluded. glim criteria and mna-sf were assessed on admission. muscle mass (one of the glim criteria) was estimated by bioimpedance (thresholds for low muscle mass: < . kg in men; < . kg in women). results: patients were included (mean age . ± . years, % women). on admission, . % were malnourished according to the glim criteria ( . % met at least one etiologic criterion, . % met at least one phenotypic criterion). . % were malnourished using mna-sf. however, there was no correlation between glim and mna-sf (correlation coefficient r=- . , p= . ). mna-sf had low sensitivity ( . %) and low specificity ( . %) to detect malnutrition diagnosed with the glim criteria (roc curve auc= . ). conclusion: more than half of the very old patients admitted to an acute geriatric unit were malnourished according to the glim diagnostic criteria. a very similar proportion of patients had a mna-sf suggesting malnutrition. however, mna-sf had a low reliability to detect patients with glim defined malnutrition. corina naughton , rachel simon , tj white , darren daly ( ( ) background: hospitalised older adults are at risk of hospital associated decline (had). optimising nutrition intake is an important modifiable factor in protecting against had and promoting recovery, but food intake and the quality of mealtimes are frequently overlooked nursing activities. objectives: the study aim was to undertake an in-depth analysis of mealtime practices and to identify patient and mealtime factors associated with low food intake (<eating % of meal provided). methods: we carried out structured observation of mealtimes using a validated tool examining patient positioning, mealtime set up and assistance. the primary outcome was food consumption categorised as , %, % % or % based on visual inspection of food trays. data were also collected on patient characteristics. data analysis used descriptive statistics and univariate logistic regression to examine the association between low food intake and mealtime and patient factors. results: we included mealtime observations on patients aged years and older. at % (n= ) of meals, patients ate half or less of the food provided. meal time factors with a weak association with low intake were: allocated red tray (an indicator of nutrition risk), modified diet, and needing assistance. breakfast time was the most important meal for food intake. nearly half of the patients ( / ) ate % or less of the food provided on two or more occasions over a -hour period. patient frailty and a moderate to high nutrition risk score were independently associated with low food intake. conclusion: patient factors are the strongest predictors of low food intake. increasing the quality of the mealtime experience and more tailored nutrition plans could improve nutrition intake in frail older adults. were hospitalized at least once, ( . %) fell at least once, and ( . %) had at least one fracture during the -year follow-up. no association was found between malnutrition and -year risk of institutionalization, hospitalization, falls, or fractures (p> . ). conclusion: malnutrition according to glim criteria was associated with a . -fold higher mortality risk; double that of the espen criteria, during a -year followup. no association was found between malnutrition according to these two criteria and incidence of other adverse health consequences. glim criteria anticipate outcome and might guide interventions, with important implications for clinical practice and research. background: older adults are at high risk of developing cardiovascular disease. pre-clinical studies indicate that resveratrol (rsv), a polyphenol present mostly in grapes and red wine, may prevent development of cardiovascular disease. objectives: our hypothesis was that rsv will reduce biomarkers of cardiovascular disease risk in obese, rather healthy older adults in a dose-dependent manner. methods: older participants ( years and older) were randomized to a day rsv treatment with mg (n= ), mg (n= ) or placebo (n= ). we measured levels of atherosclerosis development risk biomarkers i.e. oxidized low-density lipoprotein (oxldl), soluble e-selectin- (se-selectin), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ), total plasminogen activator inhibitor (tpai- ). statistical significance was set at p< . . results: changes in svcam- mg vs. mg vs. placebo: (- . ± . ng/ml vs. . ± . ng/ ml vs. . ± . ng/ml) and tpai- mg vs. mg vs. placebo (- . ± . ng/ml vs. . ± . ng/ml vs. . ± . ng/ ml) indicate significantly higher levels in a mg group compared to a mg and a placebo groups. other biomarkers ( mg vs. mg vs. placebo: oxldl, seselectin- and sicam- ) followed the same trend toward higher levels in the mg group compared to the mg and placebo groups, without reaching statistical significance. conclusion: this pilot project suggests that a higher dose of rsv may increase the levels of cardiovascular disease risk biomarkers in overweight older adults. given no change in the cardiovascular disease risk biomarkers in response to a lower dose, future studies should test the effects of different doses of rsv on reduction of cardiovascular disease biomarkers in overweight, rather healthy older adults. background: actual nutrition is a factor that continually effects physiological capacity and workability, the functional aging rate of an elderly persons. objectives: the purpose of this study was to determine the relationship between nutrition and physiological abilities, the work performance, functional aging rate, residual working capacity and frailty of the elderly. methods: it has been studied anthropometric and functional parameters of respiration, physical performance, mental capability, sensory skills, as well as the rate of functional aging in different aging groups: - years - persons, - years - persons, - years - persons. we have also analyzed the professional history, social status, and factual nutrition (according to the questionnaire proposed by the who and adapted for ukraine) of the elderly. results: the nutrition or diet factors influence on the problems dealing with working capability, reduction of the hand grip strength and endurance, independence and frailty (for elderly) in overall . % for all mentioned factors. right and left hand grip strength associate with protein consumption (r = . ; r = . ; p < . accordance) with variety of cereals (r =- . ; r =- . p < . accordance) also with variety of vegetables (r = . ; r = . ; p < . accordance)variety of fruits (r = . ; p < . ; r = . ; p < . accordance). it was studied features of an actual food at centenarians of ukraine which not only have lived to this old age, but also have the relatives who have lived to age of centenarians. it was established, that meals of ukrainian centenarians include high percentage of vegetables, fruits and dairy products. meanwhile menu has been deprived practically all basic alimentary pathology risk factors which accelerates biological age, creates certain preconditions to preservation of health and longevity. conclusion: as a result of a comprehensive study and mathematical modeling was developed a quantitative method for assessing the residual working capacity for elderly persons. background: age-related decline in olfactory function has implications for health and nutrition due to reduced appetite and decreased sensory perception of food. several studies have investigated olfactory performance in the elderly, but studied mostly single odour components often less related to food and meals. food odours are composed of multiple odorants and compensation for specific perceptual losses among elderly may occur. therefore, it is relevant to study olfactory perception of complex food odours to improve understanding of odour perception in the context of foods and meals. objectives: to develop a test method to screen young and elderly ( +) subjects on their olfactory capacity for everyday food odours. the method included a series of sniffing sticks with relevant and familiar complex food odours from primarily essential oils. methods: the olfactory sniffing sticks test kit was developed in four steps: ) selection and validation of relevant, familiar and diverse food odours, evaluated on perceived familiarity. ) standardization of an iso intensity reference level for the food odours in relation to n-butanol. ) assessment of shelf-life stability for the sniffing sticks within an weeks period. ) evaluation of test-retest reliability for intensity and identification of the odours within a weeks period. results: food odours were selected due to their diverse sensory characteristics. they were provided from a french manufacturer which may have compromised the familiarity in a danish context as only out obtained satisfactory familiarity score. however out showed reliable results in a test-retest procedure. n-butanol, in two concentrations provided a satisfactory reference frame for the iso intensity scaling. furthermore the food odours were overall shelf-life stable within an weeks period. conclusion: a new odour test kit for everyday food odours was developed and validated for screening olfactory capacity (intensity perception, familiarity and identification) in elderly subjects. based on the evaluations, odours were included in the final test kit. this olfactory test reflects the complex stimulation of the olfactory system, when stimulated by eating a food, compared to odour test kits with single or few components which makes it relevant when customizing of meals for elderly to improve nutrition and wellbeing. background: nordic nutrition recommendations (nnr) ( ) suggest protein intake >= . g/kg body weight (bw) to preserve physical function in nordic older adults. however, no published study has used this cut-off to evaluate the association between protein intake and frailty. objectives: this study examined associations between protein intake, and sources of protein intake, with frailty status at the -year follow-up. methods: participants were women aged - years enrolled in the kuopio osteoporosis risk factor and prevention -fracture prevention study. protein intake g/kg bw and g/d was calculated using a -day food record at baseline . at the -year follow-up ( ), frailty phenotype was defined as the presence of three or more, and prefrailty as the presence of one or two, of the fried criteria: low grip strength adjusted for body mass index, low walking speed, low physical activity, exhaustion was defined using a low life satisfaction score, and weight loss > % of bw. the association between protein intake, animal protein and plant protein, and frailty status was examined by multinomial regression analysis adjusting for demographics, chronic conditions, and total energy intake. results: at the -year follow-up women were frail and women were prefrail. higher protein intake >= . g/kg bw was associated with a lower likelihood of prefrailty (or= . and % confidence interval (ci) = . - . ) and frailty (or= . and ci= . - . ) when compared to protein intake < . g/kg bw at the -year follow-up. women in the higher. conclusion: protein intake >= . g/kg bw and higher intake of animal protein may be beneficial to prevent the onset of frailty in older women. background: sarcopenia is a geriatric syndrome with increasing importance due to the aging of the population. progressive resistance training and protein supplementation are currently recommended for the prevention and treatment of sarcopenia. however, elderly are less responsive to these anabolic stimuli compared to healthy adults. inflammation is considered an important contributor to this age-related anabolic insensitivity. therefore, anti-inflammatory strategies, such as omega- , are a promising strategy to combat sarcopenia. furthermore, omega- were also shown to improve muscle anabolism though activation of the mtor signalling pathway and reduction of insulin resistance. objectives: firstly, we performed a narrative review of literature that gives an overview of the current knowledge about omega- intake and sarcopenia defining parameters (grip strength, gait speed, muscle strength or physical performance). secondly, we provided an overview of data on omega- supplementation and sarcopenia defining parameters. methods: a literature search was conducted in november , using electronic bibliographic databases (pubmed and embase). the reference lists of all full texts retrieved during the search process or as identified in already published (systematic) reviews were scanned. results were published in a narrative review (dupont j. et al. aging clin exp res.) results: seven observational studies described the associations between omega- intake and sarcopenia defining parameters. four interventional studies looked at the effect of omega- supplementation alone and suggested an improved muscle protein synthesis, improved gait speed and increased muscle strength and physical performance. three studies combining exercise with omega- supplementation suggested an enhancing effect of the supplement on the exercise-induced gains in muscle mass and strength. we found one study combining omega- and protein supplementation with exercise, but omega- dosage was too low for conclusive results. conclusion: observational data on omega- intake and sarcopenia remain conflicting. from current interventional data we conclude that there is growing evidence for a beneficial effect of omega- supplementation in sarcopenic elderly, which may add to the effect of exercise and/or protein supplementation. however, the exact dosage, frequency and use (alone or combined with exercise and/or protein supplementation) in the treatment and prevention of sarcopenia still need further exploration. background: with the growing incidence of cancer in older persons, malnutrition rates have increased. tumor-related malnutrition is a risk factor of treatment side effects. it reduces the quality of life and increases morbidity and mortality. therefore, malnutrition screening and diagnosis are mandatory to implement proper nutritional support. objectives: this study aimed to evaluate and compare the short form of mini nutritional assessment (mna-sf) nutritional screening tool with the new global leadership initiative on malnutrition (glim) diagnostic criteria for malnutrition among elderly patients with cancer. methods: patients >= years old, with a g screening tool ≤ , were referred to an oncogeriatrics consultation between february and september . the data recorded comprehended, demographic variables (age, sex), type of tumor, functional (barthel, lawton index, fac) and mental (mmse, yesavage) status, nutritional (mna-sf, glim criteria) and social assessment and number of drugs. if-vig, cirs-g, rockwood-ms, cci-sf, sppb and handgrip strength were used to estimate frailty. the roc curve was used to evaluate the ability to accurately distinguish malnourished patients. to determine diagnostic concordance between the assessment and the new glim diagnostic criteria of malnutrition, retrospectively analyzed, cohen's К statistic was calculated. results: patients were included, mean age . ± . , . % were women. gastrointestinal ( . %) and gynecological ( . %) neoplasms were most prevalent. . % were independent or had mild dependence on badl, . % on iadl. . % had no cognitive impairment and . % had no depressive symptoms. frailty scales showed a pre-frail patient profile, with good social support and a . ± drugs on admission. according to the new glim diagnostic criteria for malnutrition, % of the patients were malnourished. with the use of mna-sf, . % of the patients were found to be at risk of malnutrition. the roc curve of mna-sf had an area under the curve (auc) of . . no concordance was found between the mna-sf and the malnutrition diagnostic results (К= , p< . ). conclusion: in this small sample, most cancer patients were male, > years old, with low frailty index, good functional and mental status and at risk of malnutrition. the mna-sf scale detected more risk cases so preconditioning and nutritional recommendations before specific oncological therapies could be made. concentration is associated with muscle mass and strength in healthy elderly. however, there are several confounders, including body composition, nutrient intake, physical activity level and blood parameters which may also influence muscle mass. previous studies have not thoroughly examined the relationship between serum (oh)d concentration and muscle indices by comprehensively considering the potential confounders in healthy elderly. objectives: the purpose of this study was to investigate the relationship of serum (oh) d concentration with muscle mass and strength in healthy japanese elderly. methods: this cross-sectional study included healthy elderly in shiga prefecture in japan (age: . ± . years, m = , w = ). total fat-free mass (tffm) and appendicular (affm) were measured using dual-energy x-ray absorptiometry. in addition, handgrip strength and leg extension power were measured. a blood sample was collected in an overnight fasted state, and serum (oh)d concentration was assessed. habitual dietary intake and physical activity were assessed. protein intake, carbohydrate, and vitamin d intakes were adjusted for energy by the residual method. association of serum (oh)d concentration with tffm, affm, handgrip strength, and leg extension power was assessed by hierarchical multiple regression analysis with adjustment for age, gender, weight, energy, energy-adjusted protein, carbohydrate, vitamin d intakes, serum albumin concentration, and physical activity. results: the mean serum (oh)d concentration of participants was . ± . nmol/l. low serum (oh)d status (< nmol/l) was observed in . % ( / ) of participants. the mean affm was . ± . kg, and handgrip strength was . ± . kg. serum (oh)d concentration was significantly associated with affm (β = . , p = . ), but not with tffm (β = . , p = . ), handgrip strength (β = . , p = . ) and leg extension power (β = - . , p = . ). conclusion: serum (oh)d concentration is related to affm japanese healthy elderly people, even if confounders are comprehensively considered. background: muscle quality, often defined as force produced per area or mass of muscle, declines as people age. objectives: we hypothesized that dietary protein quality will better predict muscle quality than energy, carbohydrate, protein, fat, or leucine intakes when controlling for age, bmi, composition, and moderate to vigorous physical activity (mvpa). methods: strength was measured using isokinetic dynamometry at degrees per second, leg composition (lc) was examined via dual-x-ray-absorptiometry, and mvpa was measured with accelerometry. dietary intake was estimated using three-day food logs and esha software. muscle quality was defined as right knee extensor peak torque relative to right leg lean mass. protein quality was the ratio of total leucine over total protein intake. multiple linear regression and stepwise linear regression models were used. results: ninety-four women (mean ± sd; age . ± . years; bmi . ± . kg/m ; lc . ± . % fat; mvpa . ± . min/day; energy , ± kcal/day; carbohydrate . ± . g/ day; protein . ± . g/day; fat . ± . g/day; leucine . ± . g/day) completed the assessments. only protein quality (mean ± sem; beta = . ± . ; t = . ; p = . ) was significant to the full regression model containing all covariates (r = . ; adjusted r = . ; f ( , ) = . ; p = . ). to verify the importance of protein quality, a stepwise regression analysis using the same variables was performed and resulted in a model (r = . ; adjusted r = . ; f ( , ) = . ; p < . ) that included protein quality (mean ± sem; beta = . ± . ; t = . ; p = . ) and energy intake (mean ± sem; beta = . ± . ; t = . ; p = . ). conclusion: dietary protein quality is positively associated with muscle quality when controlling for bmi, lc, mvpa, and energy, protein, fat, carbohydrate, and leucine intakes. the most parsimonious model included protein quality and energy intake, suggesting that they are most related to muscle quality. background: it has been suggested that disruption of the apoptotic process may have an effect on the incidence of sarcopenia. on the other hand, one of the dietary recommendations for seniors is to increase their daily protein intake. however, the effect of protein intake on apoptosis is not well understood. objectives: the purpose of this study was to investigate the effect of eight weeks of protein whey supplementation on the expression of genes involved in the internal and external pathways of apoptosis of long extensor muscle of thumb of aged wistar rats. methods: this is an experimental studies. statistical sample of this study consisted of male wistar rats (age: months, weight: ± gr). they were randomly divided into supplement (n= ) and control (n= ) group. supplement group received . gr per body weigh protein whey daily for eight weeks. the left thumb extensor muscle of all subjects was carefully separated and after freezing in liquid nitrogen transferred to - ° c. quantitative real time-pcr was performed to measure bax, bcl- , caspase , and gene expression levels. independent t-test and mann-whitney u test were used to compare the means and rankings. the hypotheses were tested at the significant level p< . . results: results showed that bax, caspase , caspase , and caspase genes expression increased in all samples in training group compared to the control group but this increase was only significant for bax, caspase and gens (p < . ) and also bcl- gene expression significantly deceresed (p < . ) in comparison with control group. conclusion: it seems that protein supplementation lead to activation of the internal pathway of apoptosis by increasing mitochondria permeability. background: the presence of obesity alongside with impaired aging in general, and with impaired muscular performance in particular, may result in a unique and growing phenotype of obese frail/sarcopenic, which may be hardly diagnosed by simple observation. characterizing the nutritional intake of this phenotype is of a substantial relevance. objectives: to characterize the nutritional intake among frail prone (fp) and obese subjects in a sample of community dwelling older adults in israel. methods: in this cross sectional study we evaluate the nutritional intake of frail, frail prone and robust subjects (with and without the presence of obesity), as well as their adherence to the dietary reference intakes (dri). data were retrieved a series of national studies on the status of health and nutrition in different age groups in israel (mabat zahav) for [ ] [ ] . the frailty likelihood presented here is based on a previous study from our group suggesting a non-direct validated model estimating frailty based on components. results: compared to the robust, fp subjects were more likely to have lower intake of several nutrients. among them are: iron (mg) (mean . vs. . , p < . ), vitamin c (mg) (mean . vs. . , p < . ), folate (μg) (mean . vs. . , p < . ), vitamin a (iu) (mean . vs. . , p = . ). the average overall adherence score according to the dri (based on a sum of nutritional components) was . among fp subjects, compared to . among robust subjects (p = . ). obesity either defined by bmi or by wc had a lower «effect» on the nutritional intake differences as compared to frailty status. this observation was seen when obese subject were compared to non-obese subjects and as fp subjects were more likely to show a poor nutritional status regardless of the presence of obesity. conclusion: our results show a clear association between frailty and poor nutritional intake, regardless of the presence of obesity. moreover, the functional status may better reflect nutritional gaps than obesity -challenging the concept of the frail -obese phenotype regarding to nutritional status. background: the loss of bone density during aging induces risks of falls, fractures and mobility decline. moreover, bone structure seems to be a better predictor of fractures than bone density. these phenomena are exacerbated in the presence of sarcopenia. however, dynapenia alone or in combination with obesity is more involved in falls and loss of mobility than sarcopenia. nevertheless, the impact of obesity on bone density and bone structure is still controversial. furthermore, protein intake appears to be associated with maintenance of muscle, bone density and bone structure. to our knowledges, the impact of protein intake on bone density and bone structure among dynapenic-obese older adults is not known even if this condition reached around % of elderly. objectives: to assess the influence of protein intake on bone density and bone structure among dynapenic-obese older adults. methods: twenty-six older adults (>= years), obese (%fat: men > ; women: > ) and dynapenic (relative to body weight grip strength: men < . ; women < . ) were divided into groups according to their initial protein intake : prot-: < g/kg/d (n= ; . % of women; . ± . years) and prot+: > . g/ kg/d (n= ; . % of women; . ± , years). the following measurements were performed: relative to body weight grip strength using lafayette dynamometer, body composition using dxa, femoral bone structure using ct-scan, nutritional intake using the -day food record method. results: excepted, by design, for initial protein intake, both groups were comparable at baseline. the prot-group had a higher (p< . ) marrow area ( ± ) than the prot + group ( ± ). in addition, the compressive loading strength was greater (p< . ) in the prot-group ( ± ) than in the prot + group ( ± ). finally, the total bone area was larger (p< . ) in the prot-group ( ± ) compared to the prot + group ( ± ). conclusion: surprisingly, a lower protein intake but higher than rda seems to protect bone structure but not bone density among dynapenic-obese older people. these results should be confirmed in larger studies designed to address this question. background: unintentional weight loss occurs in % to % of older adults and has been associated with morbidity, functional incapacity, risk of hip fracture, and overall mortality. while the impact of this condition is well established in frailty, studies involving sarcopenia are still insipient. objectives: to investigate the association between unintentional weight loss and sarcopenia in community-dwelling older adults. methods: a cross-sectional study was conducted among older adults (>= years) assisted in primary care. the unintentional weight loss was assessed by questions contained in three frailty assessment tools and one nutrition screening and assessment tool, described below: ( ) "have you recently lost weight such that your clothing has become more loose?" [edmonton frail scale (efs)]; ( ) "have you lost a lot of weight recently without wishing to do so? ('a lot' is: kg or more during the last six months, or kg or more during the last month)" [tilburg frailty indicator (tfi)]; ( ) "in the last year, have you lost weight unintentionally (i.e., not due to dieting or exercise)? (unintentional weight loss is: more than . kg or of at least % of previous year's body weight)" [phenotype for frailty (pf)]; ( ) «weight loss greater than kg during the last months" [mini nutritional assessment (mna®)]. sarcopenia was identified by european working group on sarcopenia in older people (ewgsop ) criteria. the data were analyzed with use of pearson chi-square test (p< . ). results: a total of older adults were evaluated ( . % female). the mean age was . ± . years ( - y). sarcopenia was identified in . % of the sample (n= ). the frequency of unintentional weight loss in sarcopenics was % in tfi (n= ; p= . ), % in efs (n= ; p= . ), . % in pf (n= ; p= . ) and . % in mna® (n= ; p= . ). conclusion: we observed that the unintentional weight loss evaluated by tfi and efs (frailty assessment tools) was associated with sarcopenia. so, different ways to evaluate weight loss (amount and time) seems to influence this association. funding: this study was financed by fapergs (process number - / - ) and capes (finance code ). background: half of older adults admitted to hospital are malnourished. malnutrition often leads to weight-loss and may lead to a loss of muscle mass, muscle strength and physical performance. nutritional interventions should individualise nutritional requirements, particularly energy and protein. objectives: to assess if energy requirements, determined by indirect calorimetry compared to usual care (predictive equations), can lead to a reduction in weight loss (primary outcome) and improvements in muscle mass, muscle strength and physical performance (secondary outcomes) in geriatric rehabilitation patients at risk of malnutrition. methods: geriatric rehabilitation inpatients were derived from the resort cohort (royal melbourne hospital, australia) and allocated by wards to either the indirect calorimetry or usual care group for the need study. energy requirements were measured using indirect calorimetry; the results were utilised by dietitians in the indirect calorimetry group and concealed for the usual care group. weights were obtained weekly. food intake assessment, muscle mass (bioelectrical impedance analyser), handgrip strength (hgs) and physical performance (short physical performance battery (sppb)) were measured at admission and discharge. within-group and betweengroup differences were calculated for the changes in outcome measures during hospitalisation. results: twenty-one patients (indirect calorimetry n= ; usual care n= ) were included (mean age . ± years; males, females). preliminary results showed that in the indirect calorimetry group, five patients gained weight, four patients maintained weight and one patient lost weight during hospitalisation; the usual care group had four patients with weight gain and five patients maintaining weight. there were no significant within-group differences or between-group differences for changes in weight ( background: many older people have difficulties in performing daily living activities such as preparing meals and food shopping, which could be partly due to cognitive and physical decline [ ]. these factors may influence food choice and represent a potential barrier to achieving good nutrition [ ] . nevertheless, the association between mealrelated difficulties and nutritional risk, as well as dietary intake, has been understudied. objectives: ( ) to examine the prevalence of autonomy in food-related activities, as measured with instrumental activities of daily living scale (iadl), among frail and pre-frail older subjects with an objective cognitive impairment ( ) to characterize the association of food autonomy with an insufficient dietary intake and nutritional risk of cognitive frail older people. methods: this is a secondary cross-sectional analysis using baseline data from the cogfrail study, which is a monocentric observational study of cognitive frail and prefrail older participants, aged >= years, with an objective cognitive decline. dietary intake is evaluated with a dietitian, using a diet history method. autonomy in food-related activities is assessed using iadl scale. nutritional status was categorized according to the mini nutritional assessment (mna). results: ongoing analyses. preliminary results show a mean energy intake of less than kcal and g of protein per day, we considered all nutritional needs cannot be covered under this threshold. conclusion: frail older people, with cognitive impairment, are particularly at nutritional risk and insufficient dietary intake. food autonomy has to be evaluated systematically to prevent nutritional risk in this population. elderly aged years or over, and this number will continue to increase. in order to extend the healthy life expectancy, disease prevention and health management of the elderly are important. preventive intervention of sarcopenia is considered to be an important issue in promoting care prevention for the elderly. objectives: the purpose of this study was to clarify the relationship of muscle weakness and physical characteristics with nutritional intakes. methods: subjects were men and women ( to years old) in the nagoya longitudinal study for healthy elderly (nls-he) in , excluding those who had missing values of the examinations. nutritional intakes were assessed by the food frequency questionnaire (ffq). low grip strength (gs) was diagnosed by asian working group for sarcopenia (awgs) criteria. the cut-off value of gs was kg for men and kg for women. results: the number of the subjects diagnosed with low gs was , ( men and women). comparison was made between the low gs group and the normal group. there were no significant differences between the two groups in age, sex, number of teeth, chewing ability and occlusal force, whereas mini nutritional assessment (mna) score, walking speed at the normal and maximum speed, exercise habits, and percent of body fat were significantly lower in the low gs group than the normal group. also, the rate of polypharmacy was significantly higher. in nutritional intakes, vitamin d and b were significantly lower in the low gs group. in the intakes by food groups, fish and meat intakes were significantly lower, but the intakes of snack were significantly higher. furthermore, the protein ratio and the amount of animal protein intakes were significantly lower in the low grip strength group. conclusion: in this study, muscle weakness was related to lower intake of specific nutrients such as vitamin d, b , and animal protein, independent of number of teeth, chewing ability, and occlusal force. background: the status of calcium intake, the main mineral of the bone has no suitable biomarker to assess it. its evaluation is relevant in clinical practice as in research. postmenopausal women should be evaluated for risk factors for osteoporosis, including poor calcium intake. objectives: to develop and validate a food frequency questionnaire (ffq) to assess the calcium intake of mexican postmenopausal women. methods: after obtaining approval from the institutional ethics committee, a pilot study was performed including mexican women whose calcium intake was assessed trough a day food diary ( dfd). the ffq was designed including the foods reported by the participants of the pilot study that provided more than . % of the calcium requirement and that were reported by at least participants. the ffq was tested through a validation study that included postmenopausal whom also completed the dfd. the validity of the ffq was assessed with the interclass correlation coefficient (icc) alongside a bland-altman analysis. results: postmenopausal women were assessed from june , to january , . participant's characteristics are shown in the table . the ffq underestimated mean calcium intake compared to day food diary (- mg ± . , p< . ). the two methods were strongly correlated by the icc (icc= . , ci . - . ). the ffq could identify individuals who consumed >= mg/ day with a high sensitivity, and a reasonable specificity (table ). figure shows the agreement between the dfd and the ffq were plotted against the average of the two measurements (figure ), the mean (solid line) and the % ci (broken lines) of the difference are shown. conclusion: conclusions: the ffq´s good sensitivity in identifying low calcium intake in postmenopausal women makes it useful also as an educational tool in diet counselling and for identifying subjects in need of supplementation. the difference between methods limits its utility as an epidemiological tool. helen yl chan , winnie kw so , regina cheung , kc choi , brenda ho , francis li , ty lee , janet wh sit , martin mh wong , sy chair ( ( ) background: nutritional status has been recognized as a predictor of the level of frailty. however, little is known about how the eating habits and dietary preferences associated with frailty, especially in the chinese elderly population. objectives: this study aims to identify dietary factors in predicting frailty among community-dwelling older adults. methods: a multicentre cross-sectional correlational study was conducted in hong kong in . frailty was defined by using fried's phenotype model. the frail scale was used to classify level of frailty and the mini-nutritional assessment (mna) was used to evaluate the nutritional status, in addition to anthropometric parameters. association between nutritional status (at risk or malnourished vs normal) and frailty status was examined using ordinal regression in a hierarchical fashion for adjusting participant socio-demographics, health status, lifestyle characteristics, eating behaviours and dietary habits. all the statistical analyses were performed using ibm spss . . all statistical tests were two-sided with level of significance set at . . results: a total of chinese older adults participated in the study. the prevalence of robust, pre-frail and frail were . %, . % and . % respectively. one third of the participants were malnourished or at risk of malnutrition. malnutrition and at-risk of malnutrition significantly increased the likelihood of frailty (or . , % ci . - . ). however, the level of frailty was not associated with age, gender, anthropometric measurements, eating behaviours, and use of dietary supplements. other nutritional factors significantly increased the likelihood of frailty were chewing difficulties (or . , % ci . - . ) and inadequate consumption of vegetables (or . , % ci . - . ). however, good appetite significantly reduced the likelihood of frailty (or . , % ci . - . ). conclusion: the findings showed that chewing difficulties and inadequate consumption of vegetables were associated with frailty, whereas good appetite was a protective factor. hence, interventions for addressing chewing problem and promoting appetite and consumption of vegetables are imperative to counter frailty in the older population. lack of energy was associated with nutritional status in nursing-home (nh) residents. methods: we performed a cross-sectional analysis of the incur study cohort. lack of energy was measured at baseline as part of the -items geriatric depression scale. nutritional status was evaluated according to mini nutritional assessment short-form (mna-sf). a -items frailty index (fi) was computed. logistic regression models were performed to test the association of lack of energy with nutritional status. results: a total of nh residents were available for analysis. the median age (iqr) was ( - ) years, with ( . %) females. at baseline, median mna-sf (iqr) was ( - ) with ( . %) patients that were malnourished. among the patients included . % ( patients) reported lack of energy. at univariate logistic regression analysis mna was inversely associated with lack of energy. at multivariate logistic regression analysis, adjusted for age, sex nursing home years and fi, we found that mna was independently inversely associated with lack of energy (or . , % ci . - . ). being malnourished is independently associated with lack of energy (or . , % ci . - . ). among mna components we found that item a (decrease in food intake), item c (reduced motricity) and item d (psychophysical stress) were inversely associated with lack of energy (or . , % ci . - . ; or . , % ci . - . ; or . % ci . - . ; for each point respectively), independently each one and from the other confounders. conclusion: in a cohort of very old nh residents, we found that an impaired nutritional status is associated with lack of energy. in particular, being malnourished bring a -fold risk of reporting lack of energy. more precisely, decrease in food intake, reduced motricity and psychophysical stress, each one were independently associated with lack of energy. a g e . m a r g u e r i t a s a a d e h , , f e d e r i c a p r i n e l l i , , anna-karin welmer , , weili xu , davide l vetrano , , serhiy dekhtyar , laura fratiglioni , , amaia calderón-larrañaga ( ( ) background: while declines in physical function are a common feature of ageing, the rate of the loss varies substantially between individuals, and has been attributed to intrinsic but also extrinsic (modifiable) factors such as diet, physical activity, and psychosocial well-being. objectives: ( ) to assess the role of food and nutrient intake in the speed of functional decline over years of follow-up. ( ) to explore whether such an association differs between levels of physical activity and psychosocial well-being. methods: we analysed data from individuals aged + from the population-based swedish national study on aging and care in kungsholmen (snac-k). the mediterranean diet score, mds (trichopoulou et al.) and the healthy diet indicator, hdi (who recommendations for saturated fatty acids, monodisaccharides, cholesterol, pufas, protein and fibre) were calculated for each participant, based on baseline data from a validated food frequency questionnaire and the corresponding transformation into nutrient intake. physical activity levels were assessed with questions about type, frequency, and intensity, and categorised as inadequate vs health/fitness-enhancing. we created a psychosocial well-being index by integrating variables linked to life satisfaction, positive/negative affect, social network and social participation. a global score of physical function was obtained by combining data on walking speed, balance, and chair stand tests. linear mixed models were used and adjusted for age, sex, education, smoking, baseline number of chronic diseases and impaired activities of daily living, total energy intake and time to death/drop-out. results: one standard deviation (sd) increase in the mds was associated with a lower functional decline both crosssectionally (β= . ; p= . ) and over the -year follow-up (β*time= . ; p= . ). higher scores of the hdi were also significantly associated with a lower functional decline, but only cross-sectionally (β= . ; p= . for one sd increase). when stratifying the analyses by levels of physical activity and psychosocial well-being, the protective effect of high mds was limited to subjects with health/fitness-enhancing physical activity (β*time= . , p= . ) and high levels of psychosocial well-being (β*time= . , p= . ), respectively. conclusion: a high adherence to a mediterranean dietary pattern, especially in combination with higher physical activity and psychosocial well-being, may slow down the age-relate decline in physical function. background: this cross-sectional study describes the application and follow-up of the self-care actions applied in a white male, years old, . m tall, a former athlete, currently sedentary, who in january presented % of glycated hemoglobin in medical consultation -between . and . %: pre-diabetes; fasting glycemia (mg / dl); (mg /dl) and the postprandial dose between and mg / dl. blood pressure between - mmhg; characterizing hypertension in stage. objectives: the objective was applying and follow-up a food re-education program associated with a resistance training program to reduce non-communicable diseases. methods: during , a program of dietary reeducation was carried out, with a few complex carbohydrates, an increase in proteins of high biological value, associated with a program of resistance exercises, which was adapted and individualized, obeying the individual's particularities. a short physical performance battery (sppb) was also applied to assess walking speed, strength and muscle balance. this program was performed three times a week, under the supervision of a physical education professional. capillary blood glucose was collected and analyzed times and blood pressure times, respectively. it was carried out a basic training for weeks aiming to rescue the muscular memory of the elderly, after beginning the adaptive phase of the physical valence training (cardiovascular endurance, localized muscular resistance); for weeks and the specified. the loads corresponded to % of rm for - repetitions with three series and to minutes intervals at each stage of the training. we used the ibm spss statistics program to perform descriptive statistics. results: the mean glycemia was (mg / dl), the glycated hemoglobin analyzes showed . ; low risk of diabetes. systolic blood pressure and diastolic blood pressure presented a mean of . ± . mmhg, and . ± . mmhg, respectively. we observed a gradual gain every months of resistance training. the sppb score changed from to points; performance between intermediate to high. conclusion: dietary re-education associated with a well-designed strength training program can result in the reduction of diabetes and hypertension, as well as strengthening the muscular system of the elderly. background: diet can be an important non-pharmacological aspect in order to prevent and/or attenuate brain and frailty outcomes in older adfults. objectives: to investigate, by a systematic review, studies associating the dietary inflammatory index (dii) with brain and frailty outcomes in older adults. methods: we searched the publications in pubmed and lilacs databases up to june . inclusion and exclusion criteria were formulated based on pi(e)cos strategy (population= older adults, >= years; intervention/ exposition= dietary inflammatory index; comparison= not applied; outcomes= brain and muscle outcomes; study type= randomized clinical trials, cohorts, cross-sectional, casecontrol studies). results: searches resulted in publications, and after exclusion due to duplicity (n= ) and not compliance with exclusion and inclusion criteria (n= ), eight studies were selected. these studies were published from to , all of them were cross-sectional, with participants above years old, and the outcomes investigated were frailty and frailty risk, survival free of disabilities (by fried's frailty criteria, sppb test, lawton and broady scales); memory, cognitive decline and risk of dementia (by meem, cerad, gds, prime-md, dsst and animal fluency test). conclusion: the data extracted from the articles showed significant association between dii and the outcomes investigated, namely, the more inflammatory diet was associated with higher odds to be frail and pre-frail, and to have any type of cognitive impairment. therefore, the dii showed to be associated to brain and frailty outcomes in older adults, however, to understand causality, longitudinal studies are still necessary. background: it is well established that reactive oxygen species (ros) are increased in skeletal muscle with age. we have recently shown that increased ros with age is associated with increased expression of the senescence-associated microrna mir- a- p (mir- a) in skeletal muscle as well as in muscle-derived extracellular vesicles. these vesicles enriched in mir- a are elevated in aged mouse serum, and can induce senescence in bone stem cells. the histone deacetylase sirt is a validated target of mir- a, and sirt plays important roles in cell survival as well as in muscle hypertrophy with functional overload. importantly, we previously found that mir- a expression was much higher in muscle from aged female mice compared to male mice, a phenomenon others have observed in mouse cardiac muscle. objectives: here we tested the hypothesis that pharmacological ablation of senescent cells could modulate mir- a and sirt bioavailability in skeletal muscle of aged mice. we utilized the senescent drug abt- (navitoclax) since previous studies have shown that oral administration of abt- removed senescent satellite (stem) cells in mouse skeletal muscle. methods: ten male and ten female c bl mice, months of age, received either abt- ( mg/kg bw, ul) or vehicle by oral gavage for ten days. tibialis anterior muscles were removed at the end of the study for examination of mir- and sirt levels using rt-pcr and elisa, respectively. results: abt- reduced mir- a expression in both male and female mice, although the effect was more pronounced in male mice compared to females. abt- significantly increased sirt levels in male skeletal muscle but not in females. the changes in sirt and mir- a levels were not associated with significant differences in muscle fiber size over the treatment period. conclusion: these findings suggest that certain senolytic compounds can modulate levels of senescence-associated mirnas and their targets in aging skeletal muscle. these data also underscore the importance of considering sex differences in the molecular mechanisms underlying age-related muscle atrophy. background: the growth of the elderly population is a worldwide phenomenon and is associated with profound changes in body composition. the purpose of this study was to describe the magnitude of the problem, to evaluate the associated factors and the relation with functional capacity in the study population. objectives: to estimate the association between demographic factors, comorbidities and muscle mass index over time until functional disability or death appears in non-obese elderly individuals. methods: longitudinal study of elderly individuals aged years or over, non-obese and absence of functional disability at the beginning of the cohort on the epidoso project database. the variables gender, age, ethnicity, medical history, functional capacity and death were investigated. the low or normal muscle mass index (mmi) was obtained through anthropometric data and a predictive equation. the functional capacity was measured using a structured and validated multidimensional questionnaire. the deaths occurred in the period were investigated with relatives through household surveys, in registries and registries of the state system of data analysis foundation. estimates of eventfree survival (functional disability or death) were calculated using kaplan-meier curves using the log-rank test in the gross comparisons. a multiple cox proportional hazards model was used to identify the independent effect of time predictors until onset of functional disability or death. results: the mean time found for the onset of functional disability or death was . years ( %ci=[ . ; . ]). in the crude analysis, there were statistically significant differences in the time to occurrence of functional disability or death, by age group (p< . ), arterial hypertension (p= . ), diabetes mellitus (p= . ) and marginal statistical difference muscle mass level (p= . background: a consequence of the ageing population is the increasing number of older adults with physical limitations. these limitations are mainly caused by decreased muscle mass and strength (sarcopenia). treatment or rather prevention of sarcopenia is necessary, as it may lead to lowered quality of life, hospitalization, loss of independence and even mortality. since older ethnic minorities are more likely to have an unfavourable health status compared to the majority population, variations in the prevalence of sarcopenia for ethnic minority groups are expected. further investigation seems imperative to be able to target preventive interventions to those at high risk of sarcopenia within the population. objectives: to examine the sarcopenia prevalence and its association with protein intake in an older multi-ethnic population in the netherlands. methods: we used cross-sectional data from the helius (healthy life in an urban setting) study, comprising the largest ethnic populations living in amsterdam, the netherlands. in total individuals from dutch, south-asian surinamese, african surinamese, turkish and moroccan origin aged years and over were included. sarcopenia was defined according to the ewgsop . in a subsample (n= ), protein intake was measured using ethnic-specific food frequency questionnaires. descriptive analyses were performed to study sarcopenia prevalence across ethnic groups in men and women, and logistic regression analysis were used to study associations between protein intake and sarcopenia. results: sarcopenia prevalence was found to be sex-and ethnic specific, varying from . % in turkish to . % in south-asian surinamese men and ranging from . % in turkish up to . % in south-asian surinamese women. higher protein intake was associated with a % lower odds of sarcopenia in the total population (or= . , % ci . - . ) and across ethnic groups. conclusion: ethnic differences in the prevalence of sarcopenia and its association with protein intake suggest the need to target specific ethnic groups for prevention or treatment of sarcopenia. background: few studies have evaluated the relationship between frailty and acute respiratory illness (ari), despite of increasing heavy burden of ari in older people. objectives: we conducted a prospective cohort study in communitydwelling older people in hong kong, to evaluate the impact of frailty on the risk of acute respiratory infections in the community setting and the potential modifying role of outdoor activities. methods: we recruited and followed up participants who were chinese and aged from to years, from december to may . frailty was measured by fried frailty index (ffi) twice during the study period. daily hours of outdoor activities were collected by a monthly activity journal (n= ) during the whole period, and by wearable gps device from some participants for one week in summer (n= ) and winter (n= ), respectively. the ari incidence was collected by monthly phone calls to the participants. we used a logistic regression model to estimate the odds ratio (or) of ari associated with frailty status (robust as reference group). results: the participants were classified into three groups according to the ffi criteria: ( . %) as robust, ( . %) as pre-frail and ( . %) as frail groups. of them, reported ari during the study period. according to the activity journals, daily hours of staying outdoors in the ari participants were slightly less than those in without ari ( . vs . in whole study period, . vs . in summer, . vs . in winter). while, the gps data showed that the participants with ari had longer daily hours of outdoors activities in summer ( . vs . ) but shorter in winter ( . vs . ), although none were statistically significant (p > . ). after adjustment for age, age, living alone or with family and daily hours of outdoor activities, we found that the frailty and pre-frailty groups had a higher risk of ari incidence compared with the robust group, with or . (p = . ) and . (p = . ), respectively. conclusion: frailty might be associated with a higher risk of ari among older people, but the role of outdoor activities remains inconclusive. background: previous studies have investigated the association between impaired muscle health and mortality. however, muscle health is a dynamic entity which change with time. objectives: to assess the effect of a short-term decline of muscle health (i.e., over year) and its association with long-term mortality (i.e., over years). methods: the sarcophage cohort follows up older belgian adults to assess consequences of sarcopenia. an assessment of muscle mass (dxa), muscle strength (handheld dynamometer) and physical performance (by means of sppb, including gait speed) are performed annually. all-causes deaths are collected annually. the association between short term (i.e. after one year) decline in muscle parameters and -year occurrence of deaths was tested using cox model. roc analyses were performed to assess performance of prediction of the different muscle components and to find optimal cut-points. missing data were handled using multiple imputations. results: from the subjects recruited ( . ± . years, . % women), were discarded from our sample because they died during the first year. therefore, the muscle decline was available on a sample of subjects. deaths occurred within the first years of follow-up. a -point decrease in performance at sppb test resulted in % higher risk of dealth (hradjusted = . [ %ci . - . ]). for each decrease of . m/s of gait speed, we observed an % higher risk of death (hradjusted = . [ . - . ]). a -kg decrease of muscle strength resulted in % higher risk of death in men and % higher risk of death in women (hradjusted = . [ . - . ] and hradjusted = . [ . - . ], respectively). we did not found any association between short-term loss of muscle mass and the occurrence of death (p= . ). then, we tried to find cutoffs optimizing the sensitivity-specificity ratio and we found following results : over year, a decline of sppb superior or equal to , of gait speed superior or equal to . m/s and of muscle strength superior or equal to . kg in men and . kg in women. conclusion: a short-term decline in muscle function is predictive of premature deaths. background: sarcopenia, the age-related progressive loss of muscle mass and function, is associated with an increased likelihood of adverse outcomes like falls, fractures, physical disability, and mortality. international consensus groups continue providing new definitions and clinical cut-off points despite over a decade of work in this area. objectives: we examined the prevalence of sarcopenia using two of the most current operational definitions (foundation of nih sarcopenia project (fnih) and the european working group on sarcopenia in older persons (ewgsop )) in a cohort of older adults (n= , >= yrs) hospitalized for an acute disease at utmb hospital in galveston (jan -may ). methods: testing included measures of: demographics (age, gender, race, education), body composition (dexa), physical function tests (sppb, tug, grip), psychological wellbeing and independence questionnaires, and chart review (comorbidity, length of stay). results: we found % had low physical performance, % had low muscle strength, and % low lean mass. we compared multiple tests and cutoffs for each of the three groupings under the fnih and ewgsop and found there to be differences depending on the test usedespecially for low performance which varied from %- %. in our cohort, the prevalence of sarcopenia was . % by ewgsop and . % by fnih. the subgroupings were found to be near identical across almost all measures despite the definitions' discrepancies in cutoff points between fnih and ewgsop . conclusion: in conclusion, recent updates to the new ewgsop make it almost indistinguishable to the older fnih standard, but the new ewgsop algorithm does provide a grading system to identify different levels of severity of sarcopenia. background: the population is experiencing a fast growth in the number of older adults, therefore determine the prevalence of frailty could help to inform future strategies to reduce its social and health burden. objectives: determine the prevalence of frailty in chilean older adults. methods: participants, aged > years, from the chilean national health survey - were included in this study. frailty was assessed by fried criteria modified, therefore people classified as frail should meet at least out of the criteria (low strength, low physical activity, low body mass index, slow walking pace and tiredness). results: the prevalence of frailty was . % ( . % for men and . % for women). the prevalence of prefrailty was . % whereas . % was classified as normal. the prevalence of frailty increased with markedly with age, . % and . % of men and women, respectively, were frail at the age of . this prevalence increased to . % and . % for men and women at the age of . the prevalence of pre-frailty increased from . % to . % for men and from . % and . % for women from the age of to years, respectively. conclusion: the prevalence of frailty increased markedly with age. with the chilean population expected to increase their life expectancy and number of older adults, it is important to implement prevention strategies that allow for early identification of high-risk individuals. a year follow-up. jair licio ferreira santos , yeda aparecida de oliveira duarte , tiago da silva alexandre background: sarcopenia has been increasingly recognized as leading to poor prognosis in health outcomes. likewise, falls -although important at older ages -have not been studied frequently and may lead to an increased risk of death. we evaluated survival of elderly people living in são paulo -brasil in a -year follow-up, considering the presence of sarcopenia at baseline and the occurrence of falls before the interview. objectives: to investigate whether sarcopenia and/or falls increase mortality among brazilian older adults. methods: data came from the second ( ) and fourth ( ) rounds of the health, welfare and aging study (sabe), which begun in , with a sample of the population over years old in the city of são paulo, brazil. after the first round, follow-up was performed every five years. sarcopenia was defined according to the consensus of the european working group on sarcopenia in the elderly (ewgsop), and the occurrence of falls was assessed by direct questions answered by the elder or his caregiver. a multivariate analysis with robust estimation and control for exposure time was done using the poisson regression model. results: mortality rates (per thousand person years) were: . (non sarcopenic, no falls) ; . (non sarcopenic with falls); . (sarcopenic no falls) ); and . (sarcopenic with falls. the poisson regression resulted in incidence rate ratios (when compared to sarcopenic, no falls) of . for non sarcopenic with falls; . for sarcopenic elders with no falls and . for sarcopenic with falls. conclusion: sarcopenia and the occurrence of falls are important risk factors for mortality. this finding highlights the importance of considering sarcopenia in health risk assessment and developing educational programs to prevent falls. ecosse l. lamoureux, , , alfred t.l. gan , ryan e.k. man , , eva k. fenwick , , bao lin pauline soh , angelique chan , david ng , chong foong-fong mary , preeti gupta (( ) singapore eye research institute and singapore national eye centre, singapore; ( ) duke-nus medical school, singapore; ( ) singapore institute of technology, health and social sciences, singapore; ( ) saw swee hock school of public health, national university of singapore, singapore) background: individually, sarcopenia and frailty are known risk factors for cognitive impairment (ci) in older adults, but information on their conjoint presence on the increased risk of ci is unavailable in this same population. objectives: we examined the association of the combined presence of sarcopenia and frailty with ci in elderly singaporeans. health profile in elderly singaporeans study (pioneer), a nationally-representative, population-based study of singaporean chinese, malays, and indians aged >= years. participants underwent body composition (dual energy x-ray absorptiometry -dxa); grip strength (hand dynamometer) and habitual m-walking speed assessments. sarcopenia was defined using the asian consensus as low appendicular lean mass (lalm; men < kg/m , women < . kg/m ) and low muscle strength (lms; men < kg, women < kg) or slow walking speed (sws; < . m/s); and frailty was defined as meeting three or more of the following components: ) unintentional weight-loss >= . kg in the past - months and/or bmi < . kg/m , ) lms, ) self-reported exhaustion in the past one month, ) sws, and ) low physical activity level. ci was determined using the montreal cognitive assessment (moca) basic scale. logistic regressionb models were used to determine the cross-sectional sarcopenia-frailty and ci relationship. results: of the included participants (mean age [sd]: . [ . ] years; . % females), ( %); ( %); and ( %) had neither sarcopenia nor frailty, either sarcopenia or frailty, and both sarcopenia and frailty, respectively. ci was present in ( . %) individuals without sarcopenia and frailty; ( . %) with either sarcopenia or frailty; and ( . %) individuals with both sarcopenia and frailty. in multivariable-adjusted analyses, presence of either sarcopenia or frailty was not significantly associated with higher odds of ci (odds ratio (or) [ % confidence interval]: . [ . - . ]), while having both sarcopenia and frailty significantly increased the odds of ci by nearly . times ( . [ . - . ]). conclusion: the co-presence of sarcopenia and frailty is independently associated with a higher risk of ci, compared to one condition alone, although longitudinal studies are needed to confirm this finding. strategies to prevent the concomitant onset of sarcopenia and frailty may be warranted to potentially reduce the risk of ci in older adults. background: car accidents related to older adults increased with aging, particularly in japan. safety driving required robust of physical function. however, the association between frailty and car accidents was still unclear. objectives: the aim of this study was to examine the association between frail status and car accidents. methods: participants were , older adults ( . % women, mean age: . years) enrolled current drivers in the national center for geriatrics and gerontology -study of geriatric syndromes. the criterion of frailty used in this study was j-chs index modified according to fried's criteria (chs index). the components of frailty in j-chs index were based on the original chs index: shrinking (weight loss), weakness, poor endurance (exhaustion), low activity level, and slowness. based on the presence numbers of these five components, our study defined "frailty" as and over, i.e., including pre frail and frail. the data of car accidents were collected from self-reported history of car accidents during years. results: among , participants, , participants ( . %) had a history of car accident. higher proportion of car accidents group was observed in shrinking ( . % vs . %, p = . ), exhaustion ( . % vs . %, p = . ), physical inactivity ( . % vs . %, p = . ) and slowness ( . % vs . %, p = . ), but not weakness ( . vs . , p = . ). in a logistic regression analysis, frailty was independently associated with car accidents in an adjusted model (or . [ %ci . - . ], p < . ). conclusion: this population study reveals frailty associated with car accidents. the findings have contribution of enhancing utility of risk assessments among older drivers. further studies were required to clarify risk of car accidents.model. background: frailty, a state of vulnerability to stressors resulting from a loss of physiological reserve across multiple systems. frailty is associated with higher morbidity, mortality and healthcare utilization. the national prevalence of frailty among us older veterans was found to be as high a %. however, little is known about the incidence of frailty in older, community-dwelling veterans. objectives: determine the incidence over years of frailty among robust or prefrail community-dwelling older veterans. methods: this is a retrospective cohort study of community-dwelling veterans years and older who had determinations of frailty from july -june and were followed until their last clinician visit before september , . a -item va frailty index (va-fi) was generated at baseline and during each subsequent primary care encounter as a proportion of all potential variables (morbidity, function, sensory loss, cognition and mood and other) with data from electronic health records. the va-fi categorized veterans into robust (fi<. ), prefrail (fi=>. , <. ) and frail (fi>=. ). using baseline and median duration of follow-up data based on event rates, incidence rates of frailty per person/years were calculated for robust, prefrail, combined (robust and prefrail) and gender groups. results: patients were . % white, . % non-hispanic, . % male, mean age . (sd= . ) years. the proportion of robust, pre-frail and frail patients at baseline was . % (n= ), . % (n= ) and . % (n= ) respectively. among robust veterans surviving a median follow-up of . (iqr . ) years, . % ( / ) became frail with an incidence rate of . cases/per person-years. among prefrail veterans . % ( / ) became frail and the incidence rate was . cases/per person-years. among the combined group, % became frail, with an incidence rate of . per person-years. the proportion of veterans becoming frail and the incidence rates were higher in women than men ( . % vs. . % and . vs . cases per person-years respectively). conclusion: this study shows a high incidence of frailty in community-dwelling older us veterans. identification of older veterans at high risk for frailty may assist in the development of interventions aimed at preventing frailty and its associated complications. background: anticholinergic drugs are prescribed to treat a variety of medical conditions through pharmacological actions opposing the actions of acetylcholine. anticholinergics and may contribute to frailty by causing cognitive, functional and physical impairment. frailty represents a state of vulnerability to stressors resulting from a loss of physiological reserve across multiple systems. frailty may potentially make patients more susceptible to the deleterious effects of anticholinergic medications on cognition. objectives: determine the crosssectional association of anticholinergics with cognitive impairment according to frailty status among communitydwelling older veterans. methods: this is a cross-sectional study of , community-dwelling veterans years and older whose frailty status was assessed october -october . the use of medications (active/inactive) with high anticholinergic burden scale (acb ) and cognitive impairment diagnoses (icd codes for mild cognitive impairment/dementia) were obtained from electronic health records. a -item va frailty index (va-fi) was generated as a proportion of all potential variables at the time of the assessment. we compared robust (fi≤. ), prefrail (fi=>. , <. ) and frail (fi>=. ) patients. after adjusting for age, gender, race, marital status, median household income, and bmi, odds ratios (ors) and % confidence intervals (cis) were calculated using binomial logistic regression with cognitive impairment as the outcome variable and anticholinergics (acb ) as independent variables. we repeated the analysis according to frailty status. results: patients were % white, . % male, mean age . (sd= . ) years, . % ( ) had cognitive impairment, . % (n= ) were taking acb medications, . % ( ) took them in the past and . % ( ) never used them. the proportion of robust, pre-frail and frail patients was . % (n= ), . % (n= ) and . % (n= ) respectively. in binomial logistic regression, active and inactive acb medications were associated with higher risk for cognitive impairment, adjusted or= . background: frailty, a state of vulnerability to stressors resulting from a loss of physiological reserve across multiple systems. the national prevalence of frailty among us older veterans was found to be as high a %. multiple studies have shown a higher prevalence of frailty and mortality in african americans. however, little is known about racial-differences in all-cause mortality in older veterans who had just transitioned to frailty. objectives: determine racial differences in allcause mortality over years among community-dwelling older us veterans who transitioned to frailty. methods: this is a retrospective cohort study of , community-dwelling veterans years and older who transitioned to frailty from july -september and were followed until death or september . a -item va frailty index (va-fi) was generated at baseline and during each subsequent primary care encounter as a proportion of all potential variables with data from electronic health records. the va-fi categorized veterans into robust (fi≤. ), prefrail (fi=>. ,<. ) and frail (fi>=. ). at the end of follow-up, we aggregated data on mortality only on those veterans who transitioned to frailty (robust/prefrail at baseline) and compared whites and african americans. after adjusting for age, gender, ethnicity, marital status and median household income, the association of race with mortality was determined using a multivariate cox regression model. results: patients were . % white, . % african-american, . % non-hispanic, . % male, mean age at frailty transition was . (sd= . ) years. over a median follow-up period of days (iqr= ) from the time they transitioned to frailty, deaths occurred (n= , in whites vs. n= in african americans). african american veterans had a lower risk for all-cause mortality than white veterans, unadjusted hazard ratio (hr) =. ( %ci: . -. ), p<. . however, these mortality differences disappeared after adjustment for covariates, adjusted hr =. ( %ci: . - . ), p=. . conclusion: our study suggests that in community dwelling older us veterans who had transitioned to frailty, race is not significantly related to overall survival when adjusting for other covariates. background: previous studies show that sarcopenic obesity (so) is associated with higher risk of mortality. however, a consensus definition of so is lacking, and more information is needed on the validity of simple measures applicable at a regular health care visit, such as anthropometric measurements and hand-grip strength or chair stand test. objectives: to examine the association between so and mortality, defining so based on body mass index, waist circumference, hand-grip strength and chair stand test, in a representative sample of finnish population. methods: this study was based on , participants aged years or over with data on anthropometrics, hand-grip strength and chair stand test from the nationally representative health survey. baseline sarcopenic obesity was defined as having bmi >= kg/m or waist circumference >= cm (men)/ cm (women), and hand-grip strength < kg in men, < kg in women, or chair stand > s for five rises. register-based follow-up data of the statistic finland containing , deaths during the years of follow-up were individually linked with the baseline data. survival analyses were based on cox proportional hazards models using age as the time scale. results: mean age was . years (sd . ) and . % were females. overall prevalence of sarcopenic obesity was . % at baseline. sarcopenic obesity was associated with higher risk of mortality (hr . , %ci . - . ) in an age and sex adjusted model. further adjustments for education, smoking, alcohol use, and physical activity did not notably change the results (hr . , %ci . - . ). conclusion: sarcopenic obesity, as defined based on anthropometric measurements as well as hand-grip strength or chair stand test, predicted higher mortality over years of follow-up. background: malnutrition and sarcopenia have a negative impact on mobility, risk of falls, fractures, physical disability and mortality. currently, limited information is available on nutritional status and nutritional interventions in geriatric rehabilitation (gr) patients. objectives: to characterize nutritional status and evidence of nutritional interventions with and without physical exercise in gr patients. methods: eight electronic databases were screened for nutritional status and interventions in patients >= years, admitted to gr, one search string was used for both topics. pooled estimates were calculated for mean bmi and prevalence of (risk of) malnutrition (mna). meta-analyses were performed to quantify intervention effects on albumin, muscle mass, barthel index (bi), and hand grip strength (hgs). results: observational and intervention studies were included out of references. pooled estimates ( % confidence interval (ci)) for prevalence of malnutrition and risk of malnutrition were ( - )% and ( - )%. pooled estimate ( %ci) for bmi was . ( . - . ) kg/m². low protein and energy intake and vitamin d deficiency were prevalent. intervention studies were heterogeneous in interventions and outcomes. meta-analyses showed no significant effects on albumin (standardized mean difference (smd) . , % ci - . : . ), muscle mass (mean difference (md) . kg, % ci - . : . ), bi (md . points, % ci - . : . ) and hgs (smd - . , % ci - . - . ), based on - studies. eight interventions tested oral nutritional supplements (ons) with protein, with or without exercise, reported protein intake and showed an increase, / studies showed increased albumin levels and / reported improved functional outcomes. conclusion: a high percentage of gr patients was affected by reduced nutritional status. intervention studies were limited and heterogeneous, but studies with ons improved nutritional outcomes, and functional outcomes in the majority of reporting studies. the results emphasize the need for malnutrition and sarcopenia screening and show benefits of protein supplementation in this population. future well-designed, well-powered trials are needed to clarify existing controversial aspects. therefore, feasibility of an intervention with a high-whey protein, leucine and vitamin d enriched ons (fortifit®), combined with resistance-type exercise in gr hip fracture patients will be investigated in a new intervention study (empower-gr). background: sarcopenia is a progressive and generalized skeletal muscle disorder associated with an increased likelihood of adverse outcomes such as falls, fractures, physical disability and mortality. the geographical region of residence (urban and rural area) may affect the prevalence of sarcopenia due to physical and environmental conditions. in , the european working group on sarcopenia in older people (ewgsop) updated the definition of sarcopenia (ewgsop ). objectives: to describe the prevalence of sarcopenia related to ewgsop and ewgsop criteria and to analyze the association between sarcopenia and geographical regions of residence. methods: this is a cross-sectional study involving elderly women ( years old or more) that were undergoing dxa in a radiology facility located in palmeira das missões (southern brazil). sociodemographic data were collected through a questionnaire. for the diagnosis of sarcopenia, we used the criteria recommended by the ewgsop (low muscle mass plus low grip strength and/or low gait speed), and ewgsop (low grip strength plus low muscle mass and/or low gait speed). the study was approved by the university ethics committee. results: out of the participants, . % was married, . % had education between and years of schooling, . % was caucasian, and . % was retired. the mean age was . ± . years old ( - ). the frequency of sarcopenia in the total sample assessed by the ewgsop and ewgsop was . % and . %, respectively. the prevalence of sarcopenia by the ewgsop was % in the urban area and . % in the rural area (p= . ) and by the ewgsop was . % in the urban area and . % in the rural area (p= . ). conclusion: in a sample of elderly women from the southern brazil, the prevalence of sarcopenia was low through both consensus (ewgsop and ewgsop ), and was higher among urban area. funding: this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior -brazil (capes) -finance code . background: patients with disuse syndrome have gradually increased with aging of inpatients in saitama medical university hospital. because these patients have been inactive in the acute phase, sarcopenia is likely to occur. sarcopenia was graded by three criteria in ewgsop ; muscle strength, muscle quantity and physical performance. muscle volume can be measured only in limited medical centers. many of patients with disuse syndrome can not walk even after the acute phase. for these reasons, muscle strength is the only quantitative factor reflecting sarcopenia, especially in old patients with disuse syndrome after the acute phase. objectives: to show ) muscle strength in old patients with disuse syndrome after the acute phase, ) effect of muscle strength on activities of daily living (adl). methods: subjects were old patients with disuse syndrome admitted in the department of rehabilitation medicine (rm) in saitama medical university hospital from january to december . inclusion criterion were as follows; ) patient age was or older ) patients could not walk independently at admission in the department of rm exclusion criterion were as follows; ) patients with motor paresis, contracture of fingers ) patients in inactivity before the onset of the disease causing disuse syndrome. grip strength (gs) was measured by handheld dynamometer. cut-off point of gs set by awgs in was adopted; kg for men and kg for women, adl was evaluated using functional independence measure motor scale (mfim) one week after admission in the department of rehabilitation medicine . percentage of gs below cut-off point was shown in men and women respectively. effect of gs on mfim was investigated using regression analysis. results: ninety nine out of patients were subjects in this study. median age was . years in men (n= ), . years in women (n= ). only two in men and one in women were below gs cut-off point. correlation coefficient between gs and mfim was . (p= . ) in men, . (p= . ) in women respectively. conclusion: gs was below cut-off point in most of the subjects. gp may affect adl after the acute phase in old patients with disuse syndrome. death, whereas measures of functional ability, physical strength and morbidity were stronger associated with time to death than with chronological age. from the age of and forwards participants have a high life-satisfaction in general, however, a decline is seen as persons get older and with proximity to death. measures of functional ability (e.g. going shopping) and morbidity (e.g. self-related health) had a significantly increasing effect on life-satisfaction with increasing age. whereas social function (e.g. living alone, meeting friends) did not significantly modify the decrease in life satisfaction with increasing age. conclusion: physical strength, functional ability and morbidity were measures mostly linked to biological aging, while social functioning was strongly correlated with chronological age. functional ability and self-related health are important factors to prevent age-related decrease in life satisfaction. background: previous studies mostly conducted in western countries support that physical frailty predicts future cognitive decline in general older populations. however, longitudinal evidence on this association is limited, especially among older japanese women. objectives: this study has investigated the prospective associations of frailty status with cognitive decline over two years among community-dwelling older japanese women, including which individual frailty components (i.e., slowness, weakness, exhaustion, low activity, and unintentional weight loss) could predict cognitive decline. methods: this study was a two-year population-based cohort study conducted in a metropolitan area of tokyo, japan. data were collected in october (baseline) and september (follow-up) and analyzed between december and january . participants were community-dwelling older japanese women, aged to years at the baseline, without any neurological diseases or cognitive impairment as measured by a mini-mental state examination (mmse) score of >= points. cognitive decline was defined as a drop of two points or more in the mmse score over two years. the physical frailty phenotype was classified by the japanese version of cardiovascular health study criteria. multiple poisson regression analyses with a robust error variance were applied to assess risk ratios (rrs) of two-year cognitive decline across the baseline frailty statuses (robust [reference category], prefrail, or frail). results: of the women analyzed, ( . %) were prefrail ( or components), and ( . %) were frail (≥ components) at the baseline. at the follow-up, ( . %) robust, ( . %) prefrail, and ( . %) frail women experienced cognitive decline. after being adjusted for various confounding factors including age, educational attainment, and baseline mmse score, the rrs of cognitive decline were . ( % confidence interval [ci]: . , . ) in the prefrail and . ( %ci: . , . ) in the frail women. among the five frailty components, slowness (rr: . , %ci: . , . ), weakness (rr: . , %ci: . , . ), and unintentional weight loss (rr: . , %ci: . , . ) were significantly associated with cognitive decline. conclusion: over the two-year period, approximately % of women experienced cognitive decline. baseline physical frailty status, particularly slowness, weakness, and unintentional weight loss, predicted this decline. intervention strategies targeting physical frailty may help delay cognitive decline in older japanese women. background: menopause leads to estradiol (e ) deficiency that is associated with decreases in muscle mass and strength. yet the mechanistic role of e in the loss of muscle mass has not been established. programmed cell death termed apoptosis has been proposed a key signaling route in skeletal muscle homeostasis, including muscle aging and sarcopenia. to date several micrornas (mirs) have been found to regulate key steps in apoptotic pathways. objectives: here we studied the effect of e deficiency on mir-signaling in skeletal muscle apoptosis. our aim was to reveal whether e -responsive mirs have mechanistic role in inducing skeletal muscle apoptosis. methods: we utilized c bl mice with three study groups; sham (normal estrous cycle, n= ), ovx (e deficiency, n= ) and ovx+e (high e supplemented by pellet, n= ). in our setup, ovx and ovx+e groups represent the extremes of e level. six weeks following the sham or ovx surgery, mice were sacrificed, gastrocnemius muscles were harvested and rna isolated. mir-profile was studied with ngs and candidate mirs verified using qpcr. the target proteins of the mirs were found using in silico analysis (target scan) and target proteins measured at mrna (qpcr) and protein levels (western blot). results: of the apoptosis-linked mirs found, four ( - p, a- p, - p and - p) indicated differential expression patterns between ovx and ovx+e groups. in qpcr verification, ovx had lower expression in all of the studied mirs compared with ovx+e (p= . ). accordingly, ovx had higher expression of cytochrome c and caspases , and compared with ovx+e at the mrna level (p< . ). at protein level, ovx had greater cytochrome c and active caspase compared with ovx+e (p< . ). conclusion: in muscle from e deficient mice (ovx vs. ovx+e group), several apoptosis-linked mirs were down regulated concomitant with higher mrna expression of the target proteins. furthermore, e deficiency was associated with higher cytochrome c and active caspase protein levels. to conclude, e deficiency down regulated several mirs related to apoptotic pathways that may lead to increased apoptosis and reduced skeletal muscle mass. background: although sarcopenia's pathogenesis is multifactorial, with its major phenotypes, muscle mass and muscle strength, being highly heritable, its genetic underpinning is not well studied. objectives: summarize evidence for use of zebrafish as a model system to decode the sarcopenia's gwas findings. methods: several genome-wide association studies (gwas) of muscle-related traits were published recently, providing dozens of candidate genes, many of them with unknown function. therefore, animal models are required not only to identify causal mechanisms, but also to clarify the underlying biology and to translate this knowledge into new interventions. over the past several decades, small teleost fishes had emerged as a powerful system for modeling the genetics of human diseases. due to their amenability to rapid genetic intervention and the large number of conserved genetic and physiological features, small teleosts, such as zebrafish (d. rerio), are indispensable for skeletal muscle genomic studies. results: we summarize the evidence supporting the utility of small fish model for accelerating our understanding of human skeletal muscle in norm and disease. the following stable mutants (mostly knockouts) exist for the «monogenic muscle» diseases (human gene, fish mutant, disease): for duchenne and becker muscular dystrophy (md), sapje/dmd (homology of human dmd gene); for limb-girdle md, popdc s f (bves); for bethlem myopathy and ullrich congenital md, col a ama (col a ); for nemaline myopathy, froto c (myo b), and tmod trg (tmod ); for merosin deficient congenital md, lama cl /cl ; candyfloss/lama (lama ); for limb-girdle md, bvesicl /icl (popdc ), heltg (ttn), and «foie gras» (trappc ); for native american myopathy, stac mi (stac ), as well as fish homologues of the acvr , cacnb , cavin , cms, dag , fhl , flnc, vcp and other human genes. these models provide evidence of muscle-related gene's conservancy and similarity of skeletal muscle morphology and physiological phenotypes. we will outline challenges in interpreting zebrafish mutant phenotypes and translating them to human disease. conclusion: we conclude with recommendations of future directions to leverage. centenarians exhibit extreme longevity and a compression of morbidity. we showed previously that centenarians display a unique genetic signature, in terms of mrna and mirna profile, which is similar to that found in young people and different from that found in octogenarians. centenarian offspring seem to inherit centenarians' compression of morbidity, as measured by lower rates of age-related pathologies such as hypertension, diabetes, strokes, and heart attacks. we therefore hypothesized that they will also display a lower incidence of frailty. in this study, we aimed to ascertain whether centenarian offspring are endowed which such "genetic footprint" and a lower incidence of frailty, when compared to their contemporaries. for this purpose, we collected plasma and peripheral blood mononuclear cells from septuagenarians, , age-matched centenarian offspring (but not sons or daughters of the centenarians included in this study) and centenarians. mirna expression and mrna profiles were performed by the genechip mirna . array (affimetrix) and genechip clariom s human array (affimetrix), respectively. frailty phenotype was determined by meeting three or more of the following criteria: unintentional weight loss, low grip strength, exhaustion, slow gait speed, and low physical activity. we found that mirna and mrna expression patterns in centenarians are similar to centenarian offspring and different to non-centenarian offspring (p< . ). importantly, we found a lower incidence of frailty among centenarians' offspring (p< . ), when compared to their contemporaries. taken together, our results indicate that centenarian offspring resemble centenarian characteristics and that they enjoy significantly less frailty than their less fortunate contemporaries that are not sons or daughters of centenarians. this lower incidence of frailty may be a key feature to achieve extraordinary ageing. background: hypoglycemic episodes increase in older patients and their consequences are more significant. objectives: the aim of this prospective observational study is to explore unknown hypoglycemic episodes diagnosed by continuous glucose monitoring in older type diabetic patients and to describe the link between the occurrence of hypoglycemia and glycosylated hemoglobin (hba c) level. methods: we included patients with type diabetes aged years or over hospitalized during consecutive months in a geriatric acute care unit in tours university hospital in france. demographic characteristics, type of diabetic treatment, mini mental state examination, hba c levels, albumin and creatinin level were recorded. continuous glucose monitoring (cgm) was used to detect hypoglycemia for a maximum of days, and capillary blood glucose measurements (cbgm) were also performed to times a day. patients with at least one blood glucose measure lower than mg/dl were compared with others for demographic, clinical and biological parameters. results: seventeen patients experienced hypoglycemia. these groups did not differ in demographic characteristics and in diabetic drug class. among these patients, had an episode of severe hypoglycemia (< mg/dl) and patients had nocturnal episodes, more often between and am. twelve patients had unrecognized hypoglycemia by cbgm. the average duration of hypoglycemic episodes was . hours. there was no difference in the hba c levels between the two groups (mean . %, p= . ). conclusion: the prevalence of hypoglycemia is underestimated in the oldest diabetic population receiving hypoglycemic drugs. measurements of cbgm and hba c level in the target may overlook nocturnal and prolonged hypoglycemic episodes. our study showed the benefit of cgm in older diabetic patients in order to detect unknown hypoglycemia. more prospective studies are needed to explore factors that predict hypoglycemia. catenacci, sophie le-gonidec, alizée dortignac, ophélie pereira, romain madeleine, jean-philippe pradère, philippe valet, cedric dray (umr inserm,universitéfédéral de toulouse -universitépaul sabatier toulouse iii, france) background: healthy lifespan does not increase proportionally compared to global lifespan leading to an increased number of disabled aged persons. to increase healthy lifespan, locomotion could be considered in the future as the main targetable outcome to fight against the frailty to dependency transition. the so-called sarcopenia, characterized as the loss of muscle mass and function, affects to % of the populations over . mechanistically, sarcopenia is associated with an imbalance between protein synthesis and degradation, an increase of muscle inflammatory processes, a reduction of mitochondria-driven metabolism and an exacerbated fibrosis. several therapeutic strategies have been proposed such as hormonal replacement but, regarding the adverse effects, these strategies have been abandoned. in this context, we hypothesize that, through a modified secretory profile, adipose tissue could play a crucial role in the muscle loss of function. we previously promoted an unbiased proteomic study and identified haptoglobin as an up-regulated cytokine overproduced by the adipose tissue during aging. objectives: in this context, our project proposes to better understand the role of adipocyte haptoglobin in age-related muscle weakness. methods: to do so, we used complementary in vitro and in vivo models of haptoglobin supplementation and strategies of adipocyte haptoglobin over-expression/deletion. impacts of such interventions have been monitored by measuring myogenesic processes as well as muscle aging. moreover, a human cohort in progress will help to constitute a new biobank by collecting blood, adipose and muscle from sarcopenic individuals in order to evaluate the role of hapatoglobin on sarcopenia (inspire cohort). results: the results obtained in vivo and in vitro suggest that haptoglobin treatments induced an age-dependent decrease in muscle mass. moreover, these protocols indicated a muscle-specific role of haptoglobin when we measured the fiber diameter. in addition, a direct effect of haptoglobin on differentiation alteration was also observed in in vitro human muscle cells. conclusion: these results suggest that haptoglobin induces effects according to the age, the muscle type and the dose on muscle physiology. thus, a better knowledge of adipocyte haptoglobin production could help to better apprehend the age-related muscular complications. background: sarcopenia contributes to loss of independence and is increases risk of mortality. mitochondrial dysfunction and loss of proteostasis are two interrelated hallmarks of aging with well-established roles in skeletal muscle function. mitochondrial dysfunction increases cellular oxidative stress and impairs atp-generating capacity. consequentially, oxidatively-damaged proteins accumulate; however, a dysfunctional mitochondrial reticulum cannot sufficiently provide energetic resources to repair the proteome. in skeletal muscle, this impaired proteostasis and mitochondrial dysfunction promote sarcopenia. thus, improving mitochondrial function by increasing endogenous antioxidants could attenuate age-related loss of muscle function. objectives: using a phytochemical nrf activator (nrf a), we sought to determine if upregulation of cytoprotective genes would improve mitochondrial function and gait, an integrative metric of musculoskeletal function. methods: we utilized dunkin-hartley (dh) guinea pigs that develop primary osteoarthritis and experiences age-related skeletal muscle dysfunction by months of age (~ % of their maximal predicted lifespan). we treated young ( mo) and older ( mo) dh guinea pigs for and months, respectively, daily with a nrf a. we assessed metrics of gait monthly to measure the effect of nrf a on agerelated musculoskeletal dysfunction. we evaluated the effect of nrf a on skeletal muscle protein turnover using the stableisotope deuterium oxide. we also assessed soleus mitochondrial function using high resolution respirometry. results: while nrf a did not affect gait in young guinea pigs, months of nrf a treatment maintained stride length (p= . ) in older male and stance width (p< . ) in older female guinea pigs compared to untreated controls. nrf a improved (p= . ) adp vmax in young females and old males compared to their respective controls. nrf a also increased uncoupled electron transport system capacity in both male and female guinea pigs of both ages (p< . ). nrf a augmented contractile protein synthesis in the soleus of old male and female guinea pigs (p= . ), but did not prevent the age-related declines in the gastrocnemius. conclusion: in summary, long-term nrf a treatment improved skeletal muscle mitochondrial function, increased contractile protein synthesis, and maintained aspects of gait. together, our findings provide evidence that targeting the transcription factor nrf mitigates the decline in musculoskeletal function in a model of osteoarthritis and sarcopenia, with concomitant improvements in mitochondrial function and protein turnover. . j a n n e k e v a n w i j n g a a r d e n , francina j dijk , miriam van dijk , lisette cpgm d e g r o o t , y v e s b o i r i e , , y v e t t e c l u i k i n g background: sarcopenia is a muscle disease rooted in adverse muscle changes that accumulate across the lifespan. multiple factors cause or worsen sarcopenia, with aging as the primary factor and malnutrition, inactivity and diseases as secondary factors. objectives: to design a nutritional strategy to manage sarcopenia. methods: our research program investigated ) specific nutrient deficiencies in sarcopenic older adults, ) muscle protein synthesis (mps) response in cells and rodent models, and ) effect of a specific nutrient combination (whey protein, leucine and vitamin d -actisyn(tm), present in the medical nutrition supplement fortifit(r), on mps in older adults. results: cross-sectional studies indicated a significantly lower intake of protein (- %) and vitamin d (- %) in sarcopenic versus healthy older adults (p< . ) [verlaan, clin nutr ], and higher prevalence of sarcopenia among those with lower blood levels of leucine, total essential amino acids ( the specific combination of whey protein, leucine and vitamin d (actisyn(tm)) provides the right environment for muscle building in sarcopenia, where these nutrients are often deficient. this combination acts through a proven anabolic mode of action with optimal nutrient bioavailability for the muscle to stimulate mps. fortifit and actisyn are trademarks of n.v. nutricia. background: age-related sarcopenia is a major responsible for premature death, poor quality of life and several adverse outcomes, which lead to higher health care costs. despite its recent incorporation as a muscle disease (icd- -cm m . ), early identification of this disease remains challenging. mostly, due to classification and diagnostic criteria, which are predominantly based on technically advanced assessment tools, which may not be available in all clinic settings. recently, a non-invasive technique to analyze variations in biological tissues considering the effect of physiological and biological properties on microwave signals is being studied for its potential to determine muscle mass, with possible applications in the early diagnosis of this disease. objectives: therefore, the principal objective of this study is to preliminarily test the potential of this technique as a new tool for early diagnosis of age-related sarcopenia in a clinical setting. methods: muscle surface area are going to be assessed by abdominal computational tomography (ct) on the third lumbar spine vertebra (l ) and bioimpedance measurements among men and women, aged >= years in the maastricht university medical center, the netherlands. participants will also be subjected to measurements done with the device under test (dut) (the proposed technique) in the same location. the data collected from the three different measurements are analyzed looking for correlation. laboratory experiments made from synthetic materials emulating human tissues and from ex-vivo porcine tissues are used for optimization and interpretation of the clinical measurements. results: up-tonow, the campaign has just started and there is no enough data to give a preliminary result. initial laboratory experiments prove that the thickness of the fat and muscle tissues is correlated to the system response. conclusion: this prospective device will estimate the muscle mass locally using microwave electromagnetic principles. the results of this study can contribute to reveal the potential of this approach as a tissueanalysis tool for early diagnosis and management of age-related sarcopenia. the results might also provide useful evidence to consider in a future planned prospective cohort study, which aims to examine the impact of dietary biomarkers and genetic factors on the incidence of age-related sarcopenia in older adults. background: sarcopenia has become a serious problem in this aging society. at present diagnosis of sarcopenia consist of physical performance and muscle quantity. dexa has been widely applied to examine muscle quantity in clinical but it's radioactive, inconvenient and unaffordable in remote area. as a result, there are more studies in ultrasound in replace of dexa. objectives: based on others researches csa might be a suitable parameter to evaluate the muscle quantity. we develop a cheaper ultrasonic imaging system to evaluate the cross-sectional area (csa) of rectus femoris (rf)muscle. methods: we use a cmos image sensor combing with digital signal processor to detect the displacement of single element ultrasonic transducer. therefore, we combine us a-mode signal with displacement into b-mode image. by circling region of interest (roi), we can obtain the csa of rf muscle. then, we use siemens s evaluating the csa in the same region to testify the reliability. results: we recruited young college students undergoing the experiment. the result shows that the correlation coefficient is up to . . conclusion: in conclusion, our device can successfully evaluate the csa of rf muscle. moreover, our system using single element ultrasonic transducer is much cheaper than linear transducer in practice .it can be affordable in remote village or somewhere lacking in medical resource. a case-control study. camille nicolay , sandra higuet , sandra de breucker (( ) geriatric department, hôpital erasme, brussels, belgium; ( ) geriatric department, hôpital isppc-charleroi, charleroi, belgium) background: ten percents of belgian population are considered to be informal caregivers. little is known about their frailty status and their physical health. objectives: we compared the frailty status, the clinical and psychosocial status of old caregivers with controls (> ). we analyzed the association of frailty status according to fried's criteria and rockwood frailty index (fi) with the characteristics of caregivers and controls in multiple regression analysis. methods: eighty six caregivers and gender and agematched controls were included. frailty was assessed by the frailty phenotype (fried) and the -deficit frailty index (fi). social data, sf- health survey, basic and instrumental adl, geriatric depression scale, mini nutritional assessment, mini-cog, cumulative illness rating scale-geriatric, usual gait speed, handgrip strength, and burden scale (zarit) were collected. results: the prevalence of frailty was similar in caregivers and controls with the fi (p= . ) but higher with the fried's criteria (p= . ). compared with the control group, caregiving was associated with a lower mental quality of life (p< . ), a higher risk of depression (p< . ), a higher consumption of antidepressant (p= . ), a lower nutritional status (p= . ), a more frequent help from health care providers (p= . ), and more problems to maintain physical contacts with a social network (p= . ). in multiple regression, the fried's criteria adjusted for age, gender, marital status and incomes were associated with the age, the grip strength, the physical quality of life, the gait speed and the nutritional status (r = . -p< . ), while fi was associated with the risk of depression, the use of antidepressants, the physical quality of life, the cognitive status and basal & instrumental adl (r = . -p < . ) in caregivers. conclusion: the prevalence of frailty is similar in caregivers and controls when using fi, but higher in caregivers with fried's criteria. compared with controls, caregiving is associated with poorer health and psychological issues. while fried's criteria focus on physical frailty, the fi is more related with geriatric syndromes like depression, cognitive disorders, loss of autonomy, and quality of life. this study could help researchers to choose between frailty scales before starting a study about older caregivers. background: nursing home (nh) residents are often undernourished and physically inactive contributing to sarcopenia and frailty. mobility is identified by older nh residents as being key to their quality of life and well-being. the combination of protein supplementation and physical exercise has been shown to be most effective to maintain and increase muscle mass. objectives: the older persons exercise and nutrition (open) study aimed to investigate the effects of sit-to-stand exercises (sts) integrated into daily care combined with a protein-rich oral nutritional supplement (ons), on physical function, nutritional status, body composition, healthrelated quality of life and resource use. methods: residents in eight nh were randomized by nh units into an intervention group (ig) or a control group (cg) (n= /group). the ig was offered a combination of sts (four times/day) and ons ( bottles/day providing kcal and g protein) for weeks. the participants resided in nh units (dementia and somatic care), were >= years and able to rise from a seated position. the seconds chair stand test ( scst) was the primary outcome. secondary outcomes were balance, walking speed, dependence in adl, nutritional status and body composition, health-related quality of life and resource use. data was analyzed using descriptive and inferential statistics including regression models. results: altogether residents ( ± years, % females) completed the study. no improvement in the physical function assessments was observed in the ig, whereas body weight increased significantly ( . ± . kg, p= . ) vs the cg. twenty-one (of ) participants with high adherence to the intervention, i.e. at least % compliance to the combined intervention, increased their fat free mass ( . kg ( . , . iqr), p= . vs cg. logistic regression analyses indicated that the odds ratio for maintained/improved scst was . (ci . , . , p= . ) among the participants with high adherence compared to the cg. waly dioh , cendrine tourette , carole margalef , amy chen , rené lafont , , pierre dilda , stanislas veillet , samuel agus (( ) biophytis, sorbonne université -bc , paris, france; ( background: sarcopenia is a geriatric condition characterized by loss of muscle mass and functions and can contribute to risks of falls, fractures and hospitalization. sara-obs is a multicenter, observational trial designed to better characterize age-related sarcopenia in a community dwelling population at risk of mobility disability. this is part of a clinical program that strives to provide more understanding of the target population in order to further develop a potential sarcopenia medical intervention. sara-obs study rationale, design and main baseline characteristics are presented. objectives: the objective is to characterize sarcopenia and sarcopenic obesity in older adults through evaluation of their physical performance and body composition. changes in baseline characteristics after a -month period will be assessed and used for development of a phase interventional study on the efficacy and safety of an investigational drug, bio . methods: participant recruitment was based on age (>= years), sppb score =< and body mass based on the fnih criteria. physical functions were assessed by two walking tests ( m walk test and the -minute walk test), the sppb, the handgrip strength test and the stair climb power test. patient reported outcomes were also assessed with the sf- and the sarqol questionnaires. results: subjects were included in this study and the main screen failures were sppb scores and body mass criteria. baseline characteristics indicated that the average bmi was high, ~ % of the participants were women and that the alm/bmi in men was lower than the fnih threshold ( . vs . ) but was similar in women ( . vs . ). m gait speed was . m/s, the mean total sppb score was . with the gait speed component of < . m/s and the chair stand sub-score of . . conclusion: this population has a similar m gait speed as the populations in life and sprint-t studies at baseline. however, the sppb total score and the chair stand sub-score correspond more closely with the sprint-t study. addressing the loss of physical function and preventing mobility disability is still an unmet need of older adults. sara-obs included a population representative of a suitable target for subsequent interventional studies aimed to fulfill this need. yen-lung chen, hui-hua chiang (department of biomedical engineering, national yang-ming university, taipei, taiwan) background: in whole world, the elderly formally entered the aging society , and the patients with sarcopenia were highrisk groups in the fall. more than % of the elderly suffered moderate injuries due to falls. the sarcopenia as defined by the eu's sarcopenia working group was refers to progressive reduction in muscle mass and decreased muscle function. objectives: it is expected to provide diagnostic tools and techniques for the rapid determination of sarcopenia and muscle strength. at the same time, it will also be developed toward portable devices to facilitate the diagnosis of the aging of muscle function in the elderly at home to take care of the health and well-being of the elderly. methods: at present, the clinical measurement part is assisted by the radiation department of the veterans general hospital to collect and measure the subjects. clinical testing methods are mainly for older people over years of age. the walking speed test is firstly performed on the method. if it is normal, then the grip strength test is performed. if the grip strength is too small, the femoral rectus femoris muscle volume test should be performed. generally, dual energy is used. dual-energy x-ray absorptiometry (dxa) is used for testing. if the walking speed is too slow, the dxa test should be performed directly. the test value is less than . (kg/m ) in woman and less than . (kg/m )in man. that is, it is determined as a sarcopenia patient. since dxa has a small amount of free radiation, high cost, and a large space occupation, we expect to obtain a wide range of data through ultrasonic scans. back-end development algorithms are calculated to determine if there is sarcopenia and how severe it is. results: at present, the rectus femoris muscle volume obtained by using ultrasound has a highly linear relationship with the appendicular muscle mass measured by dxa (r = . ,p< . ), and has the ability to distinguish whether it is sarcopenia. conclusion: the use of muscle volume of rectus femoris can improve the accuracy of sarcopenia prediction. in the near future, this plan will be used to develop automated ultrasonic scanners. background: although sarcopenia has multifactorial causes, the decline in physical activity has been considered a very important aspect for its development. since the promotion of higher levels of physical activity can attenuate the progression of sarcopenia, it is possible that the participation in a programmed training increases the spontaneous physical activity of the participants. objectives: to investigate if the participation of sarcopenic older women in a resistance training program and supplementation with fish oil leads to changes in the level of spontaneous physical activity (sedentary time and number of steps). methods: randomized, double-blind, placebo-controlled clinical trial. thirty-two older women, aged >= years, participated in the study. all participants were classified as sarcopenic based on the criteria of the european consensus on sarcopenia (ewgsop). the participants were divided into two experimental groups: ( ) exercise group + placebo (ep) and ( ) exercise group + fish oil (efo). both groups underwent a resistance exercise program over weeks, consisting of three weekly supervised sessions. all volunteers were instructed to take two capsules of food supplement at each main meal, lunch and dinner ( g/day). the ep group used capsules composed of sunflower oil as placebo, and the efo group fish oil capsules, (epa mg and dha mg). measurements of the level of spontaneous physical activity were made before and after the intervention by using the actipal® physical activity monitor (glasgow, uk), for a period of seven consecutive days, during which the volunteers were instructed to maintain their normal routine. the volume of the quadriceps muscle in the pre and post intervention periods was calculated from the images obtained by magnetic resonance imaging. for statistical analysis, a linear regression model with mixed effects was used to compare longitudinal data on mean intra-group differences between groups and moments. for all analyzes, a significance level of . was adopted. results: both groups showed an increase in muscle volume after the intervention ( . cm ( . %) and . cm ( . %), respectively). regarding the level of spontaneous physical activity, both groups had a similar sedentary time and number of steps, at both times (average . h and , steps in the pre-intervention period and . h and , steps in the postintervention period for the ep group, and . hrs and , steps in the pre-period and . h and , steps in the postintervention period in the eop group). conclusion: although sarcopenic older women supplemented with fish oil showed a higher increase in muscle volume, the level of spontaneous physical activity remained unchanged both in the pre and post intervention periods and between groups, indicating that the increase in muscle volume was not associated with significant changes in the level of spontaneous physical activity. background: regardless of improvements in surgical and anesthetic practices, older surgical patients often experience postoperative complications. the purpose of this study was to investigate the association between physical frailty and cognitive function using a validated upper-extremity function (uef) test with in-hospital outcomes in aging adults undergoing abdominal surgery. objectives: to recognize frailty and cognitive function as a risk factor for in-hospital adverse outcomes. methods: we administered pre-operative uef tests, within -hours after admission, among patients aged years and older undergoing emergent/urgent abdominal surgery. the uef involved two tests; -and -sec of respectively fast and consistent elbow flexion, while angular velocity was measured via two wearable motion sensors applied to the wrist and upper-arm of the dominant arm. uef physical score was calculated, based on slowness, weakness, flexibility, and exhaustion (range: resilient= -frail= ). uef cognitive score was assessed based on motor function variability within a dual-task performance that involved uef motor task and a cognitive task of counting backwards by threes (range: cognitive normal= -cognitive impairment= ). adverse outcomes included: length of stay, complications, and death during their hospital stay. a logistic regression model was used to assess the association between uef physical and cognitive scores (independent variables) and in-hospital outcomes (dependent variable). results: a total of participants (mean age . ± . years) completed the preoperative uef assessment. thirty-six participants with an average age of . ± . years experienced at least one adverse outcome while in the hospital. while age independently predicted in-hospital outcomes with receiver operating characteristic area under the curve (roc-auc) of %, this prediction improved by adding either the uef physical or the cognitive score. the physical score predicted in-hospital outcomes with a roc-auc of %, and the cognitive scores predicted in-hospital outcomes with a roc-auc of %. conclusion: the proportion of emergency surgical procedures increases with age, and population trends indicate that this demand will increase significantly. results from the current study showed that sensor-based measures of physical and cognitive function can provide an objective tool for predicting adverse outcomes, with potential applications for other surgical procedures. risk stratification can help to establish targeted management strategies to improve the healthcare system and patient-centered outcomes. background: while sensor-based daily physical activity (dpa) gait performance has been demonstrated to be an effective measure of physical frailty, it is not clear how repeatable the dpa gait parameters are between different days of measurement, especially across frailty groups. objectives: to evaluate the test-retest reliability (repeatability) of dpa gait performance parameters (stride time, variability, and irregularity) and quantitative measures (number of steps and walking duration) between two separate days of assessment among older adults. methods: dpa was acquired for -hours from older adults (age>= years) using a tri-axial accelerometer motion-sensor attached to the trunk. purposeful continuous walking bouts (>= s) without long pauses (> . s) were identified from acceleration data and used to extract gait performance parameters, including stride time, power spectral density (psd) slope (representing the variability of walking cycles), dominant frequency of walking, and gait irregularity (sample entropy, representing predictability of walking cycles). to assess repeatability, intraclass correlation coefficient (icc) was calculated using two-way mixed effects f-test models for day- vs. day- as the independent random effect. repeatability tests were performed once for all participants and once within each frailty group (non-frail and pre-frail/frail). results: data from older adults, non-frail (age: . ± . years) and pre-frail/frail (age: . ± . years) were analyzed. within all participants with purposeful walking bouts on both the days, gait performance parameters of stride-time and gait variability parameters (slope and dominant frequency of walking) showed excellent test-retest reliability values (icc>= %) while quantitative parameters, including number of steps and walking duration showed poor test-retest reliability results (icc< %). among gait performance parameters (stride time, dominant walking frequency and sample entropy), we observed higher repeatability among the pre-frail/frail group with icc> % compared to icc< % for non-frail individuals. conclusion: from our study, it is evident that gait performance parameters including average step-and stride-time and frequency-domain gait variability parameters provided higher test-retest reliability compared to quantitative measures. further, gait performance parameters showed higher repeatability among pre-frail/frail volunteers between the two days compared to non-frail volunteers, which may be attributed to a lack of functional capacity among frail individuals for performing more intense and more variable physical tasks. background: while evaluation methods for skeletal muscle characteristics which are necessary to know the pathogenesis of sarcopenia are being considered, ultrasonography is attracting attention as a method simultaneously evaluate quantitative and qualitative evaluation of skeletal muscle. although we have found many, the statements that examined the relation between muscle thickness, echo intensity, physical function, and sarcopenia by quadriceps muscle ultrasonography in the previous report, there are few reports for the lower leg muscles. objectives: we conducted a study to examine whether the lower leg muscle ultrasonography is useful for evaluating sarcopenia index and muscle quality (muscle strength per unit muscle mass) evaluation in comparison with the quadriceps ultrasonography. methods: the participants were patients over years old ( males, females). the muscle thickness of the quadriceps muscle, tibialis anterior muscle, gastrocnemius muscle, soleus, and echo intensity were measured by ultrasonography, and the relationship between lower extremity muscle mass, muscle strength, physical function, and muscle quality was examined. results: the muscle thickness of quadriceps muscle, tibialis anterior muscle, soleus muscle was related to lower extremity muscle mass, grip strength, leg muscle strength, and only quadriceps muscle was related to gait speed. the echo intensity of the quadriceps, tibialis anterior, gastrocnemius was related to, grip strength, leg muscle strength, and only the tibialis anterior muscle was related to gait speed. the muscle thickness and the echo intensity of tibialis anterior muscle and soleus muscle are highly correlated with the quadriceps. the echo intensity of the tibialis anterior muscle, as well as that of the quadriceps muscle, showed a high correlation with the muscle quality of lower extremity. conclusion: concerning the assessment of sarcopenia using ultrasonography, muscle thickness and echo intensity evaluation by tibialis anterior muscle showed the same utility as them by the quadriceps muscle, and echo intensity of the tibialis anterior muscle can be a marker of muscle quality. lucena germano , cristiano dos santos gomes , juliana fernandes de sousa barbosa , , raysa freitas , , alvaro campos c. maciel , ricardo oliveira guerra ( ( background: phase angle (pha) is emerging as a measure of great clinical relevance provided through bioimpedance assessment and its related to health adverse outcomes such as osteoporosis and sarcopenia. on the other hand, poor physical performance as gait speed and grip strength in elderly is associated with poor health conditions. we hypothesized it is plausible that those two measures might be related and can be used as a tool in clinical practice. objectives: to investigate the relationship between pha and physical performance measures in community-dwelling older adults from brazil. methods: this cross-sectional study enrolled older adults of both sexes who had a comprehensive health evaluation including physical performance tests (gait speed and handgrip strength) and electrical bioimpedance screening. linear regression models were used to estimate the associations between pha and physical performance measures. results: the mean age of . ± . and . ± . for men and women respectively. hand grip strength (n: , ; p-value < , ) and gait speed (n: , ; p-value < , ) were independently correlated with pha. conclusion: pha could help to easily identify elderly on the onset of present heath adverse outcome and guide specific interventions by clinicians. shosuke satake , , kaori kinoshita , yasumoto matsui , background: in japan, we have a simple yes/no questionnaire to assess multiple functions in daily living for older adults; the kihon checklist (kcl). in the questionnaire, questions to assess mobile functions are included. objectives: we examined whether the -item questions in the physical domain of the kcl (kcl-phys) could be a surrogate of validated measurements of physical functions. methods: subjects were independent and ambulatory seniors aged years or older who had been consulted in our frailty clinic. all of them received grip strength test, dual energy x-ray absorptiometry, physical performance tests, cognitive examination, and the kcl questionnaire. among them, we excluded subjects with missing data, and with moderate cognitive impairments. we examined the relationships between scores of the kcl-phys and usual gait speed, short physical performance battery (sppb), and timed up and go (tug) with the spearman's rank correlation. the score of the kclphys were counted when the subject meets any criteria with each question as previously reported. also, we evaluated the cutoff point of the kcl-phys equivalent to slow gait speed (< . m/s), low sppb score (sppb < ), and slow tug (tug >= sec) with the receiver operating characteristic (roc) curve analysis. results: the mean values of age, body mass index, and prevalence of sarcopenia were . years old (women . %), . (kg/m ), and . (%), which were no differences between sexes. on the other hand, physical functions of gait speed, sppb, and tug were all worse in women than in men. relationships between the scores of the kcl-phys and usual gait speed, sppb, and tug were moderate with the coefficients of - . , - . , and . , respectively (p< . for all). the area under the roc curve of the kcl-phys score equivalent to slow gait speed, low sppb score, and slow tug were . , . , and . , respectively. the cutoffs were thought to be the best at points of the kcl-phys to identify low physical functions based on the youden index. conclusion: physical domain of the kcl could be a surrogate of assessments of physical functions in older people. yuji hirano , izumi kondo , tetuya nemoto , naoki itoh , hidenori arai (( ) national center for geriatrics and gerontology, japan; ( ) nihon fukushi university, japan) background: we have developed a new type of grip strength measurement that addresses the time axis in evaluating physical function. it can measure the dynamic force, response in gripping performance, and maximum grip strength. the "kihon checklist" (kcl) is used to screening the frail elderly, based on the japanese long-term care insurance system. however, the relationship between the gripping performance and kcl has not been well investigated. objectives: the purpose of this study was to introduce a novel automatic reading method for dynamic force parameters in gripping performance and to evaluate their relationship with the kcl. methods: the subjects comprised patients ( men, women, average age . ± . years) who visited the integrated healthy aging clinic (locomo-frail outpatient clinic in japanese) of our hospital. the four indices of grip force response measured were: reaction start time (rst), time constant (tc), maximum value of force (mvf), and force rising slope (frs). we examined the relationship between these four indices and seven categories of the kcl; activities of daily living (adl), physical functions and fall, nutrition state, oral functions, outdoor activities, cognitive functions and mood, using spearman's correlation coefficient. results: in the female right hand, the mvf was only significantly correlated with adl and overall scores; whereas, in the female left hand, the mvf and the frs were significantly correlated with many items (adl, physical functions and fall, nutrition state, outdoor activities, and cognitive functions). the time-dependent items (rst and tc) were significantly correlated with outdoor activities in the female left hand and significantly correlated with adl and oral functions in the male left hand. however, in the right hand, the time-dependent items were not correlated with any of items in kcl in both sexes. conclusion: our newly developed grip strength measurement system could automatically calculate not only the maximum grip strength but also the time response of the grip force. moreover, their relationship with kcl was clearly indicated. the relationship between detailed grip strength response indicators and kcl items differed between men and women, and the left hand was correlated with more items than the right hand. ranyah almardawi, rao gullapalli, michael terrin (university of maryland school of maryland, baltimore, usa) background: rotator cuff (rc) tear and shoulder pain are both highly prevalent in older populations. routine medical screening for shoulder dysfunction is uncommon for community-dwelling older adults. the disabilities of the arm, shoulder and hand (dash) survey estimates self-reported dysfunction of both upper limbs in a composite score. dash offers a quick method to identify older adults with potential dysfunction in either shoulder, which otherwise may go unrecognized during routine medical visits. objectives: . to determine if dash, american shoulder and elbow surgeons (ases) and simple shoulder test (sst) surveys are related to one another in older adults. . to assess dash, ases and sst score relationships to the sf- physical functioning (pf) subcomponent score, shoulder forward flexion range of motion (ff-rom) and shoulder abduction range of motion (abd-rom) in older adults. methods: cross-sectional study: twenty-three community-dwelling-older-adult volunteers [mean age, . ± . years; range, to years; female, %] with no history of rc surgery and no history of shoulder injury or shoulder physical therapy in the prior months completed shoulder magnetic resonance imaging (mri) and dash, sf- , charlson co-morbidity index (cci), katz activities of daily living (adls) and lawton instrumental adls (iadls) surveys. for the shoulder ipsilateral to mri, participants completed ases, sst, visual analog scale for pain (vas) surveys; and shoulder ff-rom and abd-rom. descriptive statistics and spearman rank order correlation (rho) were performed. results: frequencies: rc tear (supraspinatus tendon) on mri: . %; shoulder pain >= on vas: . %; no limitation (score= ) on katz adls: . %; no limitation (score= ) on lawton iadls: . %. means: cci, . ± . ; dash, . ± . ; ases, . ± . ; sst . ± . ; . ± . ; . ± . ; . ± . . range of correlation among dash-ases-sst surveys: (|rho|= . - . , p< . ). range of correlation for dash-ases-sst with sf- pf(|rho|= . - . , p< . ), p< . ), abd-rom (|rho|= . - . , p< . ). conclusion: dash, ases and sst correlate well, and all three surveys show a consistent relationship with sf- physical functioning, ff-rom and abd-rom. next steps would be to evaluate the feasibility of dash to identify older adults with shoulder dysfunction during routine medical visits. background: physical performance is closely associated with chronic diseases and dysfunction of numerous organ systems. old persons with chronic renal failure have shown the apparent decline in physical performance, especially in the end-stage. however, it is unclear whether the subclinical kidney dysfunction is associated with skeletal muscle function deficit in the elderly population. objectives: the aim of this study is to determine the association between renal function and skeletal muscle function deficit in old persons without nephropathy. methods: eight hundred fifty-four korean elderlies (female, . %) aged to years were included in the cross-sectional analysis. of the participants, elderlies (female . %) were available for the -year follow-up test session. all participants were interviewed face-to-face and received measures of anthropometry, body composition and serum biomarkers of metabolic diseases. estimated glomerular filtration rate (egfr) was calculated using the chronic kidney disease epidemiology collaboration (ckd-epi) equation based on serum creatinine concentration. skeletal muscle function deficit was defined as a combination of weakness and slowness based on the handgrip strength to body mass index ratio (hs/bmi, men < . , women < . ) and converted timed up-and-go to walking speed (tugspeed < . m/s). results: the subjects with <= egfr < ml/min/ . m showed significantly lower physical performance for muscular strength and functional mobility than those with <= egfr < and egfr > ml/min/ . m , respectively (all for p < . ). logistic regression analysis indicated the significant association between egfr and skeletal muscle function status even after adjustment for potential confounders (p for trend < . ). moreover, the prospective observational analysis by ancova showed the significant effects of enhancement in hs/bmi [f( , ) = . , p = . ] and tugspeed [f( , ) = . , p < . ] on the improvement in egfr during -year followup. conclusion: taken together, skeletal muscle function status is associated with even moderately reduced egfr in an older population. these results suggest that maintenance of physical and functional fitness may be a contributory factor for preserving renal function in elderly persons. rn, brazil) background: sarc-f is a brief and useful test to identify older people at risk of sarcopenia-associated adverse outcomes. previous studies with older populations have suggested that it may be useful to screen those with severe sarcopenia. its ability to screen sarcopenia among low-income brazilian older adults is still unknown and its association with sarcopenia diagnostic criteria may be useful to understand its utility among this population. objectives: this study aims to evaluate the validity of sarc-f in screening low muscle strength and low physical performance among a low-income sample of older adults. methods: in a cross-sectional study, community-dwelling older-adults (>= years old; men and women) from santa cruz (northeast brazil) answered the sarc-f questionnaire and were classified as sarcopenic (>= ) and non-sarcopenic (< ) according to sarc-f scores. they were also evaluated in relation to the sarcopenia criteria of muscle strength (handgrip strength) and physical performance (sppb). the cutoff of < kg for women and < kg for men were used to classify those with low muscle strength. a sppb score of <= was used to classify low physical performance. a chi-square test was used to assess the association between the sarc-f and the objective parameters of sarcopenia. sensitivity and specificity of the sarc-f according to the objective functional parameters were also assessed. results: the sample was composed by % of women, with mean age of . (± . ) years old. according to sarc-f, . % of the sample was sarcopenic. low muscle mass and low physical performance were identified in . % and . % of the sample respectively. sarcopenia was significantly associated to low muscle mass (p< . ) and low physical performance (p< . ). the sensitivity of sarc-f in identifying those with low muscle mass was of % and specificity of %. for low physical performance, sensitivity and specificity were of % and % respectively. conclusion: sarc-f has a moderate ability to identify the sarcopenia criteria of low muscle mass and low physical function among older adults from a low-income setting. since it is a simple measure, it can be advantageous for low-income and rural communities. background: menopause marks a critical transition towards older adulthood for women and studies suggest that it is associated to several sarcopenia parameters, such as muscle mass and physical functioning. understanding how the menopausal transition associates to sarcopenia diagnostic criteria may help to direct screening tests for middle-aged populations and to identify earlier those at higher risk of sarcopenia. objectives: to evaluate the association between menopausal status and sarcopenia diagnostic criteria (muscle strength, muscle quantity and physical performance). methods: in a cross-sectional study, communitydwelling women from northeast brazil ( - yearsold) were evaluated in relation to menopausal status using the stages of reproductive aging workshop classification (premenopausal, perimenopausal or postmenopausal) , and in relation to sarcopenia diagnostic criteria according to european working group on sarcopenia in older people (ewgsop ): muscle strength (grip strength -handheld dynamometer), muscle quantity (appendicular muscle mass adjusted for height through bioelectrical impedance) and physical performance (gait speed). association between menopausal status and sarcopenia criteria was evaluated with multiple linear regression models adjusted for covariates (current age, education, family income, walking, bmi, reproductive history). results: among the participants, . % were classified as premenopausal, . % as perimenopausal, and . % as postmenopausal. menopausal status was significantly associated to grip strength, since premenopausal women were significantly stronger than perimenopausal or postmenopausal women, even in the fully adjusted analyses (b= . ; % ci= . : . ). muscle quantity and gait speed were not significant according to menopausal status. conclusion: perimenopausal and postmenopausal status are associated with less muscle strength among middle-aged women. muscle weakness may be the first sarcopenia parameter that is affected by women's aging and should be tracked among middle-aged to women for early identification of sarcopenia risk.. background: we speculate maintaining good postural stability is the key to good adl in elderly patients. this is a preliminary study to evaluate which factor relates to good postural stability. objectives: we evaluated patients ( males and females) over years old. the average age was . years old ranging to . methods: we measured index of postural stability(ips) using gravicoder gw- manufactured by anima. the ips was adovocated by mochizuki in . it was defined following this equation; ips=log[(area of stability limit + area of postural sway)/area of postural sway). larger ips means better postural stability. the average ips in each age was already known. ips was calculated automatically through gravicoda. we devided these patients into two groups by the results of ips. group a with the patients whose ips was larger, group b with the patients whose ips was smaller than the average in their age. we compared the following items between the two groups. nutrition(albumin, calcium, magnesium, ferritin, vitamin b ,b , , -d , zinc in blood test) , bone status(bone density, % of yam), spinopelvic parameters (pelvic incidence(pi), lumbar lordosis(ll), pelvic tilt(pt) using whole spine x-ray photograph. results: ten patients were classified into group a and patients were into group b. the average age was . ± . years old in group a and . ± . in group b. in group a , ll and pt were respectively . , . . in group b, . , . . ll and pt were significantly different between the two groups. pi minus ll is an important indicator to determine the spino-pelvic balance. it is known that pi-ll< means good spino-pelvic balance. in group a, pi minus ll was . ± . . in group b, it was . ± . . according to nutrition and bone status, albumin was significantly higher in group b. conclusion: our results showed spino-pelvic alignment related to the postural stability. this suggests good spino-pelvic alignment is likely the key to good postural stability. background: physical performance is of main relevance for quality of life and independence in the community. identification of deterioration of physical performance helps to start early interventions to stay independently in old age. objectives: to determine physical performance of communitydwelling older adults above years by using a comprehensive geriatric assessment to find most sensitive tests for functional decline. methods: older community-dwelling adults aged +. analysis of baseline and (t ) and months (t ) of followup data of hand grip strength (hgs), stair climb power test (scpt), timed up and go test (tug), short physical performance battery (sppb), m gait speed ( mgs), -time chair rise test ( tcr), minute walking test ( mwt) and frailty categories according to fried. results are shown in mean (± sd) in total numbers and percentage. results: participants ( , y.± , ) were included, ( %) female. overall physical performance was on high level, above geriatric cut-offs for physical disabilities at baseline: (hgs female: , (± , ) (- , (± , )%) followed by scpt (- , (± , )%). all tests showed a decline except tcr (+ , (± , )%). conclusion: physically active, communitydwelling older people show a high level of functional performance, far from geriatric cut-offs indicating physical disabilities. nevertheless, after two years a clinically relevant reduction of strength in upper (handgrip) and lower extremities (stair climb) was detected. these data may be relevant for the identification of older individuals who may benefit from early intervention exercise programs to keep them physically independent as long as possible. tcr showed divergent results and could be of special interest for continuous measurements to identify gradual decreases in functional performance. background: sarcopenia is characterized by loss of skeletal muscle mass and strength and it is a frequent finding in oncology, being associated with reduced quality of life, impairment in the response to antineoplastic therapy and increased toxicity, especially in older patients. objectives: the aim of the present study was to evaluate the association between low muscle mass (lmm) assessed by computed tomography (ct) analysis and sarcopenia considering the revised european consensus published by the european working group on sarcopenia in older people (ewgsop ) with the variables of the comprehensive geriatric assessment (cga) in older oncological patients. methods: for this purpose, patients ( . % female; mean age of . ± . years) followed at the oncogeriatric outpatient clinic of a university hospital were enrolled. clinical data were obtained from electronic medical records and the skeletal muscle mass evaluation was performed using ct (in the height of the third lumbar vertebra). for lmm and sarcopenia classification, specific cutoff points were adopted. cga variables were compared between lmm and normal skeletal muscle mass (nsmm) and between sarcopenic and non-sarcopenic individuals. groups were compared by the independent t test (r core team®, p< . ). results: the most frequent tumors were breast, intestine, stomach and lung, at different stages of the disease. the prevalence of lmm was . % and the prevalence of sarcopenia was %. of all cga variables evaluated, hand grip strength ( , ± , ) and katz scale ( , ± , ) were associated with lmm and sarcopenia. conclusion: the results highlight the importance of early geriatric clinical assessment of older cancer patients, considering the association of cga variables with low muscle mass and most important, to sarcopenia, for the possible reversal of functional and nutritional impairments and for the indication or appropriate planning of cancer therapy. lygia paccini lustosa , patricia parreira batista , jéssica rodrigues de almeida , andré gustavo pereira de andrade , aimée de araújo cabral pelizari , stephanie aguiar , leani de souza máximo pereira (( ) physical therapy department -universidade federal de minas gerais, ufmg, eeffto, belo horizonte, mg, brazil; ( ) sports department -universidade federal de minas gerais, ufmg, eeffto, belo horizonte, mg, brazil) background: functional tests in the older person reflect the integrity of the interrelationship between muscle mass and function, vascular, endocrine and neurological aspects of central and peripheral command. the reduction in functionality, muscle mass and strength associated with advancing age is related to the increase of circulating proinflammatory cytokines in plasma, which in turn predisposes the individual to negative repercussions, such as the development of chronic diseases, falls and disability. they can identify changes in the intrinsic capacity of the older people. objectives: to compare older women who reported being active or sedentary regarding functional capacity and plasma indices of inflammatory mediators. methods: participated community older women ( years or older), recruited for convenience. those unable to walk were excluded; acute musculoskeletal diseases; lower limb fractures in the last year; neurological diseases and sequelae; history of cancer in the last five years and cognitive impairment (mental state mini-exam). all responded to clinical and demographic information, performed the short physical performance battery (sppb), timed up and go (tug) and plasma tests of stnfr and il- (elisa method). correlation analysis by spearman test. % significance level. approval by the research ethics committee/ ufmg (caae: . . . ). results: older women participated, with a mean age of . ± . y; number of comorbidities . ± . and medications in use of . ± . . mean of body mass index were . ± . kg/m . there was a significant negative relationship between the sppb test and stnfr (rho= . ; p= . ) and a significant positive relationship between tug and stnfr (rho= . ; p= . ). other relationships were not significant (p> . ). conclusion: older women with better functional capacity presented lower plasma dosage of stnfr . the results suggest influence between these variables -functional capacity, mobility and inflammatory process -and no causal factor can be attributed. in these case, longitudinal studies are needed to verify functional performance vulnerability factors and their causal relationship with circulating inflammatory mediators in plasma. however, these results point to the importance of evaluating these variables in daily clinical practice. patricia parreira batista , stephanie aguiar , andré gustavo pereira de andrade , jéssica rodrigues de almeida , leani de souza máximo pereira , lygia paccini lustosa (( ) physical therapy department -universidade federal de minas gerais, ufmg, eeffto, belo horizonte, mg, brazil; ( ) sports department -universidade federal de minas gerais, ufmg, eeffto, belo horizonte, mg, brazil) background: perceptions of health and well-being in the older people are identified as subjective aspects by the international classification of functioning (icf), with direct and indirect interference with overall performance, activities of daily living, social relationships and independence. subjective well-being is associated with the form of coping adopted with a health condition, adaptability and resilience. positive and negative physiological repercussions on functionality and interaction with the family and social network may be consequences of inadequate adaptation and perception of subjective well-being. objectives: to explore the relationship between subjective well-being, functionality and plasma indices of inflammatory mediators in community older wowen. methods: participated community older women ( years or older), recruited for convenience. those unable to walk were excluded; acute musculoskeletal diseases; lower limb fractures in the last year; neurological diseases and sequelae; history of cancer in the last five years and cognitive changes (mini-mental state examination). all answered about clinical and demographic data and information about subjective well-being. they performed tests of functional capacity (short physical performance battery -sppb) and mobility (timed up and go -tug). plasma dosages of stnfr and il- were by elisa method. correlation analysis by spearman test. significance level of %. approval by the research ethics committee/ ufmg (caae: . . . ). results: elderly women participated, with a mean age of . ± . years; number of comorbidities . ± . , final sppb score . ± . , tug of . ± . seconds; body mass index of . ± . kg/m . there was a significant positive relationship between subjective well-being and sppb (rho= . ; p= . ) and tug (rho= . ; p= . ). other associations were not significant (p> . ). conclusion: the results showed a significant association of subjective well-being with functional capacity in the older women. however, this condition was not associated with inflammatory markers, suggesting the need for further studies. on the other hand, it can be thought that the identification of personal strategies and perception of health and well-being act as barriers and/ or facilitators in a functional rehabilitation process, indicating the need for a multidisciplinary approach. background: the united states census bureau projects a rise in the population aged and over from . million in to . million by . the projected rise in the elderly population represents an accompanying increase in geriatric syndromes. frailty is a common geriatric syndrome defined as a clinically recognizable state of increased vulnerability to adverse outcomes related to a decline in physiologic reserve. this decline in reserve places the individual at increased risk for poor health outcomes including falls, disability, hospitalization, institutionalization and mortality. various effective interventions for frailty are established in the literature. the body of knowledge on the role of technology in reducing frailty is less abundant. objectives: to summarize available evidence on frailty and technology use for community dwelling older adults. methods: a comprehensive search of computerized databases was conducted in the following databases published between - : cinahl, pubmed, and academic search complete. the prisma search strategy was utilized for this review. articles were included if they met the following criteria: ) focused on community dwelling adults aged and over; ) peer-reviewed; ) published in the english language; ) featured randomized controlled trials (rcts), cohort studies or qualitative research; and ) included an operationalized definition for frailty. results: the database searches yielded a total of articles. duplicates were removed. results were excluded based on title and abstract. relevant articles were retrieved for full text examination. articles were excluded based on inclusion/exclusion criteria. references of included articles were hand searched for relevant works. four additional relevant articles were identified. the final analysis included articles. conclusion: current research focuses on assessment and diagnosis as opposed to intervention studies. methodological weaknesses limit generalizability and validity of findings. few studies utilize frailty as an outcome measure thus, limiting available research directly related to frailty. emerging technologies represent potentially effective, flexible and integrative solutions for frailty assessment, monitoring and intervention in the home environment. more research is needed on the potential for technological tools as interventions for frailty in community dwelling elderly specifically, for the purpose of detection and prevention of pre-frailty. a study protocol. inae c. gadotti , raquel aparicio ugarriza , , fernanda civitella , jorge g. ruiz , , edgar ramos vieira ( ( ) background: there are several studies on the association of balance and gait impairments with frailty and falls in older adults. however, little is known about the associations between postural alterations, frailty and falls in older adults in general and among older veterans. also, inter-relations among postural alterations, balance, strength, gait impairments, falls and frailty in older adults are not well known. objectives: the objective of this study is to evaluate if postural alterations, gait and balance impairments are associated with falls and frailty in older veterans. methods: sixty veterans, years old or older, will participate on a voluntary basis. one-hour long assessments will be completed at baseline, , and months. participants will fill out a questionnaire including information on demographics (age, sex, height, and weight), health conditions, falls (history, characteristics, and fear of falls), mobility impairments, physical activity level, medication history, medication changes and adherence, and health care utilization. frailty status will be assessed based on fried's frailty phenotype. the following physical health variables will be assessed: sagittal head and neck posture using photogrammetry, spinal curvatures using flexicurve, deep neck flexors activation by performing the craniocervical flexion test with a pressure biofeedback, grip strength using a dynamometer, usual and fast gait analysis using a gaitrite, balance using a force plate, and lower limb functional strength based on chair stands in s. differences among the variables by frailty status and falls history will be assessed using manovas. results: the results will be presented at conferences and published in scientific journals. conclusion: the results of this study may inform interventions to reduce frailty and falls in older veterans and possible among non-veterans as well. background: the number of deaths caused by pneumonia is increasing. the guidelines for pneumonia recommend optimal application of antibiotics based on a pathogenoriented strategy. despite wide distribution of these guidelines, pneumonia demonstrates high mortality in aged people. thus, for developing the next strategy for pneumonia management in aged people, new targets are required. with aging, the loss of skeletal muscle mass and strength occurs, which is named sarcopenia. the sarcopenia phenotype is associated with malnutrition. little is known about relationship between muscles and pneumonia, however, we reported that aspiration pneumonia induced respiratory muscle atrophy. impaired swallowing and/or cough functions often induce pneumonia in aged people. the swallowing muscle weakness is associated with impaired swallowing function. the strong respiratory muscles generate effective cough, which clears the airways and prevents pneumonia. objectives: to investigate presently unknown relationships between onset or recurrence of pneumonia in aged people and; respiratory muscle strength; swallowing muscle strength; and malnutrition. methods: a cross-sectional cohort study consisted of patients aged -year-old and older admitted to the hospital by pneumonia, and controls. the respiratory muscle strength was measured by a hand-held multi-functional spirometer with a pressure sensing transducer. the swallowing muscle strength was evaluated by measuring tongue pressure. a bioelectrical impedance analysis evaluated muscle and body fat masses. malnutrition was evaluated by serum albumin level and body fat mass. results: the respiratory (both the inspiratory and the expiratory) and the tongue muscle strengths, body trunk muscle mass, serum albumin level, and body fat mass divided by height were lower in aged pneumonia patients than in controls. body trunk muscles include the respiratory and swallowing muscles. the multivariate logistic regression model showed the low inspiratory and expiratory respiratory muscle strengths, the low body trunk muscle mass divided by height , and the low serum albumin level as risk factors for onset of pneumonia. for recurrence of pneumonia within months after the onset of pneumonia, low body fat mass divided by height was a risk factor. conclusion: above findings suggest that the respiratory muscles and malnutrition as new targets of the new management strategy for pneumonia in aged people. background: more than % of the people with hiv are older than fifty years. data about this population are still scarce and mainly focused on comorbidity instead of on physical function and frailty. hiv-funcfrail cohort is one of the four european cohorts of older hiv adults launched in . objectives: our main objective in this work was to know the factors associated to physical impairment. methods: longitudinal prospective cohort study. patients from the "hiv-funcfrail: multicenter spanish cohort to study frailty and physical function in years or older hiv-infected patients" were included. eleven centers participated. we recorded sociodemographic data, comorbidities and variables related to hiv infection. physical function was measured by gait speed and sppb and frailty according to frailty phenotype. other components of the comprehensive geriatric assessment such as depression and cognitive impairment were evaluated too. results: were included. median age was . ( . - . ). . % were women. at baseline median cd count was . ( . - . ). viral load was undetectable in . %. % of the patients had > comorbidities and . % had polypharmacy. . % of the patients were able to walk independently and % were completely independent for the activities of daily living. more than half were prefrail, . % prefrail and . % were robust according to frailty phenotype. . % of the patients had a sppb score < and . % had a gait speed < . m/sg. in the univariate analysis we found association between physical impairment defined as sppb score < with: diabetes, copd, osteoarthritis, comorbidities number, moca test < , gds-sf > and age. but in the multivariate analyses the factors associated were just: polypharmacy ) p= . ], gds-sf > [ . ( . - . ) p= . ]. conclusion: functional impairment was prevalent among older adults with hiv in their middleage. polypharmacy doubles the risk of functional impairment and depression increases the risk three-fold. therefore, polypharmacy, depression and physical function should be assessed in all the older adults with hiv in order to implement early prevention intervention to avoid physical impairment. sophie bastijns, anne-marie de cock, maurits vandewoude, stany perkisas (university of antwerp, antwerp, belgium) background: acute sarcopenia is defined as a decline in muscle mass and muscle function within days after hospitalization or acute illnesses, sufficiently to meet the sarcopenia criteria. muscle ultrasound is an objective and non-invasive technique that can measure muscle quantity and quality. muscle elastography can furthermore measure muscle stiffness, which is regarded as an important qualitative parameter. objectives: the primary aim of the study is to assess the effect of acute hospitalization on muscle stiffness. the secondary aim is to evaluate other influencing parameters. methods: this study is a prospective, observational study. patients admitted for at least days to one of the geriatrics departments of the zna antwerp hospitals are included. rectus femoris (rf) and vastus lateralis (vl) muscle stiffness are measured through elastography on day of admission, and then every days until discharge. results: preliminary results show significant differences between rf and vl values in men, but not in women. in rf, a non-significant downwards trend is seen for elastography between day and day . in vl, a non-significant downwards trend is seen in women, but also a non-significant upwards trend is seen in men between day and day . in rf, a non-significant trend of decreasing stiffness is seen with increasing age in men, but an increase is seen in women. a significant negative correlation is seen between elastography of rf and vl on day and hand grip strength on day . conclusion: this study seeks to gain insight in parameters affecting muscle stiffness and of the evolution of muscle stiffness after acute illness or hospitalization. a trend of decreasing muscle stiffness is seen after seven days of hospitalization and illness. this study showed no direct relation between age and muscle stiffness. a decrease in muscle stiffness results in higher hand grip strength and therefore better muscle performance. more data and longer follow-up periods are needed and are expected by march . ainhoa indurain , , jennifer linge , mikael petersson , thobias romu , fredrik uhlin , , anders fernström , mårten segelmark , , olof dahlqvist leinhard (( ) departments of nephrology and medical and health sciences, linköping university, linköping, sweden; ( ) departments of acute internal medicine and geriatrics and medical and health sciences, linköping university, linköping, sweden; ( ) background: sarcopenia is a prevalent condition in hemodialysis patients and it´s associated with poor quality of life, hospitalization and mortality. recent research using magnetic resonance imaging (mri) has demonstrated the importance of proper body size-adjustment in the assessment of muscle mass, and that the addition of muscle fat infiltration reflecting muscle quality, improves functional correlations and prediction of hospitalization in sarcopenia. it is not yet demonstrated if this new mri method, combining body sizeadjusted muscle volume and muscle fat infiltration, improves the evaluation of sarcopenia in hemodialysis patients. objectives: to investigate if adverse muscle composition, defined using mri, predicts survival and comorbidity in hemodialysis patients. methods: in , patients on hemodialysis were scanned using rapid whole body fat and water separated mri. following years, survival and comorbidity index (nci) were recorded using electronic health care records. thigh muscle fat infiltration (mfi) and fatfree muscle volume (ffmv) normalized with height was assessed using amra research (amra medical, linköping sweden). a z-score describing the deviation from expected ffmv/height was calculated using sex and bmi-matched virtual controls (ffmvvcg) and mfi adjusted (mfiadj) was calculated using the sex-specific population mean. for these calculations, normative data from subjects in uk biobank was used. to estimate a combined muscle score (musclecomb), mfiadj and ffmvvcg were projected on the linear regression line describing the normal population relationship between mfiadj and ffmvvcg in the uk biobank dataset. spearman rank correlation was estimated comparing mfiadj, ffmvvcg and musclecomb to nci. wilcoxon signed-rank test was used to estimate the association to survival. roc values and confidence interval were also calculated. results: musclecomb (combined muscle score) was significantly correlated to comorbidity (p< . ) and predicted survival (p< . ) while mfiadj (adjusted muscle fat infiltration) and ffmvvcg (deviation from an individual´s expected muscle volume) did not reach significant level on either test. the roc values for predicting survival were . ( . - . ) for ffmvvcg, . ( . - . ) for mfiadj, and . ( . - . ) for musclecomb. background: frailty is a risk factor for cardiovascular disease (cvd). as declines in bone metabolism and impaired inflammatory response are often associated with frailty, bone analytes and inflammation markers involved in these signaling pathways may act as biomarkers of frailty-related disease progression. objectives: this study sought to examine differences in systemic bone analyte and inflammation marker concentrations based on cvd risk profile and frailty status. methods: females with no prior cvd were stratified into low or high cvd risk groups based on their framingham risk scores. frailty was assessed using the fried phenotype of frailty. greedy matching with pre-frailty as the exposure variable was used to identify a set of closely matched pairs in both the low and high cvd risk groups for a total of females in a case-control design. factorial anova was used to compare differences in log transformed concentrations of bone and inflammation analytes based on frailty status, cvd risk, and their potential interaction. results: differences for il- ( . ± . vs. . ± . pg/ml, p= . ), leptin ( . ± . vs . ± . pg/ml, p= . ) and tnfα ( . ± . vs . ± . pg/ml, p= . ) systemic concentrations were found with high cvd risk status compared to low. no differences in bone or inflammation analyte concentration were found based on frailty status, nor were any interaction effects. conclusion: there was a difference in inflammatory marker concentrations based on cvd risk status indicating that higher cvd risk is associated with impaired inflammatory response in females. there was no difference in bone or inflammation analytes in the pre-frail group compared to their robust peers as these females may be too early in the progression of frailty to have these signs of impaired bone health and inflammation. ( ) pancreato-biliary cancer center, gangnam severance hospital, yonsei university college of medicine, seoul, korea) background: biliary tract cancer (btc) is a highly lethal disease, and improved prognostication methods should be sought. sarcopenia (low muscle mass), poor muscle quality (low muscle attenuation) and excess adiposity (subcutaneous and visceral) can be surrogate markers of sarcopenia and related frailty. however this hypothesis has not been demonstrated conclusively in btc patients. objectives: to evaluate associations of all four body composition measures, derived from clinically acquired ct at the time of initial diagnosis, with overall survival in advanced btc patients. methods: we measured skeletal muscle index (smi), mean muscle attenuation (ma), visceral adipose tissue index, and subcutaneous adipose tissue index via computed tomography at the level of the l vertebra. clinical data were extracted from patients' charts. results: a total of patients ( % males, median age [range - ]) were included in this study, % were metastatic and % were recurrent disease. during the follow-up duration (median of . months; range . month to months), patients ( %) died. sarcopenia, defined as low l smi (lower than cm /m for women and lower than cm /m for men) was noted in patients ( %), and patients ( %) had low muscle radiodensity. for adiposity, % and % of patients had low subcutaneous and visceral fat, respectively. when we combined this four factors and grouped the patients, no risk group (n = ) had the best overall survival (median . months, % ci, . - . ), while the patients who suffered all the risk factors (n= ) showed the poorest survival (median . months, % ci, - . ) which was statistically significant (log-rank test < . ). this classification was independent factor for survival in multi-variate analysis along with other clinical factors, carcinoembryonic antigen (cea), neutrophil-to-lymphocyte ratio, white blood count, platelet, and cholesterol (hr . , % ci . - . ). conclusion: sarcopenia, ma, and adiposity independently predict mortality in patients with btc and can be utilized as surrogate markers for prognosis. background: frailty is a clinical syndrome of reduced systemic physiological reserve that phenotypically overlaps with heart failure. nt-probnp is a cardiac-specific marker that increases with ventricular stress, whereas growth differentiation factor (gdf- ) is a non-tissue specific systemic marker that increases with inflammation, tissue injury and possibly inflammageing. objectives: this study aims to determine if combination of nt-probnp and gdf- organised in a x matrix can classify cardiac dysfunction with and without frailty, non-cardiac frailty, and non-frailty. methods: this is a cross-sectional analysis of a prospective cohort study (phase ), undiagnosed heart failure in older adults (ufo), that recruited community-living older adults aged >/= years in a ratio of : : for robust, pre-frail and frail status classified by the frail scale. participants without a history of heart failure and meeting the eligibility criteria were entered into the study. nt-probnp and gdf- levels were measured using the roche cobas elecsys platform. echocardiography and -minute walk distance ( mwd) were documented. informed consent was obtained from all participants. the study was approved by the local institutional review board. ) was ascertained by correlation with abnormal echocardiographic diastology represented most prominently by increased left atrial volume index (r= . , p= . x e- ). conclusion: a x dual biomarker approach utilising nt-probnp and gdf- may assist in subclassification of cardiac (diastolic) dysfunction and frailty. background: frailty was occurred frequently in elderly and known as higher risk of mild cognitive impairment (mci) and dementia than healthy elderly. hippocampus, parahippocampus and entorhinal cortex as memory system is considered one of the key regions of dementia especially alzheimer's disease. in addition, atrophy of these regions presumably related to higher risk of alzheimer's disease. on the other hand, it is poor understood about neural substrates of relationships frailty and higher incident rates of mci and dementia. objectives: the purpose of this study, therefore, to clarify differences of atrophy level of hippocampus, parahippocampus and entorhinal cortex and total gray matter between healthy, pre-frail and frail in elderly. methods: a total , elderly were measured brain structure with t-mri, and , were fulfilled inclusion criteria in this study. structural brain images were preprocessed and total hippocampal volume was estimated using freesurfer v . . and ubuntu . lts. we classified participants into three groups as healthy, pre-frail and frail characterized by , or and or more of the following domains respectively: low activity, slowness, weight loss, exhaustion and weakness. we compared total gray matter or hippocampal volume between healthy, pre-frail and frail in elderly with one way analysis of covariance (ancova) adjusted for sex, age, educational years, drinking and smoking habit, geriatric depression scale points and estimated total intracranial volume (etiv) and multiple comparison using bonferroni correction. results: the prevalence of pre-frail and frail was . % and . % respectively. hippocampus, parahippocampus and entorhinal cortex volume were significantly decreased in elderly with frail compared healthy and pre-frail (hippocampus: p= . and p= . ; parahippocampus: p= . and p< . ; entorhinal cortex: p= . and p= . respectively). in contrast, total gray matter volume was not significantly difference between three groups. conclusion: hippocampus, parahippocampus and entorhinal cortex were atrophied in elderly with frailty compared healthy or pre-frail elderly. it might be neural substrates of higher risk of dementia in elderly with frailty. rasekh kashkosh , irina gringauz , jonathan weissmann , gad segal , , michael swartzon , abraham adunsky , , dan justo , (( ) geriatrics division, sheba medical center, israel; ( ) biomedical engineering department, israel; ( background: low alanine aminotransferase (alt) blood levels prior to rehabilitation are associated with poor rehabilitation outcomes in terms of low mobility and function in older adults following hip fracture. objectives: we have hypothesized that low alt blood levels prior to rehabilitation are also associated with -year mortality in this population. methods: included were older adults (age >= years, median age years, . % women) admitted for rehabilitation following hip fracture. alt blood levels were documented between one and six months prior to rehabilitation. excluded were patients with alt blood levels over iu/l possibly consistent with liver injury. the study group included patients with low ( iu/l or lower) alt blood levels, and the control group included patients with high-normal ( - iu/l) alt blood levels. the main outcome was all-cause mortality one year following rehabilitation admission. results: the study group included ( . %) patients with low alt blood levels, and the control group included ( . %) patients with high-normal alt blood levels. overall, ( . %) patients died within one year following rehabilitation admission. compared with the control group, patients with low alt blood levels had significantly higher -year mortality rates ( . % vs. . %, or . , %ci . - . ). cox regression analysis showed that low alt blood levels prior to rehabilitation were associated with -year mortality (hr . , %ci . - . ) together with peripheral vascular disease (hr . , %ci . - . ) -independent of age, gender, albumin serum levels, length of rehabilitation, and rehabilitation outcomes. conclusion: low alt blood levels prior to rehabilitation are associated with -year mortality in older adults following hip fracture. fawaz azizieh , dia shehab , khaled al jarallah , renu gupta , raj raghupathy (( ) gulf university for science & technology, mubarak al-abdullah area, kuwait; ( ) faculty of medicine, kuwait university, jabriya, kuwait) background: in addition to some well-characterized bone turnover markers, cytokines and adipokines have also been suggested to be linked to osteoporosis seen in menopause. however, there is much controversy on the possible association between these markers and bone mineral density (bmd). objectives: this study was aimed at measuring circulatory levels of selected cytokines and adipokines in postmenopausal women with normal and low bmd. methods: the study population included post-menopausal women, of whom had normal bmd, had osteopenia and had osteoporosis. circulatory levels of selected pro-resorptive (tnf-a, il- b, il- , il- , il- , il- ), anti-resorptive (ifng, il- , il- , il- , tgf-b) and five adipokine markers (adiponectin, adipsin, lipocalin- /ngal, pai- and resistin) were measured using the multiplex system and read on the magpix elisa platform. further, two bone turnover markers (p np, ctx) as well as estradiol levels were assayed from the same samples. results: while circulatory levels of cytokines were comparable between groups, women with low bmd had statistically significantly higher median circulatory levels of adipokines as compared to those with normal bmd. further, while levels of ctx were not different between the two groups; p np, p np/ctx ratio and estradiol levels were significantly lower in women with low bmd. levels of adiponectin, p np, p np/ctx ratio and estradiol correlated significantly with bmd of the hip and spine. conclusion: while the associations between the studied markers and bmd may be complex and multivariate, our data provide insights into the possible use of circulatory levels of cytokines, adipokines and bone turnover markers on the pathogenesis of postmenopausal osteoporosis. background: with the application of diffusion tensor imaging (dti), a few studies have found that some white matter (wm) structures were closely related to impaired gait speed. however, the evidence is still sparse and the wm structural association with overall lower-body physical function, which can be evaluated by short-physical performance battery (sppb), has never been investigated among older adults. objectives: the aim of this study is to explore the associations between wm structures (evaluated by dti parameters) and sppb scores among older adults. methods: data of participants ( ± years old), who were recruited in the multidomain alzheimer's preventive trial (mapt) study and with no dementia at baseline level, were analysed in this study. based on the functional magnetic resonance imaging data, dti parameters of fractional anisotropy (fa), mean (md), axial (ad) and radial diffusivity (rd) were calculated in wm structures that were annotated by the john hopkins university white matter parcellation atlas. linear regression was used to analyse the association between sppb score and each dti parameter while controlling for age, gender, body mass index, physical activity level, total intracranial volume, cardiovascular risk and time interval between the dti and sppb measurement. results: three dti parameters (the md and rd of left corticospinal tract, and the md of right cerebral peduncle) were associated with the sppb score at a p-value < . . conclusion: the findings indicate that wm structures of corticospinal tract and cerebral peduncle might be related to overall lower-body physical function of older adults. further studies on the changes of these wm structures with physical function alterations during ageing will be more informative. background: ct-derived skeletal muscle index and skeletal muscle density (smd) have been independently associated with mortality in older adults. although smd is a commonly used measure of myosteatosis on ct images, more novel muscle texture (i.e., radiomic) features may provide an alternative measure of muscle quality, independent of smd. there have been no prior studies on the association of ct-derived muscle texture features and mortality. objectives: to examine the association of skeletal muscle texture features with all-cause mortality in older adults from the national lung screening trial (nlst). methods: the relationship between ct-derived skeletal muscle texture and all-cause mortality over years was determined in , participants ( % women, age range - years, mean age . ) in the nlst. using ct images at the level of t vertebra, paraspinous muscle was automatically segmented using machine learning algorithm, and muscle texture features determined using pyradiomics. second order (and higher) texture features were grouped into categories: gray level dependence matrix (gldm), gray level co-occurence matrix (glcm), gray level run length matrix (glrlm), gray level size zone matrix (glszm), and neighbouring gray tone difference matrix (ngtdm). muscle texture features often indicate greater or lower heterogeneity/complexity of an image. associations between standardized muscle texture variables and all-cause mortality were determined using cox proportional hazards models, adjusted for age, sex, race, body mass index, pack years of smoking, presence of type diabetes, chronic lung disease, cardiovascular disease, cancer at enrollment, and smd. multiple comparisons were accounted for using false discovery rate testing. results: after a mean . ± . years of follow-up, ( . %) participants died. in fully adjusted models, the following muscle texture features were associated with mortality: gldm-dependenceentropy (hazzard ratio (hr) per standard deviation (sd)= . , p< . ), gldm-dependencenonuniformity (hr per sd= . , p= . ), gldmsmalldependencelowgraylevelemphasis (hr per sd= . , p< . ), glrlm-graylevelnonuniformity (hr per sd= . , p< . ), glszm-small area low gray level emphasis (hr per sd= . , p= . ), ngtdm-coarseness (hr per sd= . , p= . ), ngtdm-strength (hr per sd= . , p= . ). each of these associations were in the direction that suggested greater heterogeneity of the image was associated with increased mortality. conclusion: in a large multicenter cohort of community-dwelling older adults, ct-derived muscle texture features indicating greater heterogeneity were associated with mortality, independent of common covariates including skeletal muscle density. background: growth differentiation factor (gdf ) has been related with disease progression, mitochondrial dysfunction, and mortality. elevated gdf- level was recently reported to be associated with poorer physical performance in very healthy community-dwelling adults. however, until now, the relationship of serum gdf- level with sarcopenia in community-dwelling older adults has not been well characterized. objectives: this study aimed to investigate the association between serum gdf- levels and sarcopenia in community-dwelling older adults. methods: we analyzed participants (mean age, . ± . years; . % men) who underwent measurement of serum gdf- level and sarcopenia parameters, using their baseline data from the korean frailty and aging cohort study. participants with reduced kidney function, specifically an estimated glomerular filtration rate (egfr) from creatinine of < ml/min/ . m , were excluded. serum gdf- level was quantified with an enzyme-linked immunosorbent assay kit. appendicular skeletal muscle mass was measured using dual-energy x-ray absorptiometry. sarcopenia status was determined in accordance with the asian working group for sarcopenia (awgs) guidelines. results: according to the awgs algorithm, ( . %) of the participants in the whole study population were classified as having sarcopenia. gdf- concentration had significant negative correlations with appendicular lean mass (men, r = - . , p < . and women, r = - . , p = . ), grip strength (men, r = - . , p = . and women, r =- . , p = . ), and gait speed (men, r = - . , p = . and women, r = - . , p = . ). in the multivariate analysis adjusted for potential confounders, the highest gdf- quartile (>= pg/ml) was associated with a greater risk of sarcopenia (odds ratio [or] = . ; % confidence interval [ci], . - . ) than the lowest quartile (< pg/ml). these associations remained unchanged (or = . ; % ci, . - . ) after further adjustment for potential biomarkers (e.g., myostatin, dehydroepiandrosterone, and insulin-like growth factor- ). the or per unit increase in log-transformed gdf- level was . ( % ci, . - . ). conclusion: higher circulating gdf- levels were independently associated with a greater risk of sarcopenia in community-dwelling older adults. gdf- may be considerate a promising biomarker of sarcopenia. background: frailty has been recognized as an emerging public health problem in rapidly aging populations worldwide. use of biomarkers to identify frailty has been suggested for early frailty screening. among multiple risk factors of frailty, inadequate nutrition such as inadequate intake of protein and vitamin d has been shown to be associated with increased risk of frailty. therefore, nutritional biomarkers could be useful for early screening of frailty. objectives: to review the evidence of potential biomarkers, especially nutritional biomarkers for early screening of frailty in community-dwelling older adults. methods: a literature search was conducted using pubmed and scopus databases. studies evaluating blood biomarkers and frailty in community-dwelling older adults from to were included. information on the definition of frailty, study design, characteristics of the study populations, and the associations between biomarkers and frailty was summarized. results: in total, studies were identified in which observational studies were published since . majority of studies used physical frailty. other definitions such as multidimensional, social and frailty were also used. biomarkers were identified. cross-sectional and longitudinal studies consistently showed that low level of vitamin d was associated with frailty. emerging scientific evidence suggested that abnormal level of albumin, low levels of high-density lipoprotein (hdl), beta-hydroxy beta-methylbutyrate (hmb), vitamin b (measured by pyridoxal- -phosphate), carotenoids, or a-tocopherol (vitamin e), and high level of dp-ucmgp (marker of vitamin k) could have the potential for frailty screening. besides nutritional biomarkers, the evidence showed that inflammatory markers such as c-reactive protein (crp), interleukin- (il- ), and fibrinogen, and endocrine-related markers such as hemoglobin, dehydroepiandrosterone sulfate (dheas), and hemoglobin a c could be useful for screening frailty. additionally, there is evidence suggesting that some oxidative or immune-related markers were associated with frailty. conclusion: vitamin d could be a useful nutritional biomarker for early frailty screening in the community setting. other nutritional biomarkers, inflammatory markers and endocrine-related markers could be associated with frailty. further research is needed to validate and refine other potential biomarkers. jonathan quinlan , , , amritpal dhaliwal , , felicity williams , , matthew armstrong , , leigh breen , , , ahmed elsharkawy , , carolyn greig , , , janet lord , , ( ( ) background: end stage liver disease (esld) is associated with reduced muscle mass with a reported incidence of sarcopenia of - % (bhanji, ). loss of muscle mass in esld patients has a negative impact on clinical outcomes including mortality and recovery rates from liver transplantation (montano-loza, ) . previous research has investigated loss of muscle mass in esld via appendicular skeletal muscle mass and psoas muscle cross sectional area (csa) using dxa and magnetic resonance imaging (mri) respectively. however, the quadriceps muscle group has high functional significance and thus should be investigated in esld patients in whom function may be limited. ultrasound (us) offers a non-invasive, bedside imaging assessment of quadriceps muscle mass. however, esld may be associated with increased subcutaneous fat which can present an operational challenge for us and thus its application in esld patients requires validation. objectives: the aim of this research is to validate the accuracy of ultrasonographic measures of quadriceps muscle mass by comparison with the gold standard of mri. methods: parallel mri and us were collected from patients with an esld diagnosis and awaiting liver transplant ( patients, age ± yrs, bmi . ± . ). participants underwent us scanning of both left and right quadriceps followed directly by an mri. specifically, measures of vastus lateralis (vl) muscle thickness (mt) and quadriceps csa were obtained at % femur length during longitudinal and extended field of view us respectively. to enable direct comparison with quadriceps csa obtained during mri, an oil capsule was placed upon the leg to mark the exact location of us image collection. all procedures received research ethics committee approval and written informed consent from the participants. results: a significant (p< . , n= ) positive correlation was found between vl mt and quadriceps csa obtained via mri (r = . ). similarly, there was a significant positive correlation (p< . , n= ) between csa obtained via extended field of view us and mri (r = . ). bland-altman plots demonstrated a bias of - . ± . cm , with % limits of agreement of - . cm and . cm . conclusion: our data demonstrate that the assessment of quadriceps csa and vl mt via us may offer a suitable bedside alternative to mri in patients with esld. background: sarcopenia is defined as the gradual ageassociated loss of both muscle quantity and strength in older adults, and severe sarcopenia affects subject performance (such as reduced gait speed). it is a devastating condition, predicting an increase in mortality, falls, fractures and hospitalizations. current clinical criteria diagnose sarcopenia through dual x-ray absorptiometry (dxa) measures of muscle mass, a test that cannot be performed at the bedside and is rarely used to find this condition. point-of care ultrasound (pocus) is rapidly becoming a standard part of the physical exam, and has the potential to become a quick, noninvasive marker for both muscle mass and function. objectives: we examined the relationship between ultrasound measures of muscle mass (vastus medialis thickness, mt) and other measures of muscle quantity (appendicular skeletal mass, asm; mid-arm biceps circumference, mabc). we also examined the association between mt and measures of muscle strength (grip strength) and muscle performance (gait speed) in an older adult population. methods: older adults (age >= ; mean age . ± . years, women, men) were recruited sequentially from geriatric medicine clinics. each subject had appendicular skeletal muscle mass (asm, by bioimpedance assay), grip strength, mid-arm biceps circumference (mabc), gait speed, and an ultrasonic measure of muscle quantity (mt, vastus medialis muscle thickness) measured. our initial models contained age, sex, bmi, and mt as predictor variables, and our outcome variables were asm, grip strength, mabc and gait speed. results: in our final parsimonious models, mt showed a strong significant correlation with all measures of muscle mass, including asm(standardized ß= . ± . , r = . , p< . ) and mabc(standardized ß = . ± . , r = . , p= . ). with respect to measures of muscle quality, there was a strong significant correlation with grip strength (standardized ß = . ± . , r = . , p= . ) but not with subject performance (gait speed). conclusion: mt showed strong correlations with both measures of muscle mass (asm and mabc) and with muscle strength (grip strength). riki kosugi , yung-li hung , toshiharu natsume , shuichi machida (( ) faculty of health and sports science, juntendo university, inzai, chiba, japan; ( ) institute of health and sports & medicine, juntendo university, inzai, chiba, japan; ( ) coi project center, juntendo university, bunkyo-ku, tokyo, japan; ( ) graduate school of health and sports science, juntendo university, inzai, chiba, japan) background: loquat (eriobotrya japonica) leaves are commonly used in teas and folk medicines. recently, loquat leaf extract (lle) has been reported to promote muscle protein synthesis in vitro. additionally, resistance exercise has been shown to promote muscle protein synthesis in vivo. it is considered that lle and resistance exercise might have a synergistic effect on activating muscle protein synthesis. however, this has never been investigated. objectives: the purpose of the present study was to investigate whether lle enhances the muscle contraction-induced activation of muscle protein synthesis signaling in rats. methods: male wistar rats ( weeks old, n= - /group) were categorized into a control (con) group, an lle-administered (lle) group, an electrical muscle stimulation (ems) group , and an ems with lle (ems+lle) group. rats were administered lle ( . g/kg/ day) or distilled water once in a day by oral gavage for days. on the seventh day, h post-lle administration, the gastrocnemius muscle of the right legs of ems group and ems+lle group rats were stimulated by ems ( hz, v) through sets of isometric contractions ( s contraction, s rest) with min inter-set intervals. rats were then sacrificed and their gastrocnemius muscles were rapidly excised h post-ems. expression levels of muscle synthesis-related proteins [protein kinase b (akt), mammalian target of rapamycin (mtor), and ribosomal protein s kinase beta- (p s k)] were determined by western blotting. results: no significant differences were observed in body weight, water intake, and diet intake among the groups. akt phosphorylation at ser was found to be significantly increased in the ems+lle group compared to that in con group; mtor phosphorylation at ser did not show a significant difference. p s k phosphorylation at thr was found to be significantly increased in the ems group compared to that in con group, while the ems+lle group was observed to have significantly higher p s k phosphorylation at thr than the ems group. conclusion: our study suggests that lle enhances the muscle contraction-induced activation of p s k phosphorylation. background: metabolic aging has emerged as a new sedentarity related syndrome combining metabolic diseases and sarcopenia, a degenerative loss of skeletal muscle mass, quality, and strength associated with aging. it has been recently shown that kynurenic acid (ka), a key metabolite of tryptophan/ kynurenine pathway, improved glycemic control and lipid profile in rodents. objectives: to show that ka has a key role in metabolic aging, we have evaluated its effect on muscle function and mass in vitro and in vivo in muscle cell line and in a model of hindlimb immobilization in mouse. methods: in vitro in c c muscle cells we measured the ability of ka to inhibit myostatin gene expression (endogenous inhibitor of muscle growth), stimulate protein synthesis and enlarge muscle cell size. differentiated cells were exposed to ka for h for protein analysis, h for gene study and the days of differentiation for cell enlargement examination. in vivo, muscle mass (tibialis and soleus) was measured after a week-hindlimb immobilization in mice treated or not with ka ( mg/kg.day per os). results: in vitro, ka significantly and dose-dependently inhibited myostatin gene expression, stimulated protein synthesis and enlarged c c muscle cells. in mice, ka treatment significantly reduced tibialis and soleus muscle wasting induced by immobilization. conclusion: we demonstrated for the first time the positive impact of ka on muscle function and mass preservation offering a promising therapy for patients affected by metabolic aging, who do not currently benefit from relevant therapeutic solutions. Â n g e l a m a r i a p e r e i r a , , , a n a f r e i t a s , a n a p a c i f i c o , c a t a r i n a c o s t a , m a r g a r i d a a l m e i d a (( ) physiotherapy departement, escola superior de saúde egas moniz, portugal; ( ) centro de investigação interdisciplinar egas moniz, monte da caparica, portugal; ( )hospital garcia de orta, almada; portugal) background: as people age they are more likely to fall. although most fall-related injuries are minor, they can cause significant pain and discomfort, affect a person's confidence and lead to loss of independence. some falls can cause serious long-term health problems. one strategy to promote greater adherence and motivation to intervention in physical therapy is the use of virtual environment (ve) programs associated with a balance exercise programs as an effective method of preventing falls. objectives: the purpose of this study was to analyze the benefit of a virtual environment exercise program in non-institutionalized elderly at the end of six weeks. methods: in this randomized controlled trial non-institutionalized elderly were included. subjects, age . ± . yrs constituted the experimental group (eg); and , age, . ± . yrs constituted the control group (cg). the eg was submitted to weeks of a ve exercise program performed on a nintendo wii, and to a set of recreational activities. the cg only performed the activities. the instruments used in the present study to evaluate performance were tinetti's index, which evaluates the static balance and the gait to quantify the risk of fall, and the fullerton's functional fitness tests to assess physical parameters such as strength, aerobic endurance, flexibility and agility/ balance. results: at the end of the weeks of intervention in a virtual environment, significant improvements in upper limb strength, agility and static balance were observed. in the intragroup comparison, it was possible to verify improvements in all physical fitness battery tests. the values of functional fitness tests were significantly different (p<. ) between eg and cg groups for the following variables: -second chair stand . ± . vs. . ± . times; arm curl . ± . vs. . ± . times; -foot up-and-go . ± . vs. . ± . sec; two min. step . ± . vs. . ± . steps, respectively; as well as for the tinetti index. conclusion: this study, suggests that exercise in ve context applied to non-institutionalized elderly, promotes improvements in mobility, in lower limbs muscular strength, and may help to reduce the risk of falls by improving the static and dynamic balance. background: the small non-coding micrornas (mirs) are endogenous regulators of gene expression. they bind to complementary sequence on target messenger rna transcripts resulting in translational repression or target degradation. they are involved in the skeletal muscle response to training in animals and humans (kirby, ) . objectives: the aim of our study was to measure the effects of high intensity interval training (hiit) associated or not with l-citrulline on the expression of serum and muscle mirs in a group of men. methods: we selected men (mean age: . ± . years, men in the placebo group and in the l-citrulline group, gr/day) from a cohort of men and women submitted for weeks to hiit (buckinx, ) . we evaluated the expression of serum and muscle mirs before and after training. the quantification of mir expression was performed using the next generation sequencing (ngs) technique (exiqon). for statistical analysis, the measurements were normalized with the tmm method (trimmed mean of m-values). results: we identified mirs from serum and mirs from muscle above the detection limit (>= tpm, tags per million). after benjamini-hochberg correction, serum mirs from the l-citrulline group had a significantly different level of expression before and after training: - p, b - p, , a- p and - a- p (p < . , % fdr). no mir of the placebo group had a significantly altered expression. in muscle, our approach revealed mirs with a significantly different level of expression before and after training in the placebo group and in the l-citrulline group, of which were common to both groups. these mirs were different from those highlighted at the serum level. the most-expressed muscle mirs with the greatest difference in expression before and after training were - p, - p, - p, - p and b- p (p < . , % fdr). conclusion: with the ngs approach, we identified mirs differentially expressed before and after hiit. expression of circulating mirs appears to be influenced by l-citrulline. the next validation step will be to measure these specific mirs in the entire cohort to determine the clinical utility of these markers. background: recent interventional studies on frailty used multicomponent programs (physical exercise, cognitive stimulation, and nutritional supplementation) with some promising results. however, these emerging programs developed to counter the multidimensional concept of frailty still need methodological improvements to be completely effective. objectives: the objective of this innovative project is to develop personalized multicomponent interventions that could be easily used by frail older adults in order to reverse physical, cognitive and psychosocial symptoms associated with frailty. three original and specific action levers will be used to insure a better effectiveness: /to target a key population (hospitalized frail older adults who will be discharged to home), /to use a real multicomponent program (physical exercises simultaneously associated with cognitive and social components that mimic daily gestures), and /to encourage adherence through medical prescription. methods: one hundred and twenty frail older adults (>= ) will be recruited from the geriatrics unit of the university hospital of tours (france), and randomly assigned to one of the two study arms: the intervention group (ig), who will receive a medical prescription of an adapted multicomponent intervention, vs the control group (cg; no intervention). twelve-week programs will be adapted according to observed intrinsic capacities of the frail older adults. including exercises will be based on effective international physical programs, with original cognitive and social components added to the physical exercises. all participants will perform pre-and post-tests to compare their physical health (gait speed, balance, and strength), cognitive health (global cognition and executive functions), and psychosocial health (self-efficacy and quality of life) before and after the three-month program. results: a pilot study to this rct has already started in tours. the international conference on frailty and sarcopenia research would be the perfect opportunity to share preliminary results. the intervention will be considered as feasible if ig participants adhere to > % of the prescribed exercise and as effective if we observe significant improvements in all clinical outcomes for ig participants, compared to the cg. conclusion: final objective will be to disseminate to a large number of individuals the idea that several concrete ways exist to age well. amanika kumar, clarissa polen-de, gladys asiedu carrie langstraat, aminah jatoi (mayo clinic, rochester, minnesota, usa) background: frailty in patients with advanced stage ovarian cancer (oc) is common and associated with increased oncologic and surgical morbidity and mortality. prehabilitation is one option to reverse frailty in this subset of patients. objectives: our aim was to investigate potential barriers and facilitators of prehabilitation during neoadjuvant chemotherapy (nact) in oc patients. methods: we identified patients who underwent nact from - at a large volume single institution. patients underwent a semi-structured one-on-one phone interview. transcripts from interviews were read by independent reviewers to identify emerging themes related to patients' experience, functioning and exercise during chemotherapy. results: five primary themes emerged following analysis of the participants transcripts. participants were overall willing to participate in exercise during chemotherapy, with / patients stating they would walk or did walk at least minutes daily during treatment; this was linked to a strong motivation to improve surgical and survival outcomes. only / patients stated they were not interested in exercise during treatment. most notable, patients' motivations were tied closely to physician recommendation. patients prominently identified a shift in health as a priority following their ovarian cancer diagnosis, which subsequently lead to an increase in daily activities and exercise. surgery and improvement in mental well-being were strong motivators for patients to start or continue an exercise program. participants also identified barriers to exercise during treatment including a variety of treatment related and nontreatment related concerns, including neuropathy, nausea, pain, program availability, time and most significantly fatigue. despite this, most retrospectively thought they would have been willing to exercise with modifications. almost all participants voiced the importance of a supportive treatment community, including their medical care team, family, friends and the local community. conclusion: patients with advanced ovarian cancer demonstrated high motivation and willingness to exercise during chemotherapy when there was a perceived benefit to overall survival. prehabilitation may be a helpful to improve outcomes, but a prehabilitation strategy should be designed specifically for the patients with the most need and designed with barriers and motivators in mind. randomized control trial. kosuke fujita , , hiroki umegaki , aiko inoue , huang chi hsien , , hiroyuki shimada , masahumi kuzuya , (( ) institute of innovation for future society, nagoya university nagoya, japan; ( ) department of community healthcare and geriatrics, nagoya university graduate school of medicine nagoya, japan; ( ) department of preventive gerontology, center for gerontology and social science, national center for geriatrics and gerontology obu, japan) background: gait disorder in older adults could lead fatal consequence following falling or reducing physical activity, especially in individual with pre-clinical / clinical cognitive decline. effectiveness of exercise intervention for the gait characteristics has been examined in previous studies, however, evidence about differences between exercise modality such as aerobic training (at) and resistance training (rt) for the acute and long phase is unclear. objectives: the aim of the present study was to compare the effect of different exercise modality on the gait characteristics of older adults with preclinical cognitive decline. methods: individuals (mean age, . years) with self-reported cognitive decline were enrolled in randomized controlled trial. subjects assigned to at group (n = ), rt group (n = ) and at+rt group (n = ) underwent exercise intervention days a week for weeks. subjects assigned to control group (n = ) were provided information about healthy aging. gait characteristics were examined before, just after the intervention and after the weeks of follow-up period using an electronical walkway system. results: in the analyses about the change between pre and just after the intervention period, all of three exercise groups significantly improved gait velocity (at, p < . ; rt, p < . ; at+rt, p < . ), stride time (at, p < . ; rt, p = . ; at+rt, p < . ), cadence (at, p < . ; rt, p = . ; at+rt, p < . ), stride length (at, p < . ; rt, p = . ; at+rt, p < . ) and double support time (at, p < . ; rt, p < . ; at+rt, p < . ), and at+rt group improved significantly with cv of step width (p < . ). in the analyses about the change between pre and follow-up period, rt group only had improvements with gait velocity (p < . ), stride length (p = . ) and double support time (p = . ). conclusion: all exercise interventions could improve gait characteristics of older adults with pre-clinical cognitive decline. for the purpose of maintain improved gait characteristics for a long phase, rt is likely to be recommended. activity and a broader array of physical and psychological outcomes among nursing home residents. however, some limitation of this game should be acknowledged (e.g. too long, too bulky, exercises too simple). taking into account these weaknesses, we decided to develop and validate a new version of a giant exercising board game: the gamotion. objectives: to evaluate the impact of gamotion on physical capacity, motivation and quality of life among nursing home residents. methods: a one-month randomized controlled trial was performed in two comparable nursing homes. eleven participants ( . ± . years; men) meeting the inclusion criteria took part in the intervention in one nursing home, whereas participants ( ± . years; men) were assigned to the control group in the other institution. the gamotion required participants to perform strength, flexibility, balance and endurance activities. the assistance provided by an exercising specialist decreased gradually during the intervention in an autonomy-oriented approach based on the selfdetermination theory (ryan & deci, ) . physical capacity (i.e. quantitative evaluation of walking using locometrix; grip strength using jamar dynamometer; knee extensor isometric strength using microfet ; fall risk using tinetti test; dynamic balance using timed up and go test (tug) and physical abilities using sppb test), motivation (i.e. using behavioral regulation in exercise questionnaire- ) and quality of life (i.e. using eq- d questionnaire) were assessed at baseline and at the end of the intervention. a two-way repeatedmeasure analysis of covariance (ancova) was used to assess time*group (intervention vs. control group) effects. results: globally, during the intervention period, the experimental group displayed a greater improvement in symmetry of steps (p= . ), tinetti score (p< . ), tug (p= . ), sppb (p< . ), knee extensor isometric strength (p= . ), grip strength (p= . ), domains of the eq- d (i.e. mobility, self-care, usual activities : p< . ) and intrinsic motivation (p= . ) compared to the control group. conclusion: the effects of gamotion on physical capacity, motivation and quality of life of nursing home residents confirm the results obtained with the previous version of the giant exercising board game. in-hospital stay, even in short stays, is associated with functional impairment in older patients. objectives: the agecar plus study aims to evaluate the effectiveness of a program of physical exercise and health education to prevent the functional deterioration during the in-hospital stay. methods: randomized clinical trial. patients older than years admitted to the ace of the general university hospital gregorio marañón were included and randomized at admission in control group (cg) or intervention group (ig). exclusion criteria were baseline barthel ( days before admission) less than points, severe cognitive impairment or unable to walk. both groups received usual care, and patients in intervention group also performed simple supervised exercises (strengthening of lower limbs, walking, and inspiratory muscle training). in the preliminary analysis, we analyzed the effect of the intervention on changes in short physical performance battery (sppb) and alusti test, at admission and discharge, by t-test of repeated measures in the study periods. results: from may to february , patients were included: gc and ig. the cg and ig were homogeneous in sex (women . %), age ( . ± . vs. . ± . ), comorbidities (charlson: . ± . vs. . ± . ), cognitive impairment (pfeiffer: . ± . vs. . ± . ), fragility (fried >= : % p= . ), and functional-physical capacity (sppb: . ± vs . ± . ; alusti, . ± . vs . ± . ). p < . for all variables. a significant effect of the intervention was found, with a higher mean score in the alusti test in the ig (cg: . ± . vs . ± . ; f( , )= . ; p= . ), not finding such differences with the sppb ( . ± . vs . ± . ; f( , ) = . ; p= . ). conclusion: the preliminary analysis shows that the alusti test could be used as an evaluation test for functional capacity in hospitalized elderly patients. a physical exercise program during hospitalization in an acute unit improves the functional capacity assessed by the alusti test at discharge significantly. funding: instituto de la salud carlos iii (pi / ), ciberfes, fondo europeo de desarrollo regional (feder). the authors declare no conflicts of interest. a. sampaio , i. marques-aleixo , , j. carvalho (( ) ciafel -research center in physical activity, health and leisure, faculty of sport, university of porto, portugal; ( ) faculty of psychology, education and sports, lusófona university of porto, portugal) background: cognitive impairment is a highly prevalent, poorly managed, and disabling consequence of dementia. exercise training that improves physical fitness can represent a promising approach for managing cognitive impairment in persons with dementia. objectives: the aim of this crosssectional study investigated the association of physical fitness and balance with cognitive function. methods: sixty-four institutionalized older adults, aged . ± . years, with dementia, predominately female ( %) and with dementia due to alzheimer's disease ( . %). regression analyses were used to examine associations between physical fitness components (senior fitness test), balance (tinetti index) and cognitive function (mini-mental state examination). results: univariate regression indicates a significant association between the strength of the upper body (p= , ) and aerobic endurance (p= , ) with the cognitive function in older people with dementia. conclusion: these results suggest an association between the specific dimensions of physical fitness and cognitive function. consequently, multicomponent exercisebased therapeutic strategies aiming to improve physical fitness could be an important nonpharmacological strategy for dementia management. satoshi kurita, takehiko doi, kota tsutsumimoto, sho nakakubo, hideaki ishii, hiroyuki shimada (section for health promotion, department of preventive gerontology, center for gerontology and social science, national center for geriatrics and gerontology, aichi, japan) background: women had higher risk of cognitive impairment or dementia compared to men. although studies reported physical activity (pa) and/or cognitive activity (ca) had protective association with cognitive impairment among older adults, it is unknown whether the association is depended on sex or not. objectives: the purpose of the present study was to examine the sex differences in the association of pa and/or ca with cognitive impairment in community-dwelling older adults. methods: a community-based cohort survey was conducted in a total of participants (mean age . ± . years; . % female) who met the study criteria. time of moderate-to-vigorous intensity pa was measured using an accelerometer. ca was assessed by the frequency of engaging in activities using a ca scale including reading, doing crossword puzzles, and playing board games or cards. participants were categorized into four groups based on quartile (low) and to (high) values of pa and ca. cognitive impairment was defined by at least out of neuropsychological tests having a result at least . standard deviation below the reference threshold. results: in both sex, the prevalence of cognitive impairments showed significant differences among groups; that of low pa/low ca group, low pa/high ca group, high pa/low ca group, and high pa/high ca group were respectively . %, . %, . %, and . % for male (p < . ) and . %, . %, . %, and . % for female (p < . ). in binomial logistic regression models for male, all groups showed a low odds ratios of cognitive impairment compared to the low pa/low ca group (odds ratio = . to . , all p < . ), while for female, only high pa/high ca group had significant association with cognitive impairment (odds ratio = . , % confidence interval = . to . , p = . ). conclusion: in male, pa and ca are associated with cognitive impairment even in the case of low engagement in either pa or ca. in female, higher engaging in both activities are associated with cognitive impairment. female older adults may need to engage in more activities than male to acquire benefit on preventing cognitive impairment. ( interventions) were included in the systematic review and in the meta-analyses ( interventions). there was considerable heterogeneity in the number for interventions that detected significant increases in muscle mass ( / , %) and muscle strength ( / , %). of those muscle strength interventions / ( %), / ( %), / ( %) and / ( %) interventions reported a significant increase in handgrip strength, lower body muscle strength, upper body muscle strength and whole body muscle strength respectively. ret factors associated with the greatest gains in muscle mass and muscle strength were: use of combination of equipment, seven to eight exercises per session with three lower body exercises, a volume of three to four sets and to repetitions per exercise, a frequency of two-three days per week, intervention length of greater than six weeks, progressive intensity, intervention duration of - minutes, and in a supervised individually training structure. these results align with current guidelines provided by american, australian, japanese, british, canadian and japanese societies. conclusion: not all ret interventions are effective for improving muscle mass and strength, but our meta-analysis suggests that adhering to the current ret guidelines for older adults are likely to be most effective. duarte barros, andreia pizarro, arnaldina sampaio, joana carvalho (research center in physical activity, health and leisure, faculty of sports, university of porto, portugal) background: sedentary time (sed) and low physical activity (i.e. low levels of moderate-to-vigorous physical activity [mvpa] ) are different behaviours associated with negative health outcomes, but how synergetic combinations of these behaviours impact the risk of frailty are still unexplored. objectives: to examine the relationship between different combinations of sedentary time and mvpa in the risk of being frail. methods: a cross-sectional study including community dwelling elders ( . ± . years; . % female) accessed frailty through the phenotype of frailty. daily sed and mvpa were objectively measured using accelerometry. sed and mvpa were ranked by the median and then participants were categorized into one of four groups: lowsed+lowmvpa, l o w s e d + h i g h m v p a , h i g h s e d + l o w m v p a a n d highsed+highmvpa. results: overall, . % of the participants were frail. mvpa was associated with reduced odds of being frail (or . ic: . - . , p < . ). moreover, compared to the highsed+lowmvpa, the groups lowsed+highmvpa (or . ic: . - . , p = . ) and highsed+highmvpa (or . ic: . - , p < . ) were associated with reduced odds of being frail. conclusion: mvpa seems associated with reduced odds of being frail, irrespective of sedentary time. background: sarcopenia is central to frailty and the strongest evidence for reversal lies in the combination of resistance exercise and protein supplementation. unfortunately, uptake amongst older adults remains low, partly due to a lack of suitable exercise programs. delivery by health professionals alone will not achieve widespread participation. objectives: defrail aims to develop a novel exercise program (focused on resistance training), feasible for delivery to frail older adults in a group setting without the input of health professionals, and to examine its effect when combined with commercially-available protein-supplemented milk. methods: a multi-component exercise program was designed by expert consensus using a modified delphi process. participants were recruited from geriatric medicine clinics and primary care, with assessments at baseline, after eight weeks of regular activity and then after the eight-week intervention. the primary outcome measure was the change in the fried frailty criteria (ffc) during the intervention compared with the period of regular activity. secondary outcome measures included the timed up & go (tug) and -second sit-to-stand ( sts) tests. results: the first participants to complete the program ( females, males, mean age , range - ) had a median ffc score of (interquartile range (iqr) , ), i.e. frail, both at baseline and after the period of regular activity period, but had improved to (iqr , ), i.e. pre-frail, following the intervention. similarly, the median tug was . (iqr . , ) at baseline, increasing to . (iqr , . ) after the period of regular activity, improving to . (iqr . , . ) following the intervention. the median sts was (iqr , ) at baseline, (iqr , ) after the period of regular activity, improving to (iqr , ) following the intervention. conclusion: median frailty improved from frail to pre-frail for the first defrail participants. this program could allow increased community-based participation in resistance exercise for frail older adults. further work now includes completion of the intervention and analysis of data on a range of secondary outcome measures (assessments of cognition, mood, pain, body mass composition and biochemical markers of frailty). background: exercise interventions have been shown to improve functional status and quality of life of frail older people, and in some cases to reverse frailty status. it is important that such interventions are targeted to those people who would benefit the most. objectives: the objective of this pilot study was to assess the effectiveness of a physical activity intervention given to mildly frail older people, who were identified using electronic health records (ehr). methods: the electronic frailty index (efi) was used to identify mildly frail older people and offer them a physical activity intervention of their choice. the pilot study was offered in one area of luton (uk), with invitation letters sent by the participants gp. participants were tested before and after a -week programme of strength, balance and mobility, delivered in a weekly session lasting one hour. participants were assessed at baseline for motivation using the patient activation measure (pam), physical function using the short physical performance battery (sppb), and fear of falling using the falls efficacy scale international (fes-i). each test was carried out in a follow-up test after the programme had concluded. bootstrapped paired t-tests were used to assess the effect of the intervention. results: twenty-seven people aged . ± . years took part in the intervention. the pam scores improved from . % to . % ( . , % ci: . , . ), which is twice the minimal clinically important difference (mcid) of . for sppb, there was an improvement from . to . ( . , % ci: . , . ). the average increase was greater than the mcid for a substantial improvement of . . when fes-i was assessed, only three people ( %) had high concern about falling. there was no significant improvement in fes-i after the intervention (- . , % ci: - . , - . ). after the intervention, % of participants choose to pay for the continuation of the programme. conclusion: the findings of this study suggest that a targeted exercise programme including strength and balance training can significantly improve motivation and functional status among mildly frail older people identified using the efi, with the majority choosing to continue exercising. background: despite frailty has traditionally been examined from a physical standpoint, recent studies advocate for the existence of cognitive frailty ( ), and suggest that both physical and cognitive frailty are interrelated. thus, interventions should aim to prevent or attenuate the effects of frailty from a multidimensional perspective. objectives: to evaluate the effects of three different exercise programs on frailty among older adults living in long-term nursing homes (ltnh). methods: participants ( . % female) met the following criteria: aged years, scored on the barthel index, scored on mec test (an adapted version of mmse in spanish) and capacity to stand up and walk m independently. participants were randomly assigned to a progressive multicomponent group (mcg; n= ), a multicomponent dual-task group (dtg; n= ), or to a walking group (wg; n= ). the mcg underwent a -month moderate intensity strength and balance exercise program twice a week. the dtg performed simultaneous cognitive training (attention, inhibitory control, calculations and semantic memory) to the mc program. the wg walked up to minutes per day for days a week. frailty was measured though the following tests: fried frailty index (ffi), the tilburg frailty index (tfi) and the study of osteoporotic fractures (sof). results: the ffi revealed reductions in frailty in all groups, although only the mcg and the wg reached statistical significance (p< . ). as for the tfi and sof tests, no statically significant differences were found in any of the groups. however, there was a positive trend in tfi in the dtg (p= . ). no group-by-time interactions were found in any of the frailty tests used (p> . ). conclusion: our study showed no differences between interventions regarding frailty. however, the mcg and the wg showed significant reductions in phenotypic frailty, whereas the dtg showed a positive trend in the tfi, which takes into account physical, psychological and social domains. therefore, further studies should explore the effects of different exercise modalities on frailty from a broad perspective in older adults living in ltnhs. references: kelaiditi et al . j nutr health aging. ( ) : - . noirez , , iraj hashemi , deborah kopoin , pierrette g a u d r e a u , m a r c b é l a n g e r , g i l l e s g o u s p i l l o u , josé a morais , aubertin-leheudre ( ( ) background: aging leads to a loss of muscle strength and functional capacity. these phenomena can be slow down by daily exercise practice or resistance training intervention. objectives: the aim of this study was to investigate in elderly men muscle fiber size and type after resistance training. methods: among sedentary older men who completed a -week mixed power training program, were biopsied in the vastus lateralis before and after the program. cross sections were performed on these muscles, followed by triple immunohistochemical staining with antibodies directed against laminin, myosin heavy chain (myhc)- and myhc- a coupled with staining with secondary fluorescent antibodies. immunostaining analysis of laminin allowed us to determine fiber size and these of myhcs to determine fiber type. results: the size of the muscle fibers remained the same between before and after the mixed power training (p= . ).there was no significant difference in the percentage of expression of myhc- , a, x (p= . , p = . , p = . ) between before and after intervention. in addition, there was no difference in the size of fiber expressing myhc- between before and after the training (p = . ). however, significant increase in the sizes of fiber expressing myhc- a and myhc- x (respectively p = e- , p <. ) after the mixed power training was observed. conclusion: in elderly men, an increase of the size in fibers both expressing myhc- a and myhc- x in vastus lateralis muscle could explained the improvement on muscle mass observed previously (carvalho et al. acer ) . to confirm the mechanism explanation of this promising exercise modality, mitochondrial parameters should be also analyzed. background: muscle (in)activation related with sedentary behavior (sb) and physical (in)activity (pa) is a risk for sarcopenia in older adults. although age is not yet a risk factor for sarcopenia in adulthood, other factors such as lifestyle may significantly contribute to its progression. objectives: considering the primary and secondary prevention of sarcopenia, the aim of this study was to analyze associations of sb and pa with markers of muscle strength (lower limb muscle power) and muscle mass (fat mass (fm) to fat free mass ratio (ffm) in adult women and men with and without deficits in these markers. methods: participants were apparently healthy adults ( women) with a mean age . ± . yrs, employed in activities requiring office work. fm and ffm were evaluated by bioelectrical impedance analysis (bia, khz bia rjl, akern bioresearch, florence, italy akern). muscle power relative to body mass (pmax/mass) was assessed during a single two-legged jump on a force platform (leonardo mechanograph, novotec medical, pforzheim, germany) . sb and pa were assessed by accelerometry (actigraph, gt x model, fort walton beach, fl, usa) during four consecutive days ( -week+ -weekend days). the variables analyzed were time spent per day in sb, in light-, moderate-, vigorous-, moderate to vigorous-intensity pa, total pa and breaks per day of sb. multiple linear regressions were performed by stepwise to examine associations of sb and pa with muscle power and fm/ffm, separately for men and women with and without muscular deficits. for the identification of deficits (<- . sd), muscle power and fm/ffm were standardized separately for men and women having as reference their respective mean. results: linear regressions by stepwise evidenced an association of sb with muscle power in women with muscular deficit (β = - . , p = . , adjr = , %%) and an association of vigorous pa with fm/ffm in men without muscular deficit (β = . , p < . , adjr = , %). no associations were observed between sb or pa with muscle power or fm/ffm in other groups. conclusion: sb was negatively evidenced in women with muscle power deficit while vigorous pa revealed to be associated with fm/ffm in men without ffm deficit. funded by portuguese science and technology foundation; project c mup-eri/hc i/ / patricia parreira batista , andré gustavo pereira de andrade , jéssica rodrigues de almeida , aimée de araújo cabral pelizari , leani de souza máximo pereira , lygia paccini lustosa (( ) physical therapy department, ufmg -eeffto, belo horizonte, brazil; ( ) sports department ufmg -eeffto, belo horizonte, brazil) background: the practice of regular physical activity in the older people leads to the decreased of the loss of muscle mass and function with advancing age, and enhances the functionality in activities of daily living and social interaction. in addition, exercise promotes gains in the quantity and quality of muscle fibers and improves muscle strength and power, acting as a protective factor for negative health-related outcomes such as falls, frailty, and hospitalizations. regular practice of physical activity is known to modify the chronic proinflammatory condition common in the older people. probably, exercise reduces the drive of catabolic stimuli from this proinflammatory cascade, modifies the metabolism and production of cytones in tissues and organs, promoting protective and anti-inflammatory effect in the body. objectives: to compare older women who reported being active or sedentary regarding functional capacity and plasma indices of inflammatory mediators. methods: participated women ( years or older), recruited for convenience. those unable to walk were excluded; acute musculoskeletal diseases; lower limb fractures in the last year; neurological diseases and sequelae; history of cancer in the last five years and cognitive impairment (mental state mini-exam). all informed clinical and demographic data and performed the tests short physical performance battery (sppb) and timed up and go (tug). plasma dosages of stnfr and il- were by elisa method. comparison was by independent student t test. approval by the research ethics committee / ufmg (caae: . . . ). results: fiftytwo sedentary older women participated ( . ± . ys.); number of comorbidities of . ± . ; body mass index of . ± . kg/m . from the active group were elderly women ( . ± . ys.); comorbidity number of . ± . ; body mass index of . ± . kg/m . there was significant difference between groups in sppb (p = . ), tug (p = . ) and stnfr (p = . ). conclusion: the results showed that the active older women had better functional and mobility performance and worse plasma stnfr levels. in this case, one can think about the possible influence of body mass index in these older women, which should be explored in future studies. background: our research group designed a comprehensive geriatric intervention program (cgip) consisting of resistance exercise, physical activity increments, oral functional care, and a nutritional guide. we conducted a -week intervention and investigated the effects. after the short-term intervention, we followed up the all participants. we hypothesized that the follow-up could mitigate the loss of short-term intervention effects. objectives: the aim of this study was to compare physical functions before and after the -week intervention, and the end of the follow-up. methods: a total of were willing to participate in the -week cgip. we encouraged them to increase their daily steps and to carry out the program by using daily self-monitoring logs. the participants were randomly assigned to two groups [class-styled session (cs) group ; home-based (hb) group ] based on their residential districts. while cs group attended -minute weekly sessions and independently executed the program on other days, hb group did not attend the weekly sessions but received instructions on program execution. after the shortterm intervention, all participants were instructed to carry out the gcip habitually. also, three optional sessions for all participants were held in order to recommend implementation of the program. physical functions, such as knee extension strength (kes), maximum walking speed (mws), and anterior thigh muscle thickness (mt) were measured before and after the short-term intervention, and the end of the follow-up. results: of the participants identified, (cs ; hb ) took part in the measurements after the follow-up. thus, we analyzed their data. a significant interaction were observed in mws (p= . ). the -week cs intervention significantly improved mws (p< . ). but, mws in cs group significantly decreased after the follow-up (p= . ). there was no significant difference between before the intervention and after the follow-up in mws in cs group. on the other hand, no significant change was observed in hb group. significant time effects were observed in kes and mt (p< . ). both -wk interventions significantly improved kes and mt. while kes was maintained even after the follow-up, mt was significantly decreased. conclusion: the results suggested that appropriate follow-up helps to preserve short-term intervention effects. background: with the increasing prevalence of alzheimer disease and the current absence of drugs therapeutic, nonpharmacological strategies are definitively necessary. physical intervention is often proposed to aid in preventing or slowing cognitive decline. recent studies suggest that combining physical exercise with cognitive stimulation may have more global effect. objectives: we aimed at assessing effect of aerobic exercise alone or combined to intellectual exercises on major cognitive functions: attention (stroop), problem solving (hanoi tower) and working memory (digit span). subjects were trained twice a week for eight weeks. cognitive functions were assessed before training (base line), at the fourth and at the eighth weeks. to evaluate persistency of the effect, subjects were assessed one month after the end of training. methods: two groups were randomly constituted mild cognitive impairment subjects (mci) and alzheimer disease moderate patients (adm). each group was subdivided into three sub groups according to the task to be performed. aerobic exercise (pedaling) alone or combined to cognitive games presented on screen. control groups performed a reading task. results: an effect of training on cognitive functions was observed in adm as well as in mci subjects. however, only adm patient's performances were further improved by adding cognitive games. after four weeks, the observed effects were still maintained in both groups. mci results were obviously better than those of adm. there was no significant change in performances for control groups. conclusion: aerobic exercise induce cognitive improvement in adm and mci patients. combined physical exercise and cognitive games potentiated this effect mainly in adm group. this procedure has long lasting beneficial effect. this supports the necessity of regular aerobic exercise to prevent cognitive deficits in aging cognitive deficits. background: increasing physical activity represent a key therapeutic intervention to prevent the loss of mobility disability for enhancing health related quality of life. hence, we have set up a primary and secondary prevention care path through exercise training and nutrition to improve mobility and physical performances. objectives: our primary goal is to integrate a prevention care path into daily life of elders who may present a mobility disability risk. we aim to improve quality of life and mobility. methods: our program includes years or more who present a risk of developing a mobility disability. initially, we identify and screen a risk of mobility disability in wide elders communities. we diagnose mobility disability risk factors, sarcopenia and frailty, in day hospital (dietician, geriatrician and a kinesiologist). we use the ewgsop algorithm to diagnose sarcopenia. the patient then attend a -months training program, including sessions per week. sessions combine resistance exercises and balance training during minutes. we support the patient for his own project of long-term maintenance quality of life between physical activity and nutrition. results: patients have been seen after sessions. physical performance was significantly improved after months of intervention (sppb p< , , gait speed p< , and time-up-and-go p< , ) likewise grip strength (p< , ). the "sarqol" score was also significantly higher (p< . ). sub-group sppb ≤ with severe sarcopenia improve significantly more its score (+ . ± . p< , ) comparing to the overall population (+ . ± . ). moreover, there was a significant difference (p< , ) for sppb at baseline between responders ( . ± . ) and nonresponders ( . ± . ). conclusion: our intervention enhances mobility through physical performance benefits. we can make the assumption that adverse events will be occurring less and physical dependence will be delayed, regarding gait speed improvement. patients with lower physical performance are responding better than the overall population meaning that our intervention is more specially indicated for patients with severe sarcopenia. furthermore, our program sustains motivation for physical activity and exercise after months. we were able to show that it was possible to set up a comprehensive and effective care path for frail and sarcopenic elderly people. background: middle-aged adults who are pre-sarcopenic are at the highest risk of developing sarcopenia due to the progressive nature of the syndrome. objectives: to determine whether high intensity interval training (hiit) results in greater improvements in body composition, compared to a control group, in middle-aged adults with pre-sarcopenia. methods: eighty-two sedentary adults ( - yrs) with a low appendicular skeletal muscle mass index (asmi) were randomized into control (n= ) or intervention group (n= ) using stratified randomization based on age, sex and bmi. low asmi (asm/ht ) was determined by dxa (lunar prodigy, ge healthcare) using age-and sex-specific cut-scores as proposed by prado. the control group received one education session on general physical activity recommendations. the intervention was supervised, group-based, high-intensity aerobic and resistance interval training (hitt), times weekly for -weeks. an intention-to-treat mixed model linear regression, with a random effect, was used to analyse group differences for body composition. results: . % of the sample were female, the mean age was . yrs ( . ) and the mean bmi at baseline was . kg/m ( . ). people ( %) completed the intervention, people in the hitt group and in the control group. no adverse events were reported. significant group differences were observed for total muscle mass ( . kg, %ci: . - . ), leg muscle mass ( . kg, %ci . - . ), asmi ( . kg/m , %ci . - . ) and visceral fat mass our study indicated that group-based hiit is an effective, tolerable and safe exercise modality to increase total body and appendicular muscle mass, and to decrease visceral fat, in middle-aged adults with pre-sarcopenia. background: aging is related to body composition modifications and functional capacities declines. it is recognized than being active can prevent these changes and improve quality of life. however, it is unclear if gender or age influence this relationship and if a sub-type of voluntary physical activity is more efficient to maintain these physical parameters. objectives: to assess the association between current physical activity level or type and functional capacities and body composition among elderly people and to examine if age (< or >= yrs old) or sex modulate the relationship. methods: functional capacities using different validated tests (i.e. grip strength, timed up and go, sit-to-stand, muscle power, alternate step test, leg extension, vo max), body composition (fat & fat-free masses) using dxa were assessed. current global (total) and specific (aerobic, resistance or body and mind) physical activity levels (duration) were obtained through a questionnaire. multiple regressions, adjusted on age, sex and bmi, were performed to assess the relationship between current physical activity level and functional capacities or body composition. sub-group analysis, according to the sex and age (< y vs. >= y) were also performed by means of pearson correlations. results: a total of subjects ( . ± . years; women: . %; bmi= . ± . kg/ m²) were enrolled. after adjustment on confounding factors, total current physical activity level has positive impact on total fat mass (%; β=- . , p= ) and balance (β= . ; p= . ). moreover, current body & mind activities influence total fat-free mass (kg; β=- . , p= . ) and balance (β= . ; p= . ) whereas resistance activities influence fat-free mass (kg; β= . ; p= . ), fat mass (%; β=- . ; p= . ) and sitto-stand test (β=- . ; p= . ). sub-analysis shows that total physical activity level was significantly associated with fat mass, sit-to-stand test, balance and vo max in women but not in men. moreover, among people under y, the time spent on cardio activities does not affect functional capacities and body composition. nonetheless, among people aged y and over, the time spent on resistance activities is associated with functional capacities and body composition. conclusion: being active is associated with body composition and functional capacities, especially among women aged years and over. itxaso mugica-errazquin , nagore arizaga , janire virgala , julen gomez , garbiñe lozano , yune aranburu , udane elordi , maider kortajarena , ana rodriguez-larrad , jon irazusta ( ( ) background: low physical fitness, frailty and dependency are highly prevalent in people living in long term nursing homes (ltnh). multicomponent physical exercise, including strength, balance and endurance, has demonstrated to be effective for improving physical fitness and reducing frailty in ltnh. however, there is no evidence that this type of programs are capable to improve or even maintain the levels of autonomy in activities of daily living (adl) of this population. objectives: the major aim is to ascertain whether a new approach of months, individualized and progressive multicomponent program focused on functioning maintains autonomy in older adults living in ltnhs; the secondary aim is to assess the effects on frailty and physical fitness. methods: people living in ltnh, between and years, participated in this single group interventional study. inclusion criteria were: >= years, >= barthel index, >= mec- and be able to stand up from a chair and walk meters with or without one person/technical assistance. the intervention consisted of months of a progressive multicomponent physical exercise program (ep) aiming to improve the physical condition, followed by months of physical exercises focused on functional adl with the objective of maintaining/improving autonomy of the participants. barthel index was used to assess autonomy level in adl, frailty was measured by fried frailty index and short physical performance battery (sppb) was used to assess physical fitness. the study is registered in u.s clinical trial (nct ) and approved by the committee on ethics in research of the university of the basque country (m / / ). results: during the first months of ep participants lowered the score in the barthel index (p< , ). however, participants showed significant improvements in frailty (fried frailty index p< , ) and in physical fitness (sppb p< , ) . from the rd to th months, while physical fitness of participants did not change, they improved autonomy in adl, and decreased frailty non-significantly. when comparing the effects of the entire intervention, barthel index did not change significantly and physical fitness and frailty improved (sppb p< , ; fried p< , ). conclusion: this new approach of months of individualized and progressive multicomponent program focused on daily functioning maintains autonomy in activities of daily living, improves physical fitness and reduces frailty in older adults living in ltnhs. shuji sawada , hayao ozaki , , toshiharu natsume , daiki nakano , pengyu deng , toshinori yoshihara , takuya osawa , shuichi machida , hisashi naito (( ) juntendo university, chiba, japan; ( ) tokai gakuen university, aichi, japan; ( ) japan women 's college of physical education, tokyo, japan) background: in previous study, we found that low-load resistance training using own body weight and elastic band even only biweekly could induce muscle hypertrophy in older adults after weeks of training. however, it is unclear whether levels of different blood parameters before training associated with the effects of training. objectives: this study aimed to clarify whether levels of different blood parameters before training influenced the effect of low-load resistance training on lower limb muscle thickness (mt). methods: sixty-nine communitydwelling japanese subjects aged . ± . years ( women and men) volunteered for this study and participated in a lowload resistance training program using their own body weight and elastic band. the training was performed biweekly for weeks. each participant's mt at the anterior aspects of the thigh (at) was measured using a b-mode ultrasound device. further, the levels of the following blood parameters were assessed before and after the training program: serum albumin (alb), hemoglobin (hb), total cholesterol (tc), and hemoglobin a c (hba c). we checked the first quartile value of each blood parameter to establish the cutoff criteria for reduced levelsserum alb = . g/dl, hb = . g/dl, tc = mg/dl, and hba c = . %. participants were divided into low or normal groups in each blood parameter, and their data were analyzed using two-way analysis of variance. results: when using the abovementioned criteria, biweekly low-load resistance training increased mt at the at in every group after training. the interaction between time and groups was only detected with low (< . g/dl) versus normal (>= . g/dl) serum alb levels. in this case, there was no difference in mt at the at before training, but participants in the normal serum alb level group had greater mt after training than those in the low serum alb level group. conclusion: the effect of low-load resistance training on lower limb mt appears to be limited in participants with low pre-training serum alb level. objectives: it was to estimate the affect of complex -week treatment with kinesiotherapy methods on body weight loss and muscle function in patients with obesity. methods: men and women aged - years old with alimentary obesity were enrolled in the study (mean age . ± years, weight . ± . kg, bmi . ± . kg/m , waist circumstance wc . ± cm, hip circumstance hc . ± cm). the complex kinesiotherapy administered daily for week and included interactive sensorimotor trainings on double unstable platform, kinesiohydrotherapy in a pool, special complex of physical exercises in a gym and ergocycle trainings. weight, wc, hc, fall number for last weeks were measured at baseline and after the treatment was completed. muscle strength and walking speed functional tests results assessment ( -meters-walk test, up-and-go test, special tests for back and abdomen muscle endurance to static and dynamic loading) were performed at baseline and in weeks. results: there was a significant reduction in body weight ( . ± . kg at baseline vs . ± . kg in weeks; p= , ), in bmi ( . ± . vs . ± . kg/m ; p= . ), in wc ( . ± . vs . ± . cm; p= . ) and in hc ( . ± . vs . ± . cm; p= . ) in treated obese patients. -meters-walk speed increased from . ± . m/sec at baseline to . ± . m/ sec in weeks (p= . ). up-and-go test results improved from . ± . to . ± . sec (p= . ). we registered statistically significant elevation of the endurance to static loading in abdomen muscles from . ± . to . ± . sec (p= . ) and in back muscles from . ± . sec to . ± . sec (p= . ). the endurance to dynamic loading increased in abdomen muscles from . ± . to . ± . times (p= . ) and also in back muscles from . ± . to . ± . times (p= . ). fall namber markably decreased from . ± . at baseline to . ( %ci: . ; . ) after completion of treatment. conclusion: investigated complex treatment with kinesiotherapy methods promotes body weight loss, wc and hc reduction in obesity. -week special training of obese patients is associated with increasing in gate speed and lower extremities muscle strength, and it also causes improvement in static and dynamic loading endurance of back and abdomen muscles. those changes may probably improve balance function and decrease risk of falling in obese patients. thaiana pacheco, candice medeiros, rummenigge dantas, inae c. gadotti, edgar r vieira, fabrícia costa cavalcanti (department of physical therapy, florida international university, miami, usa) background: integrating technological advances into clinical practice can be challenging. physical therapists have been developing serious games/exergames for a variety of rehabilitation purposes, but uptake has been slow. games with virtual scenarios are an engaging and affordable way to encourage and increase physical activity levels. serious games have been developed to adapt virtual gaming environments to patients' needs and evolving capabilities. games can improve adherence and therapy effectiveness. the sensory and motor stimulation while playing serious games can help geriatric rehabilitation to improve mobility and balance. objectives: this study analyzed the effects of a new serious game on the balance of older adults. methods: this was a pilot quasiexperimental design study in which older adults completed six sessions of dynamic balance training using the virtualter serious game that uses the kinect sensor for motion capture. this game was developed by researchers from the federal university of rio grande do norte in brazil. the game consists of static and dynamic tasks for training balance. it involves stationary walk, lateral reaching and climbing steps up and down. it has phases with increasing the level of difficulty. the participants were evaluated before and after the program using the berg balance scale (bbs) and the short physical performance battery (sppb). t-test for dependent samples was used to analyze the pre vs. post data. results: twenty three participants participated in the study (age = ± ; sex = % women). the results indicate improvement in bbs scores (pre: ± ; post: . ± ; p = . ) and sppb scores (pre: ± ; post: ± ; p = . ). conclusion: playing the virtualter serious game improved balance in older adults. helen chan , duncan wong , cindy fan (( ) the nethersole school of nursing, the chinese university of hong kong, hk; ( ) silver yoga lab, hk) background: evidence showed that both frail and prefrail significantly increase the risk of developing or worsening disability in activities of daily living, poor quality of life and institutionalisation. yoga has been consistently reported as effective intervention in improving physical functioning in terms of balance, lower limb strength, mobility and body flexibility. objectives: to assess the feasibility of silver yoga in older adults and to examine the preliminary effects of silver yoga on their physical health. methods: this was a one group pre-test post-test study conducted in a community centre. people who aged and above, were mentally competent, home-living, and classified as prefrail based on physical phenotypes using fried criteria, were eligible to the study. the silver yoga class included eight . -hour weekly sessions delivered by two experienced yoga instructors with specialized training in silver yoga. senior fitness test (sft) was conducted to assess changes in physical health. paired t-test was used to compare the within-subject differences across -month time. results: a total of older adults were recruited. there were significant improvement in six dimensions of the sft, including upper extremity muscle strength, lower extremity muscle strength, upper body flexibility, lower body flexibility, agility and dynamic balance, and aerobic endurance (ps < . ). all participants except one completed the yoga programme, with high level of satisfaction. in addition to the effects of physical conditions, the participants also appreciated it as mind-soothing and relaxing. conclusion: the findings showed that silver yoga is well-received by older adults generally, with significant effects in improving their physical fitness. more rigorous study is needed to examine its effects in a longer term and also in a more holistic manner. ku leuven, leuven, belgium; ( ) physical activity, sports and health research group, department of movement sciences, ku leuven, leuven, belgium) background: with aging skeletal muscle tissue becomes less responsive to anabolic stimuli, eventually contributing to muscle wasting. inflammation is considered an important player in this age-related anabolic insensitivity. recent reports provide a promising role for omega- polyunsaturated fatty acids (ω- ) in (muscle) health, as they possess systemic anti-inflammatory properties and stimulate muscle anabolic signaling. objectives: we investigated whether ω- supplementation improves the systemic inflammation and muscular adaptations (i.e. strength, mass, molecular signaling) to resistance exercise in an elderly population. methods: twenty-three elderly ( - y; ♀) were randomized to receive either ω- (~ g/d) or an isocaloric amount of corn oil (plac) during weeks. after two weeks of supplementation, participants engaged in resistance exercise (re; x/week) for weeks. prior to and after completion of the intervention, muscle and blood tissue, parameters of body composition, muscle strength and functionality were assessed. results: upon re, -rm significantly improved in plac (+ . %) and in ω- (+ . %), irrespective of condition. isometric strength significantly improved in ω- (+ . %), but not in plac (- . %). muscle volume did not change following re. plasma crp levels decreased, though not non-significantly, in ω- (- . %), whereas only a small increase was observed in plac (+ . %). ω- supplementation nor re affected the muscle anabolic sensitivity (akt phosphorylation) in response to a protein bolus. conclusion: this study confirms that ω- pufas improve the gains in isometric but not in dynamic muscle strength upon re in elderly. however, this was not associated with changes in anabolic sensitivity or systemic inflammation. further analyses will investigate whether the ω- induced gains in strength can be related to systemic hormones or muscle molecular signaling (mtor signaling, inflammation). meera suresh, clarence chikusu, caroline goodger (nutrition and dietetics, st. peter's hospital, chertsey, uk) background: deconditioning is a common phenomenon in patients over years old in acute settings. it is well known that poor nutritional status has a major impact on adverse outcomes in frailty and can exacerbate sarcopenia ( ). currently, there is limited research exploring the impact of dietitians on optimising nutritional status in acute settings in older populations for frailty and sarcopenia. objectives: compare the impact of dietetic intervention on the change in frailty scores between a patient group (n= ; mean age . years) who received dietetic intervention (di) and a patient group (n= ; mean age . years) who did not receive dietetic intervention (ndi). methods: a -month retrospective study (august-december ) was undertaken at the older persons short stay unit at a district hospital in england. frailty scores were calculated based on the rockwood model of clinical frailty. dietary intake was recorded and analysed using a standardised nutritional profile of hospital meals. the di group was given standardised dietetic care including oral nutrition support and build up dietary advice. descriptive statistics were used to determine frequencies. results: the di had higher frailty scores (mean of . ; range: - ) and a higher mortality rate ( %).the ndi had a mean score of . (range: - ) and mortality rate of %. the average oral intake for energy and protein for patients in the di group prior to dietetic intervention was % lower than the espen recommendations. despite the higher frailty scores and mortality rates in the di group, progression in their frailty score was slower compared to the ndi group ( % vs %). conclusion: the results highlight the importance of a timely referral for early dietetic intervention which is crucial for optimisation of better clinical outcomes in these patients. a dietitian is a key member of the mdt and can prevent further deterioration in muscle mass and the impact on patients' frailty and independence and also slow down the progression of sarcopenia and frailty. this has long term impact on health and social services by reducing length of stay, hospital re-admissions and the increasing burden on social care. uz leuven, leuven, belgium) background: while the protein recommended dietary allowance (rda) for healthy adults is . g protein/kg bodyweight (bw)/day (d), expert groups recommend a protein intake up to . g protein/kg bw/d for older people with chronic diseases. in addition, at least - g protein (whereof at least . g of leucine) is recommended per meal. objectives: we aim to assess in (pre)sarcopenic older people the daily energy and protein quantity and quality intake, and their change due to supplementation. methods: dietary protein quantity, and quality (plant/animal source, amount of amino acids, amount of leucine and leucine distribution over a day) and dietary energy intake were calculated from four day estimated dietary records of (pre)sarcopenic participants of the enhance study (clinicaltrials.gov nct ) before and after a -week supplementation period. participants received an individualized protein supplement (resource® instant protein, nestlé) , to achieve a total (dietary + supplemental) intake of . g protein/kg bw/d. results: (pre)sarcopenic adults ( . ± . years, % female) had an average dietary protein intake of . ± . g/kg bw/d, which is higher than the rda, but below the . g/kg bw/d recommended by experts. (pre)sarcopenic adults were supplemented with protein powder, which improved the total protein intake to . ± . g/kg bw/d without affecting dietary protein or energy intake. moreover, supplementation increased the protein intake to at least g protein/meal without affecting dietary intake. more than % of dietary protein intake was of animal origin. leucine intake at baseline was insufficient at all meals, but increased to at least . g at lunch and dinner by supplementation without affecting dietary leucine intake. conclusion: community-dwelling (pre)sarcopenic older people do not reach the recommended protein intake proposed by expert groups. individualized protein supplementation results in adequate intake of protein without substantial change in dietary intake. nutrition and dietetics, internal medicine, amsterdam university medical centers, amsterdam, the netherlands) background: weight loss is a main treatment goal in obese older adults with dm . combined lifestyle interventions (cli) may be more effective in preserving muscle mass during weight loss. whether severe obese benefit similar to less obese is unknown. objectives: our probe-study showed an increase in muscle mass during cli in obese older adults ( +) with dm . do severe obese (bmi > kg/m ) benefit similarly to less obese. methods: in a post-hoc analysis, out of enrolled older adults had both body weight and protein intake data before and after a -month cli consisting of dietary advice (- kcal/day) and resistance exercise. a selection of assessments were appendicular skeletal muscle mass (asmm, by dxa), physical performance (wmax; by cycle ergometer steep ramp test), quality of life (rand- physical component summary score (pcs), visceral adipose tissue (vat, by dxa), crp, insulin sensitivity and resistance (matsuda, homa-ir; by ogtt), blood pressure (sbp, dbp). linear regression analysis was used with protein intake (g/kg, except for asmm being included in kg) as independent and assessments after -months as dependent (with assessment before intervention as confounder) for both groups bmi> (severe obese n= ) and bmi<= (n= ). results: mean age was , mean bmi was . , sex m/ f and protein intake during intervention was + gram/day. mean weight loss was - . + . kg and fat loss - . + . kg. per g protein intake increase + g muscle was preserved (p= . ). however, this appeared + (p= . ) vs + g (p= . ) for severe obese vs not severe obese. severe obese showed higher response for wmax (+ . + . (p= . ) vs - . + . ) and pcs (+ . + . (p= . ) vs - . + . ), for vat (- . + . (p= . ) vs + . + . ) and crp (- . + . (p= . ) vs + . + . ), for insulin sensitivity (matsuda + . + . (p= . ) vs + . + . ) and insulin resistance (homa-ir - . + . (p= . ) vs + . + . ), sbp (- . + . (p= . ) vs - . + . ) and dbp (- . + . (p= . ) vs + . + . ). while whole group and not severe obese group showed no significant effect. conclusion: these results suggest that severe obese might benefit even more from combined lifestyle intervention compared to less obese older adults with dm . further investigation is needed to confirm these findings and identify potential mechanisms. background: nutritional interventions have been shown to stimulate muscle protein synthesis. to optimize muscle mass preservation and gains, several factors, including type, dosage, frequency, timing, duration and compliance have to be considered. objectives: this systematic review and meta-analysis aimed to summarize these factors influencing the efficacy of nutritional interventions on muscle mass in older adults. methods: data sources: a systematic search was performed using the electronic databases medline, embase, cinahl, cochrane central register of controlled trials and sportdiscus, from inception date to nd november , in accordance with the prisma guidelines. inclusion criteria included randomized controlled trials, mean/median age >= years and reporting muscle mass at baseline and post-intervention; exclusion criteria included genetically inherited diseases, anabolic drugs/hormone therapies, neuromuscular electrical stimulation, chronic kidney disease, kidney failure, neuromuscular disorders and cancer. data extraction: extracted data included study characteristics (population, sample size, age, sex), muscle mass measurements (method, measure, unit) , effect of the intervention versus the control group, and nutritional intervention factors i.e. type, composition, dose, duration, frequency, timing and compliance. data analysis: standardized mean differences and % confidence intervals were calculated from baseline to post-intervention for the intervention and control group. a meta-analysis was performed using a random-effects model and grouped by the type of intervention. results: twentyeight articles were included encompassing participants (mean age . years, sd . ). amino acids, creatine, betahydroxy-beta-methylbutyrate, and protein with amino acids supplementation significantly improved muscle mass. no effect was found for protein supplementation alone, protein and other components, and poly-unsaturated fatty acids. high inter-study variability was observed regarding the dose, duration and frequency, coupled with inconsistency in reporting timing and compliance. conclusion: overall, nutrition alone is an effective intervention to improve muscle mass in older adults. due to the substantial variability of the intervention factors among studies, the optimum profile is yet to be established. background: physical and functional capacities decline with age. one new potential intervention is oral citrulline supplementation (cit) since cit seems to increase muscle protein synthesis, mass, size and strength, improve mobility but also decrease adipose tissue mass, particularly visceral depot in old rats. furthermore, exercise is known to be another efficient intervention. however, studies assessing cit supplementation combined or not with exercise on muscle function and mobility in older human adults are emerging and literature conclusions are needed to help health professionals. objectives: establish the potential effectiveness of citrulline supplementation combined or not with exercise on muscle function and physical performance via a systematic review of randomized controlled trials (rcts) in human aged years and older. methods: the preferred reporting items for systematic reviews and meta-analysis (prisma) statement has been followed. medline, cochrane central register for rcts and scopus databases have been searched. studies selection and data extraction have been performed by two researchers independently. methodological quality of each included studies was assessed using the quality assessment of diagnostic accuracy studies- (quadas- ) tool. results: based on prisma guideline, references have been identified. among this number, only rcts ( participants) matched the inclusion criteria (e.g rcts, age> yrs, human, cit supplementation, muscle or physical parameters) and were included in the systematic review. among these studies, / reported beneficial effects of cit on muscle mass. effects on muscle strength is reported on / studies but when cit is combined to exercise better improvements in upper muscle strength are observed. finally, / studies reported beneficial effect of cit on physical performance but suggested that cit with exercise displayed greater improvements in walking speed than exercise or cit alone. the overall quality of studies was rather high. conclusion: cit supplementation seems able to improve muscular and physical factors in specific elderly people (malnourished, women, hypertensive, obese, dynapenic-obese) compared to placebo. more importantly, cit with exercise is more efficient than exercise or cit alone. however, due to the small number ( ) and heterogeneity (dose, duration, population) of the studies, further investigations are needed to confirm its promising intervention for health professionals. background: the medical nutrition supplement fortifit (r), containing the specific nutrient combination actisyn™, is designed to support muscle building in sarcopenia (muscle loss). actisyn (whey protein, leucine and vitamin d) provides high bioavailability of leucine and essential amino acids for the muscle; the nutrients in actisyn act together to optimize the muscle protein synthesis response in a state of sarcopenia where these nutrients are often deficient. preclinical and acute human studies confirmed this mode of action. objectives: to demonstrate the longer-term effects of fortifit supplementation on muscle building in healthy and sarcopenic older adults and on muscle preservation in obese (diabetic) older adults during a weight-loss lifestyle intervention. methods: our clinical research program investigated the effects on muscle mass, strength and function in healthy and sarcopenic older adults and in obese and type diabetic patients. muscle mass was measured by dexa; strength and function by handgrip strength, -times chairstand test and short physical performance battery (sppb). all studies were randomized-controlled trials with an intervention duration of to weeks. results: a significant increase in appendicular lean mass and leg lean mass was observed in healthy older adults after weeks supplementation (p< . vs non-caloric control) [chanet, jnutr ]. in sarcopenic older adults, -week intervention increased appendicular lean mass ( . kg, %ci . - . kg; p= . vs iso-caloric control) [bauer, jamda ] . moreover, during a -week lifestyle intervention of energy restriction and resistance exercise training in obese older adults with or without type diabetes, fortifit preserved appendicular lean mass (p< . vs iso-caloric control) [verreijen, ajcn ; memelink, clin nutr ] . a significant improvement was observed in chairstand time after -week intervention in sarcopenic older adults (- . s, %ci - . to - . s; p= . vs isocaloric control), but improvements in handgrip strength and sppb (primary outcomes) were only significant versus baseline (p< . ) and not versus control [bauer, jamda ] . conclusion: the medical nutrition supplement fortifit effectively supports muscle building in healthy, sarcopenic and obese older adults. moreover, the improvement in chair-stand time observed in sarcopenic older adults is clinically relevant. background: chronic kidney disease (ckd) is commonly found in older persons and it affects the quality of life and economic burden. knowledge and health literacy have been reported as fundamental factors for persons with chronic illness to perform health behavior. however, from a literature review, relationships among knowledge, health literacy, and health behavior in older persons with non-dialysis ckd have rarely been reported. objectives: to examine relationships among knowledge, health literacy, and health behavior in older persons with chronic kidney disease. methods: nutbeam's conceptual framework of health literacy was used to guide the study. the sample recruited by purposive sampling consisted of older persons with non-dialysis stage to ckd, who sought healthcare services at a ckd clinic in a university hospital, thailand. data were collected by interviews using the questionnaires about the demographic data, knowledge about care of ckd, health literacy, and health behavior of older persons with ckd and then were analyzed using descriptive statistics and spearman's rho correlation coefficients. results: the sample consisted of men and women with their age ranging from to years (m = . , sd = . ). the analysis revealed that the sample had the mean scores of total knowledge about care of ckd, health literacy, and health behavior at a high level. health literacy was positively associated with health behavior (r = . , p = . ), but knowledge about care of ckd was not significantly associated with health literacy (r = . , p = . ), nor health behavior (r = . , p = . ). conclusion: only health literacy was significantly positively related to health behavior. although knowledge is fundamental of health literacy, it was not significantly related to health literacy nor health behavior in this study. it is explained that health literacy is the ability and skills that might link knowledge of individuals to perform behaviors. thus, healthcare providers should find strategies for enhancing health literacy of older persons with ckd to promote appropriate health behavior, thereby delaying complications. background: handgrip strength (gs) is linked to the vitality domain of the intrinsic capacity (ic) construct and is a marker of sarcopenia and frailty. low gs is a predictor of adverse health outcomes like disability onset and mortality. small increases in gs have been reported after exercise interventions, suggesting that life-course determinants rather than short-term determinants influence gs. objectives: to assess social inequality in the distribution of gs and the association of gs levels with a proxy of social determinants of health (sdh) among adults and older adults. methods: secondary analysis from wave ( - ) of the world health organization (who) study on global ageing and adult health (sage), which is nationally-representative of six countries, including , participants aged >= years and , < y. gs was computed in kg. wealth quintiles were assigned according to ownership of household assets. the last level of education of the participant and his/her mother was self-reported (the latter was used as a marker of early life sdh). social inequality was estimated using pairwise comparisons among the average of gs of the extreme social groups; and gradient inequality by the slope index of inequality (multivariate linear regression to adjust for age, sex, body mass index). estimations were weighted to consider the complex design of the sample. results: average gs was . kg for participants >= y and . kg for < y. participants >= y who reported a postgraduate level of education or higher showed % ( . kg) higher gs than their illiterate counterparts ( %, . kg, for participants < y). gs was on average % higher in participants >= y in the most top wealth quintile compared to those in the lowest quintile ( % in < y). in the multivariate models, gs was . kg higher in urban than rural participants and . kg higher among participants whose mothers had completed >= years of education compared to those whose mothers were illiterate. slope coefficients were significant after controlling for confounders. conclusion: grip strength displayed an unequal distribution among social groups and also among groups of early life exposures, which suggests that vitality as a domain of ic is shaped by the sdh and built through the life course. background: intrinsic capacity (ic) is the composite of the physical and mental abilities of an individual. the distribution and correlates of ic in older adults (oa) have not been reported using an integrative score with routinely-collected clinical data. it is not clear how ic is associated with multi-systemic biochemical age-related processes captured by alterations in standard clinical laboratory tests. objectives: to describe the distribution and correlates of ic in a population of older adults from the frailty day hospital of toulouse and to test its cross-sectional association with low or high haemoglobin or high crp, accounting for frailty status. methods: using routinely collected cross-sectional data of , first visits of oa aged + to the frailty day clinic of toulouse ( - ), we calculated an index of ic (biomarkers and validated scales for five who domains). low/high haemoglobin levels or high crp levels served as indicators of acute and middleterm multisystem disruption. we used descriptive statistics to learn the distribution of ic across sex, age, education and fried frailty categories. multivariate linear models were used to test the hypothesis that higher ic holds a negative association with the multi-system deficits depicted by altered laboratory tests. results: % of the population was female, and % was frail. our ic score has theoretical limits ( - ). overall, the ic was: mean= . ,sd= . ,min= . , max= . . on average ic men scored . (ic % . , . ) and women . (ic % . , . ). the relationship found between ic and age was not linear. frail older adults displayed % less ic than their robust counterparts and % less ic than their pre-frail counterparts. if frail oa would return to robust in this population, the average ic would potentially* rise %. disruption in haemoglobin or crp was inversely and significantly associated with the ic score after adjusting for age , sex, level of education and fried frailty status. conclusion: the population attending the toulouse frailty clinic displayed highly-heterogeneous ic levels, with frail oa showing significantly lower levels than robust oa. the association between ic and age is not linear. sex, age, education, frailty status and disruption in haemoglobin or crp levels were all significantly associated with ic in a multivariate model. background: older persons tend to be hospitalized increasingly because of the complex interaction among acute problems, age-related changed, and chronic diseases. qualified nursing care needs knowledge, understanding, and a positive attitude towards the care of older persons. however, little is known factors predict the caring behavior of nurses to care for hospitalized older persons. objectives: to examine the predictability of selected factors to explain intention to care and caring behavior for older persons of professional nurses. methods: the theory of reasoned action was used to guide the study. the proportionate stratified random sampling was used to recruit a sample of professional nurses from clinical wards providing care for older patients in a university hospital. data were collected using questionnaires and then, analyzed with descriptive statistics, pearson's product-moment correlation, and multiple regression analysis with the enter method. results: almost all of the sample were female, with their age ranged from to years (m = . ). factors related to professional nurses' intention to care were perceived caring climate in organization and attitude toward caring for older persons. also, factors related to caring behavior for older persons were perceived caring climate in an organization, intention to care, and attitude toward caring for older persons. through multiple regression analysis, perceived caring climate in an organization, attitude toward caring older persons, and basic knowledge about older persons jointly predicted . % of the variance in intention to care. together, perceived caring climate in an organization, intention to care, attitude toward caring for older persons, and basic knowledge about older persons accounted for . % of the variance in caring behavior for older persons of professional nurses. the perceived caring climate in an organization was the strongest predictor of caring behavior, whereas basic knowledge about older persons was not a significant predictor. conclusion: the findings support the notion of the theory of reasoned action. it is suggested that strategies to promote perceived caring climate in an organization, attitude toward caring for older persons, and intention to care should be established and maintained to promote caring behavior for older persons of professional nurses. background: environmental and social conditions play a major influence in the development and progression of negative health-related outcomes. they represent crucial elements when taking clinical decisions and planning the care plans of frail patients. nevertheless, they still often remain overlooked because priority is given to the clinical manifestations. objectives: the aim of this study is to explore the importance of social support in the definition of major health-related outcomes among hospitalized patients compared to other critical factors of older persons (i.e., frailty, age). methods: data were retrospectively collected from the medical records of patients aged years and older admitted to the geriatric unit of the fondazione irccs ca' granda ospedale maggiore policlinico (milan, italy). a -items frailty index (fi) was computed from clinical variables recorded during the first days of hospitalization (i.e., medical history, cognitive, functional and social assessment, physical examination, laboratory tests). mortality, length of hospital stay above the median, and risk of institutionalization were the outcomes of interest. results: we included patients (mean age . , sd . years, women . %). six patients died during the hospital stay ( . %). the median duration of hospital stay was (iqr - ) days. twenty-seven patients were discharged to other institutions ( %). the mean fi was . (sd . ). the fi showed a statistically borderline association with mortality (or . , % c.i. . - . , p= . ), and was predictive of longer length of stay (or . , % c.i. . - . , p= . ), even after adjustment for confounders. the presence of a caregiver was the only factor significantly associated with the discharge at home of patients (or . , % c.i. . - . , p= . ) at the multivariate analysis. age had no significant association with the three studied outcomes. conclusion: health systems should be organized according to an integrated model of care in order to adequately address the complex health needs of older people. social and environmental context plays a critical role in determining the person's health trajectory. social factors (as the presence of a caregiver) may play a stronger role in clinical decisions than biological or clinical aspects. background: the acute therapy team was formulated after the integration of an older persons assessment and liaison team (opal) with medical ward therapists. the team was spread across all acute areas. this team worked closely with the acute geriatric and frailty clinical team and it was recognised that length of stay, and improved patient experience and overall outcomes would be improved with earlier assessment and cga planning at the front door allowing closer collaborative working between the clinicians and therapists. objectives: to enhance service improvement and prevent the impact of sarcopenia and frailty syndromes leading to greater hospital stay and disability as a consequence of a delay to assessment by clinicians and therapists in the acute setting. through the screening of frailty syndrome risk and sarcopenia risk patients by the ed geriatrician and junior doctor, there would be a speedier response to therapy led interventions thereby reducing the conversion rate from ed and also therefore improving overall outcomes in length of stay and reduced disability through prolonged hospital stay. methods: consultant geriatrician and junior doctor (opssu team) to go to the emergency department in the mornings and see up to patients in cdu/a&e beds; the use of a the rockwood frailty score template identified those patients at risk of frailty syndrome and likely to benefit from early therapy intervention. these patients would have been highlighted as having the potential to be discharged within hours. a month data collection period from was chosen with data collected monday to friday only. data examined was categorised as follows: new patients, follow-ups; how many patients were seen on day of ed attendance vs after day of attendance?; number of patients seen by therapists same day of ed attendance number of patients not seen by therapists day of attendance; which team was looking after the patient from a clinically; how much time spent with patients; therapy led plan after initial assessment; an integrated assessment too was instrumental in the cga component of the therapy and clinical assessments. results: % of patients seen by therapists in ed are new patients referred. % of patients referred are seen on the actual date of ed attendance. the rest are seen later admission episode. % of therapy time is spent doing non-face to face tasks such as documentation. but up to % of patients have a discharge plan put in place after being seen by therapists in the ed. conclusion: a great deal of time is spent by therapists on documentation during assessment. this has a negative impact on the amount of time dedicated to clinical assessments and physiological and functional assessments required in the cga. there is a large number of patients referred by the clinical team to the therapists for review but a majority of patients are seen elsewhere during an admission episode and not in the ed. streamlined assessments and screening tools are recommended & planned for the future model of care. yi-chun cheng , li-ning peng , (( ) center for geriatrics and gerontology, taipei veterans general hospital, taipei, taiwan; ( ) aging and health research center, national yang ming university, taipei, taiwan) background: older people with frailty are at risk of adverse outcomes, such as falls, disability, hospital admission, long term care placement, poorer quality of life, and mortality, which denotes the importance of sarcopenia in the health care for older people, and integrated intervention program may prevent those. objectives: to evaluate the effectiveness of an integrated intervention program among those communitydwelling frail older people in north taiwan. methods: a total of participants over years old mild to moderate disability and mild cognitive impairment persons were recruited from a community-dwelling frail older people in north taiwan during august and july , frail older people were invited for the study. a weeks integrated intervention program was provided for all participants. they attended the hours program once per two weeks and physical activity, high protein diet education, and cognitive stimulation activity were included in the integrated intervention program. comprehensive geriatric assessments were performed before and after the intervention program, including basic demographic data, risk for malnutrition (by mna-sf), mood condition (by gds- ), cognitive condition (by mmse), weakness (by handgrip strength), exhaustion (by self-report in chs) slowness (by gait speed) and time-up-go test. pretest on the st week before intervention and post-test on the th week to compare the difference between twice evaluate consequence. results: overall, participants were identified as having pre-frailty ( . %) and frailty ( background: low appendicular skeletal muscle mass (asm), an integral component of current sarcopenia definitions, is commonly measured using bioimpedance analysis (bia). bia equations for estimation of asm are not generalizable across population groups and instrument types, potentially giving rise to inaccurate results when applied inappropriately. there is a lack of bia prediction equations for asian populations, none of which have been developed or validated for singaporean older adults. objectives: to develop a bia prediction equation for estimation of asm in communitydwelling older singaporean adults. methods: we studied healthy community-dwelling subjects (mean age . years) from the gerilabs- cohort. bia was performed using a single-frequency instrument. the reference method used for asm measurement was dual-energy x-ray absorptiometry (dxa). we first identified independent asm predictors by assessing the correlation of demographic, anthropometric and bia variables with dxa-measured asm. the best-fitting prediction equation was derived from these variables using stepwise (backward elimination and forward selection) linear regression with bootstrap validation. using asian working group for sarcopenia (awgs) cutoffs, we then compared anthropometric, strength and physical performance parameters between normal and low bia-derived asm groups. results: the derived bia equation incorporated predictorsimpedance index, weight, gender and body mass index (bmi), i.e. asm(kg) = . + ( . x impedance index) + ( . x weight) + (- . x gender) + (- . x bmi), where males = , females = and impedance index = height(cm )/resistance. the r and standard error of the estimate of this regression model were . and . kg respectively, with impedance index accounting for . % of its variability. individuals with low bia-derived asm have significantly smaller mid-arm and calf circumference and weaker grip strength, compared to individuals with normal bia-derived asm (p< . ). physical performance was similar in both groups. conclusion: we have developed a valid single-frequency bia prediction equation which can provide good estimates of asm in communitydwelling older singaporean adults. validation of this prediction equation in an independent sample of population is required to establish its accuracy and precision. ( ) faculty of sport sciences, waseda university, tokorozawa, japan) background: it has been well known that appendicular lean mass (alm) and skeletal muscle mass index (smi), which is the ratio of alm to height (m), is positively proportional to regional bone mineral density (bmd) in elderly men. however, there is limited information about these relationships in middleaged men. objectives: the purposes of this study were to investigate the difference in bmds (arms, lumbar spine, pelvis, legs, and subtotal: total body without head area) in middleaged men with low and normal smi (alm/height ≤ . kg/ m from asian working group for sarcopenia: awgs), and to determine the associations between alm, smi, and bmds. methods: three hundred and two middle-aged japanese men between and years of age participated in this study. alm and bmd measurements were taken using dual-energy x-ray absorptiometry (dxa, delphi a-qdr, hologic). results: based on the definition from awgs, the prevalence of low smi was approximately % in middle-aged men. the subjects with low smi (low smi group, n = , . kg/m ) had significantly lower body weight ( . vs. . kg), bmi ( . vs. . kg/m ), and fat mass ( . vs. . kg) compared to the normal group (n = , . kg/m ), although there were no differences in age ( vs. years), standing height ( . vs. . cm), and body fat percentage ( . vs. . %) between the two groups. bmds were significantly lower in low smi group than normal group for regional body parts (arms . vs. . g/cm ; lumbar spine . vs. . g/cm ; pelvis . vs. . g/cm ; legs . vs. . g/cm ) and subtotal ( . vs. . g/cm ). moreover, body weight, fat mass, alm, and smi were positively correlated with bmds using partial regression analysis controlling for age in all subjects, except for fat mass vs. lumbar spine bmd. in a stepwise multivariable model, alm was more closely related to bmds, except in the case of pelvis. conclusion: these results suggest that in order to maintain the regional bmd in middle-aged men, a key factor is to maintain or increase both alm and smi. background: the societies on sarcopenia have recently accepted the use of bioelectrical impedance analysis (bia) in the assessment of appendicular skeletal muscle mass (asm). several bia equations and devices have been introduced, which analyze the whole body composition, including the trunk and excluding the left arm and left leg at khz. it is necessary to measure the appendicular body segments of impedance parameters with a specific frequency (hz) that optimally analyze the muscle for valid assessment of asm. prior our study, literature-based bia equations and the two devices estimated asm at > % of r (coefficient of determination) with the significant constant-errors rated as «poor». objectives: thus, the aims of this study were ( ) externally cross-validate the equations and devices of bia on the appendicular skeletal muscle mass and ( ) develop valid equations based on appendicular bioimpedance parameters at the specific frequency (khz) that reflects the muscle for estimating asm; methods: community dwelling koreans over -year-old ( + . yrs, females and males) participated. asm was predicted using bia-based equations available in literature and bia devices and compared to dxa outcomes which is the gold standard. we conduct internal cross-validation and stepwise multiple linear regression to develop asmformulas with segmental multi-frequency bias. results: our new prediction formulas were developed by the appendicular impedance(z) index = height / (z of right arm + z of left arm + z of right leg + z of left leg)) at higher than khz and the appendicular reactance(xc) = xc of right arm + xc of left arm + xc of right leg + xc of left leg at khz. r s were over %, see wes under . kg of asm with the subject rating as «excellent» for men and «good» for women. conclusion: we found that our new protocol resulted in higher agreement with dxa and improved bia accuracy for this specific age group. clinicians can use this lower cost protocol and equations to better diagnose sarcopenia in larger cohorts with comparable to measurement of dxa. background: greater protein intake throughout the lifespan may be related to better body composition through the preservation of lean body mass during aging. objectives: we sought to determine whether an association between dietary protein intake (pi) and body fat percentage (bf) exists among women when controlling for dietary and lifestyle factors. methods: body composition and lean body mass were examined via dual-energy x-ray absorptiometry, grip strength (gs) was assessed using a hand grip dynamometer, and moderate-to-vigorous physical activity (mvpa) was measured by accelerometry. dietary intakes were estimated via threeday food logs and esha software. multiple linear regression and stepwise linear regression models were used. results: a total of women (mean ± sd; age . ± . years) finished all assessments. a full regression model (i.e., containing all covariates; r = . ; adjusted r = . ; f( , ) = . ; p < . ) was created using fat, carbohydrate, protein and leucine intake (g/day), protein quality (g/day of leucine over g/day of protein), energy intake (kcal/day), age (years), lean body mass (kg), bmi (kg/m ), gs (kg), and mvpa (min/day). only bmi (mean ± sem; beta = . ± . ; p < . ), gs (mean ± sem; beta = - . ± . ; p < . ), and pi (mean ± sem; beta = - . ± . ; p = . ) were significant to the full regression model. to verify their importance, a stepwise regression using the same variables was performed and resulted in a model (f( , ) = . ; p < . ; r = . ; adjusted r = . ) that included bmi (mean ± sem; beta = . ± . ; p < . ), gs (mean ± sem; beta = - . ± . ; p < . ), and pi (mean ± sem; beta = - . ± . ; p = . ). conclusion: greater protein intakes are associated with lower bf in women when controlling for various covariates. we theorize that greater protein intakes preserve lean body mass which results in improved body composition. more specifically, a one gram per day increase in dietary protein is predicted to decrease bf by . % when controlling for all other variables. background: muscle aging and the increased prevalence of obesity in the geriatric population create a new area of research: sarcopenic obesity. in prospective cohorts of nonhospitalized subjects, it is associated with an increased risk of developing physical limitation. hospitalization is an event with high risk of loss of independence. the impact of sarcopenic obesity during this episode isn't known yet. objectives: analyze the evolution of functional independence during a hospitalization in an acute geriatric ward, looking for a link between the presence of sarcopenic obesity and a decline of independence. early readmission, length of stay and changes in body composition during hospitalization were also examined. methods: prospective descriptive monocentric cohort study carried out in an acute geriatric ward of the pau hospital. sarcopenia was diagnosed using the european working group on sarcopenia in older people algorithm by an impedancemeter. a bmi over was used to report obesity. functional independence was rated on the adl katz scale. results: patients were included. sarcopenic obesity was diagnosed in . % of cases, sarcopenia and obesity in % and % of patients, respectively. the greatest variation in functional independence during hospitalization was observed in sarcopenic obese patients (mean variation of out of points, p= . ). a total of early readmission at month were counted, with the highest rate for sarcopenic obese ( %, but % at the sample level) (p= . ). the average length of stay was . days. conclusion: sarcopenia is common in patients hospitalized in geriatrics, and when associated with obesity, there is greater variation in functional independence and more readmissions. background: known that is sarcopenic obesity, excessive accumulation of adipose tissue is detected, with a decrease in muscle mass and strength, which is already over the age of years. modern diagnostic methods have their drawbacks for the diagnosis of sarcopenic obesity. bodpod quality and timeliness of diagnosis of signs of sarcopenia in obese patients is improved, which ultimately will contribute to an earlier targeted treatment of sarcopenia and an improvement in its prognosis. bodpod methodology can be recommended for use in complexes for the diagnosis of sarcopenic obesity. objectives: to compare the effectiveness of three methods of body composition assessment such as bioimpedans analysis (bia), air-replacement bodyplatismography (bodpod) and dual x-ray absorptiometry total body program (dxa total body) in the verification of reducing of skeletal muscle mass as sign of sarcopenic obesity in obese patients. methods: the study group included patients aged - y.o. (average age , ± , years) with bmi>= . kg/m . the control group included patients aged - y.o (average age , ± , years) of the same age without obesity with bmi . - . kg/m . body composition was tested using bia, bodpod and dxa with calculating fat, lean and skeletal muscles mass (kg) and % in all the patients. (bodpod) is the most sensitive in the verification of skeletal muscle mass reduction in obese patients. this method shows that patients with obesity have a significantly reduced muscle mass compared with normal weight or overweight subjects. background: in overweight and obesity excess energy and changes in body composition may favor the onset of metabolic derangements. combined with excess adiposity, the age-related decline in lean body mass can accelerate the development of insulin resistance and the consequences in terms of cardiovascular risk. objectives: the aim of our study was to investigate the association between the phenotype of sarcopenic obesity and cardio-metabolic risk in postmenopausal women. methods: postmenopausal women were recruited among subjects admitted to the high specialization centre for the care of obesity (casco), at the sapienza university, rome, italy. fat mass (fm) and fat-free mass (ffm) were assessed by dxa. obesity was defined as body fat >= %. appendicular skeletal muscle mass (asmm) was calculated. sarcopenia was defined as asmm/weight < sd than the sex-specific mean of a young population. the cut-point was asmm/weight< . . the lipid accumulation product was calculated: lap = (waist circumference cm - ) × triglycerides mmol/l]. the estimated glucose disposal rate (egdr) was calculated. high-sensitivity c-reactive protein (hs-crp) was measured. results: women were included (age: . ± . years, bmi: . ± . kg/m ). sarcopenia was diagnosed in . % of study participants. sarcopenic obese women were older than nonsarcopenic women ( . ± . vs. . ± . years, p= . ). lap was higher in sarcopenic obese women compared to their nonsarcopenic counterparts ( . ± . vs. . ± . , p= . ) after adjustment for age, body fat, and hs-crp levels. estimated gdr was significantly lower in sarcopenic obese women ( . ± . vs. . ± . , p= . ) after adjustment for age and body fat. an inverse association emerged between the index of sarcopenia, asm/weight, and lap (beta: - . * - , se: . * - , p= . ), independent of age, body fat, and hs-crp levels. a positive association was observed between asm/weight and egdr (beta: . * - , se: . * - , p= . ) adjusting for age, body fat, and hs-crp levels. conclusion: postmenopausal sarcopenic obese women exibithed a high lap and a low egdr, indicating increased cardiometabolic risk and decreased insulin sensitivity, respectively. l e a t h a a . c l a r k , , , todd m. manini , nathan p. wages , , janet e. s i m o n , , d a v i d w . r u s s , , b r i a n c . c l a r k , , , ( ( ) background: muscle weakness strongly contributes to mobility limitations and physical disability. the role of neural mechanisms contributing to age-related weakness have not been fully delineated to sufficiently target interventions that enhance strength and physical function in older adults. objectives: we sought to compare differences in voluntary inactivation and measures of motor corticospinal excitability in older adults with clinically meaningful muscle weakness compared to young adults and stronger adults without muscle weakness. methods: maximal voluntary isokinetic and isometric leg extensor strength, electrical stimulation of the leg extensors, and transcranial magnetic stimulation (tms) of the motor cortex were performed in older adults and young adults. outcome measures of leg extensor strength relative to body weight, voluntary inactivation (via), motor evoked potential (mep) amplitude and silent period (sp) duration during isometric leg extension contractions at %, %, and % of maximum voluntary contraction (mvc) were obtained. older adults were classified into three weakness groups based on previously established isokinetic leg strength/ body weight cut points (severely weak, moderately weak, or not weak). group differences were examined after controlling for sex. results: the older adults had % lower isokinetic strength/body weight when compared to the young adults. the severely weak older adults were % and % weaker than the moderately weak and older adults who were not weak, respectively. severely weak older adults exhibited higher levels of leg extensor via than older adults who were not weak ( . + . % vs. . + . %). severely weak older adults exhibited % longer sp's compared to the older adults who were not weak, but this difference was not statistically significant (p= . ). the severely weak older adults' mep's were approximately half the amplitude of the older adults who were not weak. regression analyses demonstrated that mep amplitude and sp duration -indices of hypoexcitability-were associated with relative strength. conclusion: weak older adults have significant deficits in their nervous systems' ability to fully activate their leg extensor muscles. additionally, motor corticospinal hypoexcitability is associated with age-related weakness, suggesting that interventions targeting the nervous system could be used to enhance muscle strength and prevent future health risks in older adults with muscle weakness. model. results: we evidenced oxidative stress in a mouse model of the pathology at different ages ( , and months) and aimed to identify the consequences of opa inactivation on redox homeostasis. increased ros levels were observed in cortices of the murine model opa +/-as well as in opa down-regulated cortical neurons. this increase is associated to a decline in mitochondrial respiration and an increase of antioxidant enzyme levels. upon exogenous oxidative stress opa -depleted neurons did not further up-regulated antioxidant defenses. finally, low levels of antioxidant enzymes were observed in fibroblasts from patients supporting their role as modifier factors. moreover, the simulations obtained with our mathematical model of complex i are able to reproduce biological experiments of quantification of ros production by complex i. conclusion: our study shows: (i) the prooxidative state induced by opa loss can be considered as a pathological mechanism (ii) differences in antioxidant defenses can contribute to the variability in expressivity and (iii) antioxidant defenses can be used as prognostic tools to gauge the severity and the evolution of the disease. (iv) furthermore, our mathematical model model of ros porduction by complex i will help to understand the dysfunctions of oxidative metabolism in opa gene related disorders. we will present the last results of our algorithm and wet laboratories experiments. amanika kumar, deepa m narasimhulu, michaela e. mcgree, amy l.weaver, aminah jatoi, nathan k lebrasseur (mayo clinic, rochester, mn, usa) background: patients with advanced ovarian cancer (eoc) are often frail and require multi-agent chemotherapy. objective: to evaluate the relationship between frailty and adjuvant chemotherapy tolerance and toxicity among women with advanced epithelial ovarian cancer. methods: women who underwent primary debulking surgery for stage iiic or iv eoc and received adjuvant chemotherapy at the same institution were identified. a frailty deficit index (fi) was derived from items representing comorbidities and activities of daily living. frailty was defined as a fi ≥ . . if data were unavailable for frailty index calculation, patients were excluded. relative dose intensity (rdi) for carboplatin and paclitaxel was calculated as the percentage of the standard dose that was actually administered and compared between frail and non-frail using the wilcoxon rank sum test. results: of the women who met inclusion criteria, . % ( / ) were frail. frail women were older ( . vs . years, p= . ), had a higher bmi ( . vs . kg/m , p= . ), and were more likely to have american society of anesthesiologists (asa) score ≥ ( . vs . %, p= . ) compared to nonfrail women. frail patients were less likely to complete cycles of adjuvant chemotherapy, ( % versus %, p< . ). despite the decrease in total cycles of chemotherapy, we did not observe significant differences in dose delays ( . vs. . %), dose reductions ( . vs . %), and severe neutropenia ( . vs. . %) between frail and non-frail women. we analyzed a subset of patients ( frail and non-frail) women received both intravenous carboplatin and paclitaxel. we observed that frail women were less likely to have a carboplatin rdi of % or higher ( . % vs. . %, p< . ) and less likely to have a paclitaxel rdi of % or higher ( . % vs. . %, p= . ). conclusion: frail women with advanced eoc undergoing adjuvant chemotherapy receive reduced rdi and are less likely to complete cycles of chemotherapy despite no increase in dose reduction, delays, and neutropenia. physician bias and patient choice may influence chemotherapy intensity decisions. further studies are needed to explore the association between frailty, chemotherapy, and survival. background: gait speed is a core component of physical frailty (pf) and, as a single measure, is correlated with important health outcomes, including mortality. immune dysregulation has been previously associated with pf -including increased il- production in peripheral blood mononuclear cell (pbmc) lipopolysaccharide (lps) stimulation assays. it is not known whether gait speed is associated with lps-stimulated cytokine production. objectives: this pilot study evaluated whether gait speed is correlated with dysregulated immune response in two populations of older adults undergoing procedures -knee osteoarthritis (oa) scheduled for knee replacement, and chronic kidney disease (ckd) approaching hemodialysis initiation. methods: older adults with ckd and older adults with knee oa underwent preoperative evaluation including gait speed (usual pace, -meter walk, best of two trials) and immune stimulation testing (in vitro, thawed pbmcs stimulated with lps at doses , . , and ug/ml, with il- quantified by elisa at , , , and hours; reported as area under the curve (auc)). correlation coefficient and p-value were calculated. results: for ckd, the il- auc of lps stimulated pbmcs was negatively associated with gait speed (lps . ug/ml r = - . , p= . ; lps ug/ml r= - . , p= . ). for oa, the correlation between il auc and gait speed was positively correlated for lps dose . ug/ml (lps . ug/ml r = . , p= . ; lps ug/ml r= . , p= . ). none of these associations were statistically significant. similar results were obtained when age was included as a covariate. conclusion: in people with ckd, increased cytokine production was correlated with decreased gait speed. in people with knee oa, results do not support this hypothesis. further studies with larger sample size are warranted. for participants with knee oa, future studies should account for severity of knee pain at time of gait speed assessment. background: skeletal muscle drives fuel utilization, and carbohydrate (cho) is a major fuel source. metabolic flexibility describes the ability to balance cho and fat oxidation efficiently in response to changes in metabolic demands or conditions. despite its role in long-term metabolic health, little is known about cho oxidation or metabolic flexibility in sarcopenic older adults. objectives: to examine resting metabolism and metabolic flexibility from a fasted to fed state after a cho-rich meal in sarcopenic versus nonsarcopenic older adults. methods: twenty-two men and women (age ± sd= ± y) were enrolled into this pilot study with either normal (non-sarcopenic, n= ) or low (sarcopenic, n= ) handgrip strength, gait speed and relative skeletal muscle index. resting metabolism was assessed in a fasted state at baseline, and metabolic flexibility was assessed after ( min, post-prandial) consuming a meal containing g of fat, g of protein, and g of a rapidly-digestible cho. respiratory quotient (rq), cho, and fat oxidation were measured with open-circuit spirometry, indirect calorimetry. fat and fat-free mass were measured with dual x-ray absorptiometry. blood glucose was assessed from venous samples using glucose oxidase methodology. results: rq was - % higher (p= . - . ) in sarcopenic participants throughout the experiment. after adjusting for fat-free mass, fat oxidation was % lower (p= . ), while cho oxidation was % higher (p= . ) at baseline for sarcopenic men and women. sarcopenic participants also exhibited delayed and limited (p< . ) postprandial increases in cho oxidation, despite greater (p< . ) increases in blood glucose. conclusion: sarcopenic individuals are more reliant on cho and less reliant on fat oxidation than non-sarcopenic adults, which is generally consistent with poorer metabolic health. when compared to non-sarcopenic adults, sarcopenia delayed and truncated cho utilization after a meal, indicating impaired metabolic flexibility in this population. impaired metabolic flexibility could be a mechanism underlying the losses of strength and physical function accompanying sarcopenia. anton de spiegeleer , , , hasan kahya , , nele van den noortgate , evelien wynendaele , tine decruy , srinath govindarajan , dirk elewaut (( ) unit for molecular immunology and inflammation, vib-center for inflammation research, ghent, belgium; ( ) department of geriatrics, faculty of medicine and health sciences, ghent university hospital, ghent, belgium; ( ) drug quality and registration (druquar) group, faculty of pharmaceutical sciences, ghent university, ghent, belgium) background: acute and chronic muscle wasting represent an important unmet clinical health problem. most pathophysiological studies suggest an effect of the immune system, primarily through catabolic cytokine productions such as il- . also endoplasmic reticulum (er) stress is considered to be an important pathway favouring muscle wasting. er stress in turn plays an important role in innate-like t cells, particularly invariant natural killer t cells (inkt cells), by controlling their cytokine production [govindarajan et al., nat. commun. ]. as such we reasoned that inkt cells may play a pivotal role in muscle homeostasis through their excessive cytokine production. previous studies have already highlighted the importance of these cells in a wide range of diseases such as cancer and metabolic disorders such as obesity. objectives: the aim of this study was to investigate the in vivo role of inkt cells in muscle homeostasis. methods: we compared wild-type (wt) versus inkt cell depleted mice (jα ko) for clinical, histological and gene expression differences in lower limb skeletal muscle. results: interestingly, we found that inkt cell depleted mice (jα ko) had a lower relative muscle weight, i.e. a muscle wasting phenotype, compared to wt mice. this clinical muscle wasting was associated with a decrease in oxidative enzymatic activity (succinate dehydrogenase histology). moreover jα ko mice showed a decreased transcription of genes involved in skeletal muscle growth and differentiation (follistatin and myogenin), sarcomere assembly (myosin- ) and neuromuscular junction function (neuronal acetylcholine receptor subunit alpha- ). conclusion: taken together, our results suggest a role for inkt cells in muscle wasting diseases and put innate-like t cells at the centre stage of immune cells controlling skeletal muscle biology. a r m a n d a t e i x e i r a -g o m e s , , s o l a n g e costa , , bruna lage , , dietmar fuchs , vanessa valdiglesias , , blanca laffon , joão paulo teixeira , ( ( ) background: frailty is a multidimensional geriatric syndrome characterised by increased vulnerability and functional decline that may be reversed if addressed early. it has been identified to be the most common condition leading to disability, institutionalisation and death in older adults. despite its known biological basis, no particular biological trait has been consistently associated with frailty syndrome so far. objectives: on this basis, the main objective of the present work was to evaluate the possible association between immunological: biomarkers and the frailty status in a group of community dwellers. methods: a group of older adults (>= years old) was engaged in this study. frailty status was assessed via fried's frailty model. the levels of several immune activation molecules -neopterin, tryptophan, kynurenine -were analysed. results: the classification of the study population was . % robust, . % pre-frail and . % frail. no significant differences were found between robust and pre-frail groups regarding serum concentrations of neopterin. although, the kynurenine/tryptophan ratio was significantly higher in pre-frail individuals as compared with robust subjects. conclusion: the preliminary data obtained suggest the activation of immunobiochemical pathways and are in agreement with previous studies that report alterations of the immune response in frail older adults. nevertheless, further investigation is encouraged and required to consistently demonstrate these findings. in future studies physical activity, nutritional, psychological, sociological and clinical features should also be considered when evaluating changes in immune biomarkers and frailty. the work developed by armanda teixeira-gomes and solange costa is supported by fct under the grants sfrh/bd/ / and sfrh/ bpd/ / , respectively. vanessa valdiglesias was supported by beatriz galindo research fellowship beagal / . background: frailty and hemoglobin count, above what would be considered clinical anemia, are two common findings in older patients and lead to an increased risk of negative health outcomes. objectives: evaluate whether hemoglobin concentration is an independent predictor of frailty and investigate possibe causal pathways in particuliar the relationship between inflammation and nutrition with hemoglobin concentration. methods: communitydwelling participants aged years or older who visited the toulouse frailty clinic between and were included in this analysis. patients underwent a comprehensive geriatric assessment and had a blood sample. a series of multivariate logistic regression models were perfomed after minimizing potential influence from age, gender, kidney function, inflammation, cognition, nutritionnal status and certain socioeconomic factors. results: hemoglobin count and frailty are significantly associated after minimizing potential influence from other covariates (p< . ). an increase in one point of hemoglobin concentration is associated with a % risk decrease of being frail (or= . , %ic= . - . ). there were no evidences of significant impact of inflammation and nutritional status in the relationship between hemoglobin concentration and frailty status (p> . ). conclusion: hemoglobin concentration is strongly associated with frailty in older adults. these results can have potentially important implications for prevention policies targeting frailty, by identifying potential patients with high risk of adverse outcomes and functional outcomes. juliette tavenier , line jee hartmann rasmussen , jan nehlin , morten baltzer houlind , aino leegaard andersen , ove andersen , janne petersen , , anne langkilde ( ( ) background: chronic inflammation is thought to be involved in the development of frailty. we hypothesized that increased monocyte inflammatory activity plays a role in chronic inflammation and thereby in frailty. objectives: to study the potential role of chronic monocyte inflammatory activity in frailty. methods: two groups of elderly adults (>= years) were included: patients with a recent admission to the emergency department (ed) and age-and sex-matched controls, without recent ed admission. data was collected at baseline and after year. participants were considered frail if they had or more of the following: hand grip strength ≤ kg for men or ≤ kg for women, gait speed ≤ . m/s, unintentional weight loss of > kg within the last months. frailty was also assessed using the frailty index (fi)-outref. we measured cognitive function (mini mental state examination -mmse) and chronic inflammation (soluble urokinase plasminogen activator receptor -supar). monocyte inflammatory activity was assessed by nf-κb phosphorylation (pnf-κb) using flow cytometry. results: participants had a mean age of . years (range: . - . ) and % were women. preliminary results show that at baseline, the patient group had a greater proportion of frail individuals compared to the control group ( vs. , p< . ). fi-outref was on average . points higher (p< . ) and supar levels % higher (p< . ) in the patient group, however, there was no difference in mmse score between the groups (p= . ). at year, although the proportion of frail individuals decreased in the patient group, it was still greater than in the control group ( vs. , p= . ). fi-outref remained elevated in the patient group (p= . ), but there was no difference in supar levels (p= . ). pnf-κb was positively associated with age in the control group (p= . ), but not in the patient group (p= . ). pnf-κb was % higher in the patient group compared to the control group (p< . ), and this was unchanged when adjusting for frailty, supar, and mmse. conclusion: the patient group was more frail and had elevated monocyte inflammatory activity compared to the control group. however, none of the frailty measures were confounders for the difference in monocyte inflammatory activity between groups. background: aging is most often accompanied by a loss of body weight: a decrease of fat deposits and muscle body weight. body mass index (bmi) in adults is considered normal if it is in the range of . to . kg / m (according to the who classification). bmi is widely used in the diagnosis of obesity. the association of bmi and cardiovascular and cerebrovascular diseases is known. objectives: the purpose of research is to identify the relationship of bmi with physical abilities and cognitive functions in long-livers. methods: long-living subjects aged . ± . years were examined. in long-livers, height, body weight were measured, calculated bmi. the level and direction of cognitive disturbances was determined by the mmse test (mini mental state examination). physical abilities were determined by the questionnaire and physical tests (tests the muscular strength in forearms and of the hands, chair stand test). results: bmi in long-livers had a normal distribution. the median bmi was . kg / m , the minimum value was . kg / m , and the maximum value was . kg / m . . % of long-livers had a bmi ranging from . to . kg / m . . % of long-livers have lost weight during the past year, including . % by kg or more. . % of long-livers could stand up of the chair. however, only . % of long-livers were able to complete the test correctly. amongst them, . % had a normal bmi. indicators of muscular strength in forearms and of the hand in long-livers who completed the chair stand test were significantly higher compared to long-livers who did not completed the chair stand test (r = . , p < . ). bmi had a positive correlation with the ability of a long-lived to wash without anyone's help (r = . , p < . ), go up and down the stairs (r = . , p < . ), do light housework (r = . , p < . ). mmse indicators also positively correlated with bmi (r = . , p < . ). the average mmse . ± . was observed with average bmi . ± . . conclusion: against the background of a decrease in the bmi indicator in long-livers, a decrease in physical abilities and cognitive functions is observed. however, there is a problem in determining the boundaries of the ratio of height and body weight for elderly people. in all likelihood, there are not linear, but more complex dependencies between bmi and functional abilities of long-livers. suparb aree-ue , inthira roopsawang , jansudaphan boontham , surinrat baurangtheinthong , yuwadee phiboonleetrakun (( ) ramathibodi school of nursing, faculty of medicine ramathibodi hospital, mahidol university, bkk, thailand; ( ) faculty of graduate studies, mahidol university, bkk, thailand) background: depressive symptom results in increasing poor outcomes and care dependency in older adults. the prevalence of depressive symptoms is common with its associated multiple factors. however, this conundrum problem is underestimated, particularly in older people living in rural areas. to promote healthy aging, understanding of the conundrum problem is essential in strengthening care quality and enhancing the quality of life in this population. objectives: to determine the relationships of the number of medication use, pain, frailty, and locomotive syndrome and their effects on depressive symptoms among community-dwelling thai older adults. methods: a cross-sectional study was employed. the sample consisted of community-dwelling thai older adults who met the inclusion criteria. data were assessed by using demographics questionnaire, thai version -question geriatric locomotive function scale: glfs- ; numeric rating scale; the reported edmonton frailty scale: refs-thai version; and the -item geriatric depression scale, tgds- . a path analysis was employed to determine the pathways linking the number of medication use, pain, locomotive syndrome, frailty to influence depressive symptoms. results: there were significant positive direct paths from pain (beta = . , p <. ) to locomotive syndrome and from locomotive syndrome to the number of medication use (beta = -. , p <. ). an inversely, the locomotive syndrome was a negative significant direct to depressive symptoms (beta = -. , p <. ). pain had an indirect effect on depressive symptoms (beta = -. , p <. ). additionally, the model explained . % of the variability in depressive symptoms. conclusion: the locomotive syndrome is a major factor influencing depressive symptoms. the complex relationship among pain, number of medication use, locomotive syndrome, and depressive symptoms should be taken into account for designing an appropriate intervention to reduce depressive symptoms among community-dwelling thai older adults. background: total knee arthroplasty (tka) is a clinical curative treatment for severe knee osteoarthritis. however, the outcomes are differences in each patient's perception. preoperative patients' expectations to functional abilities are one of important factors influencing on postoperative outcomes and satisfaction. objectives: to investigate the association among preoperative patients' expectations, postoperative functional abilities, and satisfaction to functional abilities among older adults undergoing tka at -week after surgery. methods: participants were older adults who were diagnosed with knee osteoarthritis and required to receive tka at a university hospital in bangkok, thailand. the sample was purposely selected based on the following criteria: were aged years or over, received tka for the first time, and had no cognitive impairment. the data were collected at preoperative and postoperative tka by using the demographic data questionnaire, the hospital for special surgery knee replacement expectations survey, and the knee and osteoarthritis outcome score in the part of function in daily living (koos adl) thai version. the data analysis was performed by using descriptive statistics, paired t-test, and pearson product moment correlation coefficient. results: before surgery, patients' expectations to postoperative functional abilities had a high level with the total mean score of . (sd = . ), and the item of improving ability to walk in a short distance was rated as the highest expectation. at -week after surgery, the overall functional ability had a significant improvement (t = - . , p = . ). satisfaction to functional ability also had a high level (mean ± sd = . ± . ), and the improving ability to walk in a short distance item had the highest. patients' expectations to functional abilities had a significantly low positive correlation to postoperative functional ability and satisfaction (r = . , p < . ; r = . , p < . , respectively). moreover, there was a significant moderate positive correlation between functional abilities and satisfaction to functional abilities (r = . , p < . ). conclusion: a better understanding of expectations may be beneficial in gaining knowledge, paving expectations on possible outcomes, and developing trust resulting in enhancing quality of care for thai older adults undergoing tka. background: identifying low muscle strength is a key step in many operational definitions of sarcopenia including the one recently proposed by the european working group on sarcopenia in older people- (ewgsop ). grip strength is widely used to identify people with low muscle strength. however, it is unclear what impact variation in the type of hand-held dynamometer used to measure grip strength has on the prevalence of low muscle strength. objectives: we aimed to assess the impact of estimated differences of between and kg in the measurement of grip strength when using different types of hand-held dynamometer on the case-finding of low muscle strength. methods: study participants were men and women aged - from a randomised, repeated measurements cross-over trial. maximum grip strength was assessed using four hand-held dynamometers (jamar hydraulic; jamar plus+ digital; nottingham electronic; smedley) in a randomly allocated order. ewgsop recommended cutpoints (< kg men; < kg women) were applied to estimate prevalence of low muscle strength for each device. agreement between devices was assessed using kappa statistics. results: prevalence of low muscle strength varied by dynamometer type ranging between % and % for men and, % and % for women. of the men identified as having low muscle strength by at least one of the four dynamometers, only % were identified by all four and % by just one. of the women classified as having low muscle strength by at least one of the four dynamometers, only % were identified by all four and % by only one. when comparing pairs of devices, kappa statistics ranged from . to . suggesting poor to moderate agreement. conclusion: case-finding of low muscle strength is influenced by the type of hand-held dynamometer used. it is important to identify the sources of variation in the measurement of grip strength and consider the implications of these for sarcopenia. further research is required to understand how best to standardise the assessment of each of the different components of commonly used operational definitions of sarcopenia and take account of sources of variation in these measures where standardisation cannot be achieved. background: sarcopenia is characterized by a progressive loss of skeletal muscle mass and strength associated with mortality and severe adverse events on health. for a healthy aging, the quality of life (qol) is essential and it is associated to autonomy of persons, social relations, and socioeconomic factors. objectives: to compare the qol of chilean older people with sarcopenia living in santiago de chile, according to an adapted version of the european working group on sarcopenia. methods: community-dwelling older people (mean ± sd: . ± . years; . % females) were interviewed, registering self-reported chronic diseases and the questions of short-form- health survey (sf- ). anthropometry, dynamometry and physical performance were measured. qol was measured using sf- , validated in chilean older adults. norm-based score of subscales and two summaries components -mental and physical (mcs and pcs; respectively)-were calculated using the chilean-specificscoring for older people. low score was defined as having a score ≤ th percentile of mcs and pcs. logistic regressions were estimated. results: sarcopenia was identified in . % of the sample ( . % women; . % men; p= . ). the average score of the subscales were significantly higher in non-sarcopenic adults than sarcopenic. the average of mcs and pcs were also significantly higher in non-sarcopenic adults than sarcopenic (mcs: . vs . ; p= . ; respectively; pcs: . vs . ; p< . ; respectively), and were significantly higher in men than women non-sarcopenic (mcs: . vs . ; p= . ; respectively; pcs: . vs . , p= . ; respectively). there were non-significant differences in sarcopenic adults by sex. logistic regressions demonstrated an association between sarcopenia and low mcs and pcs (or = . ; %ci: . - . ; or = . ; %ci: . - . ; respectively), adjusted by age, sex, multimorbidity, body mass index and lean/fat mass ratio. conclusion: sarcopenia was associated with a worse quality of life, which shows the impact of this pathology and the importance of developing programs for its prevention, delay or reversal. funded by fondef i p -munich sarcopenia registry (idsar): first results. uta ferrari , , marina schraml , ralf schmidmaier , , navina röcker , , sigrid adler-reichel , , christian lottspeich , , martin bidlingmaier , , benedikt schoser , , sabine krause , , martin reincke , , michael drey , (( ) department of medicine iv, university hospital, lmu munich, germany; ( ) friedrich baur institute at the department of neurology, university hospital, lmu munich, germany; ( ) preventive geriatrics study group, germany) background: since sarcopenia can be coded as disease in germany (icd-gm . ). in the same year we established the first sarcopenia registry linked with a biobank to identify modifiable, crucial risk factors for sarcopenia and its adverse outcomes. objectives: objectives of the registry are (i) how to optimize and standardize the diagnosis over in-and outpatient settings for musculoskeletal health, (ii) identification of clinical and molecular modifiable risk factors (iii) improvement of interdisciplinary treatment and prevention of sarcopenia as a new icd-code-based geriatric syndrome here we present the design as a practical approach for diagnosis in out-and inpatient care and a first descriptive analysis of influencing factors and comparison between in-and outpatients data. methods: patients older than years of age from outpatient clinic and acute geriatric ward at munich university hospital were consecutively screened by the sarc-f questionnaire. patients with high risk (sarc-f score >= ) were further assessed for sarcopenia in line with the european consensus definition (ewgsop ). among further factors assessed in the registry, we retrieved presence of further comorbidities, daily medication, nutritional status, sppb, frailty, and quality of life. results: at time of analysis, patients have been screened and within the first patients with high risk ( % women) % had sarcopenia. patients screened positive for sarcopenia have lower quality of life, even in a subclinical condition (mean euroqol (eq d-vas) = . ± . ). lower bmi ( . ± . , p= . ) and sex (p= . ) were statistically significant different for sarcopenia status, but not age (mean . ± . years, p= . ) or number of medication (p= . ) and comorbidities (p= . ). but the latter two were the most significant factors for inpatient status (both p< . ). the results underline the need for an early screening for sarcopenia in all patients older than years of age, suggested by hand grip strength in inpatients and sarc-f for outpatients. sex differences and further laboratory factors are necessary to add in sarcopenia diagnosis for precision medicine approaches. hospitalised older adults. we consider acute sarcopenia to be the last remaining acute organ insufficiency, with potentially devastating impact on function. characterising this condition will enable development of targeted interventions to ameliorate these changes. mobility disability, and incident mobility disability over . + . years. factor was associated with incident and prevalent mobility disability only, and factor was associated with only prevalent mobility disability. conclusion: muscle mass by d cr co-segregated with strength and physical performance measures, and together was associated with mobility and disability outcomes in older men. body composition measures (including dxa alm) did not co-segregate with strength and physical performance measures and together was associated with only mobility disability. background: currently, there are no registered drug treatments for the loss of skeletal muscle mass, strength and function that occurs during sarcopenia and cachexia. moreover, they are only limited relevant pharmacological screening options available. objectives: to improve in vitro pharmacological screening options, we developed a model of muscle wasting using donor primary muscle cells and our myoscreen™ platform that generates standardized myotubes for high-throughput phenotypic screening (young et al., slas discov. ( ) : - ). methods: myoblasts from four donors aged , , and years were compared in terms of proliferation, differentiation, size of formed myotubes and achr cluster formation using imaging and high content analysis. we then established an assay for muscle wasting: in each of the four donors various molecular pathways implicated in the pathogenesis of sarcopenia were activated using tnfa, tgfb or dexamethasone. results: myotubes formed from elderly patient's myoblasts displayed a reduced capacity to proliferate and differentiate, thinner myotubes and fewer acetylcholine receptor clusters. therefore, myotubes cultured using the myoscreen system continue to reflect age-related properties of donor muscle. interestingly, we also found that myotube sensitivity to atrophy stimulation increased with increasing age. myotubes were then co-incubated with growth/ repair factor igf- or hdac inhibitor, trichostatin a (tsa). both agents attenuated tnfa-induced myotube atrophy and differentiation inhibition in a dose-dependent manner. the extent of fusion index and myotube size increase was highest in myotubes from elderly subjects while myotubes from young subjects were more resistant to the protective effects of igf- and tsa. conclusion: myoscreen can be exploited to quantify age-dependent modifications in skeletal muscle fibers in vitro and identify candidate compounds that counteract the muscle wasting phenotype. andreas friedberger , alexandra grimm , wolfgang kemmler , klaus engelke , (( ) institute of medical physics, friedrich-alexander-universität erlangen-nürnberg, erlangen, germany; ( ) department of internal medicine; ( ) friedrich-alexander-universität erlangen-nürnberg and university hospital erlangen, erlangen, germany) background: sarcopenia is characterized by a progressive loss of skeletal muscle mass, which is infiltrated by adipose tissue. dual energy x-ray absorptiometry can only differentiate overall lean and fat mass. a local muscle analysis requires d imaging like magnetic resonance imaging (mri). usually, t weighted images are used for a visual grading of the amount of intermuscular adipose tissue (imat). however, a quantitative analysis requires segmentation of the fascia lata (fl, deep fascia of the thigh). objectives: our aim was to develop a highly reproducible d segmentation method in oder to quantify imat and the fat fraction of the thigh muscles using a combination of t weighted turbo spin echo (t wtse) and corresponding pt turbo spin echo (tse) dixon fat fraction (ff) images. methods: mri scans were acquired on a t scanner (magnetom skyrafit siemens) at the midthigh (length cm, slices, voxel size t w . x . x . mm³, dixon . x . x . mm³). since the fl is difficult to detect in the ff images, the t wtse images were used for segmentation. this process involved several steps, starting with a fuzzy c-mean clustering followed by several filtering steps to enhance d surface like structures representing the fl. finally, a level set algorithm was applied to obtain a closed d surface. if necessary, results were corrected manually. segmented masks were transferred from the t w to the ff images by rigid registration. imat was then segmented using a threshold determined from the histogram of the ff values within the intra-fascia region. sarcopenic ( ± y) and healthy ( ± y) male subjects were analyzed by three operators once (interoperator reproducibility) and three times by one operator (intraoperator reproducibility). results: inter-and intra-operator variability results of imat are shown in the table as mean / root mean square of the standard deviation (rms-sd) in units of the measured variable / coefficient of variation (rms-cv) in %. overall precision was excellent with errors below . %. conclusion: a semi-automatic d segmentation for the fascia of the thigh was developed. the operator impact on imat was almost negligible. background: sarcopenia a muscle disease that causes muscle mass loss and weakness. the calf circumference is a good screening test for sarcopenia in older adults in primary care. the most commonly used cutoff point is cm, but it is derived from north american studies and it may not be adequate for screening different populations that have lower height, weight and bmi. objectives: the objective of this study was to determine the ideal cutoff point for calf circumference for sarcopenia in community-dwelling older people in northeastern brazil. methods: this was a cross-sectional study of community-dwelling older people with a mean age of ± years ( % women). data on sociodemographics, anthropometrics, grip strength, gait speed, and skeletal muscle mass (bioimpedance) were collected. sarcopenia was assessed based on the diagnostic criteria suggested by european working group on sarcopenia in older people (ewgsop ). the area under the roc curve (auc) was calculated for different calf circumferences to identify the best cutoff point to determine sarcopenia among the participants. results: the prevalence of sarcopenia was %. the most appropriate calf circumference cutoff point was cm, with an auc of . , % sensitivity and % specificity. conclusion: it was found that the most appropriate calf circumference cutoff point to diagnose sarcopenia in older northeastern brazilians was cm. this is a more accurate cutoff point and will reduce the number of false positives and optimize health services in brazil. background: osteosarcopenia is a new geriatric syndrome defined as the presence of both sarcopenia and osteopenia or osteoporosis. this musculoskeletal disorder is related to higher prevalence of disabilities, falls and fractures and higher risk of mortality among community-dwelling older adults. therefore, the early diagnosis of this condition must be considered in order to reduce costs and negative impact on function. objectives: to explore the use of the infrared spectroscopy as a potential screening tool for osteosarcopenic older women (>= years old). methods: sarcopenia was identified by observing the presence of both reduction of muscle strength (grip strength) and mass (appendicular skeletal muscle mass) as suggested by the revised algorithm of the european working group on sarcopenia in older people ( ). reduction on bone mineral density was identified through bone densitometry and a t-score of <- , was adopted to classify the older women as osteopenic/osteoporotic. infrared spectroscopy through attenuated total reflection-fourier transform infrared spectroscopy (atr-ftir) was used to collect the sample information and to perform a multivariate analysis model. vibrational spectrum was obtained from serum. six samples of each group (osteosarcopenic and non-osteosarcopenic) were used to test the model and thirteen ostesarcopenic samples and fifteen non-osteosarcopenic samples were used for training. results: the most suitable model was the ga-svm with an accuracy of . %, % of sensibility and . % of specificity to differ osteopenic to non-osteopenic women. the more important selected variables found in the model were at the spectral regions: ~ cm- for carbs, ~ to cm- for nuclei acids and ~ to cm- for proteins. conclusion: infrared spectroscopy may be a promisor future method to early and easily diagnosis osteosarocopenia and prevent the harms this health condition may cause to the elderly population and minimizing costs to treat them. background: the modified european working group on sarcopenia in older people (ewgsop- ) algorithm to identify older people with sarcopenia contains three steps after initial clinical suspicion. the chair stand test, also known as the fivetimes sit-to-stand test ( sts), is one of two tests that can be used to assess muscle strength. the sts is also a component of the short physical performance battery (sppb), which is used as a measure of severity in the ewgsop- algorithm. objectives: the objective of this study was to determine whether the sts could be used to assess both muscle strength and physical performance in the ewgsop- algorithm to detect sarcopenia. methods: one hundred and ten older people aged . ± . years participated in the study. all participants were evaluated using the sppb score, as well as the timed-upand-go (tug). the ewgsop- algorithm specifies cut-off points of ≤ points on the sppb, ≤ . m/s for gait speed, and ≥ s for the tug. each participant was classified for tug and gait speed using the ewgsop- cut-offs, with stepwise discriminant function analysis used to predict the classification of participants. the remaining participants were used for cross-validation. prediction of sppb classification used the sts score in combination with predicted balance and sppb gait scores from stepwise linear regression. the total sppb score obtained using this method was used to predict sppb classification for the ewgsop- cut-off for sppb. results: the sts scores were able to predict tug and gait speed classification with % and % accuracy, respectively for the learning set of participants. the predicted sppb score had a classification accuracy of %, with % sensitivity and % specificity. when the remaining participants were evaluated, the sppb classification was correctly predicted for participants ( %), with % sensitivity and % specificity. conclusion: the sts can be used to accurately predict sppb classification in the ewgsop- algorithm to detect sarcopenia, meaning that the sts test could be used as a standalone test in an initial screening for sarcopenia. barrientos-calvo (nutritional support department and geriatric department, geriatric national hospital , san josé, costa rica) background: obesity is a disease characterized by increased adiposity with negative impact on patient health. aging process is associated with a progressive loss in muscle function, that may lead to functional decline and frailty. there are only few studies that have compared the prevalence of sarcopenia and dynapenia in obesity. objectives: the aims of this study were to determine the prevalence of sarcopenic and dynapenic obesity in elderly using the european working group on sarcopenia in older people criteria. methods: we conducted a cross-sectional study that included elderly patients with obesity from the obesity clinic since january to june . sarcopenia was defined according to the european working group on sarcopenia in older people (ewgsop ) criteria, and obesity with body mass index (bmi) > kg/m . handgrip strength was assess using a hydraulic dynamometer (jamar). bioimpedance analysis (bia) was performed. results: we evaluated persons, but only had bia data ( %). a total of older ( . ± years), % were women. mean body mass index, waist circumference, weight and calf circumference were . ± . kg/m , . ± . cm, . ± . kg and . ± . cm respectively. all patients had elevated body fat (mean %) and % had abdominal obesity. patients showed higher frequency of hypertension ( %), diabetes ( %), dyslipidemia ( %). sedentary was present in % and falls in %. mean handgrip strength and muscle mass for men and women were . ± . kg; . ± . kg and . ± . kg; . ± . kg respectively. there were ( . %) individuals fulfilling criteria for sarcopenic obesity, all women. but, dynapenic obesity was present in . % men and % women. conclusion: although the loss of muscle mass is associated with the decline in strength during aging, the decline in strength is more prevalent than the loss in muscle mass in our obeses. a large difference in prevalence of the two conditions was observed, sarcopenia obesity . % and dynapenic obesity %, respectively. barrientos-calvo (nutritional support department and geriatric department, geriatric national hospital, san josé, costa rica) background: sarcopenia is a geriatric syndrome characterized by progressive and generalized loss of skeletal muscle mass, strength, and function. several operative definitions for sarcopenia have been proposed over the past two decades. objectives: the aim of this study was to determine the prevalence of sarcopenia in costa rican longevity and health aging study (creles) using the ewgsop and ewgsop criteria. methods: to carry out the analysis, all the available cases of the creles study database in which belong to the cohort that follows in the period - were used. we analyzed community-dwelling older adults. low muscle mass was assessed using calf circumference < cm and low strength if < kg in men or < kg in women (ewgsop) vs < kg in men or < kg in women (ewgsop ). results: according to the ewgsop . % of the participants had sarcopenia, while according to the ewgsop sarcopenia was present in , % of participants. there was an increasing trend of sarcopenia by age group, it was more prevalent in women. mean handgrip strength was , kg in men and , kg in women with sarcopenia. mean calf circumference was , cm. sarcopenia was positively associated with age (or= . ; ci: . - . ), incomplete primary education (or , ; ic , ) , perceived as unhealthy (or , ; ic , - , ), antecedent of ischemic vascular event (or , ; ic , - , ), arthritis (or , ; ic , - , ), and falls ( r , ; ic , - , ). conclusion: the overall prevalence sarcopenia were significantly lower in ewgsop . prevalence of sarcopenia varies widely depending on the grip strength cut-off points applied. based on a -hour dietary recall, and poorer nutritional status as determined using must compared to their non-sarcopenia counterparts (all p<= . ). conclusion: the high prevalence of sarcopenia in community-dwelling older people who are at risk of malnutrition highlights the importance to devise targeted exercise and nutrition interventions to improve muscle health, physical performance and nutritional status. these interventions are essential to reduce the risk of progression to frailty and disability in this population group. v i n c e n z o m a l a f a r i n a , l e t i z i a s u e s c u n p u e r t a , a r a n t z a z u b i a i n u g a r t e , i ñ a k i a r t a z a a r t a b e , virtudes niño martín ( ( ) s o p h i e g u y o n n e t , c a t h e r i n e t a k e d a , philipe de souto barreto , yves rolland , sandrine andrieu , bruno vellas and the inspire study group ( ( ) background: the new geroscience field should not only be focusing on preventing age-related diseases, but should investigate the optimal maintenance of intrinsic capacity (ic): mobility, cognition, psychological, vitality and sensorial (hearing and vision) capacities as defined by the w.h.o. a better understanding about how to measure biological aging is an indispensable step that may lead to the definition of the best putative markers of aging capable of predicting healthspan. objectives: the main objective of inspire bioresource research platform for healthy aging is to build a comprehensive research platform gathering biological, clinical (including imaging) and digital resources that will be explored to identify robust (set of) markers of aging, age-related diseases and ic evolution. methods: the inspire platform will gather clinical data and biospecimens from subjects in the occitania region of different ages (from years or over -no upper limit for age) and functional capacity levels (from robust to frail to disabled) over years (inspire human translational research cohort). data are collected annually. between two annual visits, ic domains are monitored (with or without the help of a caregiver) each -month. once ic declines are confirmed, participants have a thorough clinical assessment and blood sampling to investigate the response of markers of aging at the time declines are detected. biospecimens includes blood, urine, saliva, and dental plaque that are collected from all subjects at baseline and then, annually. nasopharyngeal swabs and cutaneous surface samples are collected from all subjects at time-points (baseline visit and follow-up visits at m , m , m , m and m ). feces, hair bulb and skin biopsy are collected optionally at the baseline visit. results: recruitment started in october for a two years period. the identification of markers of aging will take advantage of three complimentary approaches to look for the best markers of aging: without a priori approach (transcriptomics, proteomics, lipidomics); semi a priori approach (metabolism, inflammation, cell cycle, mitochondrial network…); and targeted approach (pre-identified targets). the inspire platform will also aim to develop an integrative approach to promote novel new technologies for the assessment and monitoring of functional capacities. *acknowledgments: the inspire plateform is supported by grants from the occitania region and the european regional development fund (erdf), and co-funding by the apoc, the ctad, and the edenis, korian, pfizer, and pierre fabre groups. the promotion of this study is supported by the university hospital center of toulouse. background: energy balance is usually regulated by silent information regulator related enzyme (sirt ) and adenosine monophosphate-activated protein kinase (ampk). caloric restriction (cr) can postpone the pathological process of aging-related diseases and has a neuroprotective effect on nervous system degenerative diseases, but the mechanism is complex and not yet fully elucidated, although some of the cr effects may be mediated by sirt and ampk. objectives: to evaluate the beneficial effects of a cr diet on learning and memory ability. methods: six-week-old male c /bl mice were fed ad libitum for week before the experiment began. animals were weight-matched and randomly divided into three different groups: normal control group (nc group, n = ), high-energy group (he group, n = ), and cr group (n = ). the energy of nc diet, he diet and cr diet caloric ratio was : . : . . the total experimental duration was months. results: cr improved spatial learning and memory ability and decreased body weight and serum glucose. nissle staining showed the cell density was significantly decreased in the he group and increased in the cr group. cr decreased the expression of insulin signal pathway-related proteins such as igf- , ir, irs- , pi k, akt/pkb, and p-creb. more sirt -immunoreactive cells and fewer mtor-and s k immunoreactive cells were observed in the hippocampal in the cr group than in the nc group. cr decreased hippocampal mtor and s k protein activation and mrna expression. the expression of beclin , lc and cat b was increased and p was decreased in the cr group. the number of gfap-positive and iba- -positive cells in the cr group was significantly reduced compared to the nc group. conclusion: cr may prevent age-related learning and memory impairment via suppression of pi k/akt pathway and activation sirt / ampk/ mtor pathway in brain. background: head-down ( °) bed rest (hdbr) is a wellaccepted model to understand the pathophysiology of disuseinduced sarcopenia. human centrifugation as a measure to counteract muscle wasting during spaceflight is discussed. previous studies have observed decreases in maximal voluntary contraction force of the knee and hip-extensors of up to % following weeks of hdbr. muscle force is regulated by the recruitment of motor units (mus) and the modulation of mu firing rate. objectives: the aim of this study was to assess whether long-duration hdbr alters motor unit properties as one cause for disuse induced sarcopenia and whether human centrifugation can attenuate this decrement. methods: twelve healthy participants ( . ± . yr; ± cm & . ± . kg) were confined to -days ° hdbr in the frame of the first campaign of the agbresa bedrest study. eight received mins of artificial gravity (ag) daily via human centrifugation whereas four belonged to a control group. estimations of mu number (munix) and size (musix) in the abductor digiti minimi (adm) and tibialis anterior (ta) muscles were made using the motor unit number index method from on day preceding bed rest (bdc ) and on days (hdt ) and (hdt ). mean compound muscle action potential (cmap), munix and musix as a percent change from bdc were compared using repeated-measures anova, where muscle and time were ascribed as within-group factors and intervention a between-group factor. significance was denoted by p< . . results: both cmap and munix were unaltered over time in both muscles, irrespective of the intervention. although musix was also indifferent over time for both muscles, a significant muscle*time interaction was observed, indicating that the changes over time differed between the two muscles. conclusion: the preliminary data from the ongoing study indicate that neurodegeneration due to bedrest might affect muscles differently. there does not seem to be an effect of ag on mu number. analyses have to be repeated when the study is completed with a larger number of participants. additional histological and biochemical data will give further insight in the pathophysiology. living. soumaya msaad , geoffroy cormier , guy carrault (( ) univ rennes, inserm, ltsi -umr , f- rennes, france; ( ) neotec vision , rennes , france) background: several models have been proposed for elderly frailty detection. there is a consensus on two of them: the fried model, and the rockwood model. however, daily monitoring of the elderly is impossible with these models, whereas it is very important to detect any change as soon as possible to prevent dependency, since frailty is reversible only if early detected. objectives: the objective of this study is to propose a non-intrusive and low-cost method that anticipates frailty using depth images. crucial hypotheses are that regularity of daily activities is important for the elderly and that any prolonged change is considered as an indicator of frailty. methods: the proposed method consists in three steps: ) extraction of parameters from depth images: lying and sitting time percentage during the day, walking speed, and number of falls, visits, and exits. ) classification of the daily state using logistic regression and the extracted parameters. the daily state is considered as normal if the daily routine is maintained and abnormal if it is broken. ) computation of the weekly percentage of maintaining routine based on the classification of the nature of the day. results: tracking frailty is a difficult task that requires recording data over several months. as real data has not been collected yet, the feasibility of our approach was assessed on simulated data. in the latter, we reproduced variations of the parameters we would have extracted from real images of a patient after investigating his or her daily life. the classification of the days (normal/abnormal) led to an accuracy of % (training dataset: days, test dataset: days). a patient is considered frail when the weekly percentage of maintaining routine decreases steadily. conclusion: the preliminary results prove that in addition to being non-intrusive, a depth-imaging based approach can be a promising tool for frailty detection. anna franke , ellen freiberger , robert kob simon moskowitz , david w. russ , , leatha a clark , , , nathan p. wages , , dustin r. grooms , , brian c. clark , , ( ( ) background: one putative mechanism explaining mobility limitations (mls) in older adults (oas) is a reduction in the central nervous system's (cns) ability to rapidly drive muscle force/torque production. rapid movements can be mathematically expressed as the time derivative of force/ torque, also termed 'yank' (y). muscles are ultimately responsible for generating y, but cns input (ni) to the muscles clearly influences y. the time derivative of the voluntary electromyogram during maximal efforts is associated with gait speed (gs) and chair rise time (crt). however, since the electromyogram is influenced by non-physiological factors (e.g., subcutaneous adipose tissue acting as a low pass filter), it is difficult to fully ascribe this finding to cns deficits. theoretically, normalizing y to the time derivative of electrically evoked force/torque controls for musculoskeletal factors contributing to y (ymsk), which yields a value representing the cns's ability to rapidly produce force/torque (yni=y/ymsk). objectives: to better understand the role of the cns in mls in oas we ) compared leg extensor yni between young and oas, and ) examined the association between leg extensor yni and measures of mobility. methods: twenty-one young and fifty-nine oas ( . +/- . and . +/- . yrs) were instructed to "kick out as fast and hard as possible" against a fixed lever arm attached to a torque motor, and we quantified y between onset and -msec. next, we quantified ymsk from a supramaximal electrically evoked torque-time recording (potentiated -hz doublet) and calculated yni as described. on a separate visit six-minute walk ( mw) gs, stair climb power (scp), and x crt were measured. results: oas had higher yni vs. young adults reflecting a % reduction in central neural activation during rapid torque development ( . +/- . vs. . +/- . ; p< . ). significant associations were observed between yni and mwgs (r= . ), scp (r= . ), and x crt (r=- . ). conclusion: oas have a slower rate of volitional neural activation during rapid leg extensor torque production relative to young adults. in addition, yni explained ~ - % of the variability in measures of mobility, thereby supporting the notion that age-related reductions in the ability of the cns to rapidly activate muscles contribute to mls. background: opa mutations cause dominant optic atrophy (doa), an incurable retinopathy with variable severity and which mechanisms are still unknown. more than % of patients will endure a doa plus syndrome with ataxia, deafness or parkinsonism. the hypothesis of an oxidative stress has been proposed to explain the variability of these symptoms. objectives: that's why our goal is to improve understanding of the physiopathological mechanisms involved in this disease by developing mathematical models of the production of reactive oxygen species (ros) by the mitochondrial respiratory chain. methods: we monitored the levels of mitochondrial respiration, reactive oxygen species (ros), anti-oxidant defenses and cell death by biochemical and in situ approaches using in vitro and in vivo models of opa related disorders and model the complex i functioning with a detailed stochastic background: the sarc-f is a -question screening tool for sarcopenia. we present results for reliability and validity of the german version of the sarc-f. objectives: translation, adaptation and validation of the german version of the sarc-f for community-dwelling older adults in germany. methods: design: cross-sectional. setting and participants: community-dwelling outpatients with a mean age of . ± . years were included in the study, ( . %) of them were female. ( . %) had a positive sarc-f score of >= points. according to the definition for sarcopenia from the european working group on sarcopenia in older people (ewgsop ), eight patients ( . %) were identified as sarcopenic and ( . %) as probable sarcopenic. methods: translation and cultural adaption was composed of seven different steps that were in general based on the guidelines put forward by the world health organization. validation include test-retest and the inter-rater reliability (intra-class correlation coefficient) as well as internal consistency (cronbach's alpha). further, sensitivity, specificity, positive predictive value, and negative predictive value of the sarc-f were calculated. receiver operating characteristics (roc) analysis was performed to calculate the area under the curve. results: the translated and culturally adopted version of the sarc-f for the german language has shown excellent interrater reliability and good test-retest reliability. the internal consistency is acceptable. sensitivity ( %) and specificity ( %) for sarcopenia is low. for detecting patients with probable sarcopenia, the sarc-f in the german version has shown % sensitivity and % specificity. conclusion: due to a low sensitivity for detecting sarcopenia but an acceptable sensitivity for identifying probable sarcopenia, the german version of the sarc-f is a suitable tool for case finding of probable sarcopenia. background: skeletal muscle is a vital component of the locomotor system necessary for physical function. however, there is increasing evidence that skeletal muscle acts as a secretory organ in itself, communicating with other organ systems. acute sarcopenia is an emerging condition affecting adults following hospitalisation, which should be considered akin to organ insufficiency elsewhere. however, acute sarcopenia remains poorly characterised to date. objectives: • to characterise changes in muscle quantity, strength, physical performance, and patient-reported physical function in hospitalised older adults at one week and three months. • to determine what biological and clinical factors are predictive of changes to enable further research towards targeted interventions. methods: planned recruitment will include hospitalised patients aged years and older; elective colorectal surgery patients, emergency surgery patients, and general medical patients with acute bacterial infections. patients will be recruited to the elective cohort in pre-operative assessment clinic with repeat measures within hours of surgery, at one week, and at three months. emergency surgery patients will be recruited pre-or post-operatively with repeat measures at one week, and at three months. medical patients will be recruited within hours of admission, with repeat measures at one week, and at three months. muscle quantity will be measured by bilateral anterior thigh thickness using ultrasound and bioelectrical impedance. muscle function will be measured by handgrip strength and short physical performance battery. serum and plasma samples will be obtained prior to admission in the elective cohort, within hours of surgery in both surgical cohorts, and within hours of admission in the medical cohort. background: sarcopenia is common in old age and is associated with various diseases. as human life expectancy is projected to increase, this will pose a challenge for the global healthcare industry. since sarcopenia is highly heritable, study of its genetic underpinning can help its etiology. in the past decade genome wide association studies (gwas) have allowed the identification of new genetic markers for various conditions. identification of new genetic markers through gwas requires functional validation using cellular models in order to both prioritize and validate the potential loci/genes. objectives: demonstrate that a locus identified in gwas may affect muscle health, which is approximated by lean mass and hand grip strength. methods: gwas results are screened using a two-step scoring system which utilizes publicly available databases such as genecards, ensembl and coxpresdb to assess the relevance of a certain locus. relevant genes are then knocked out using crispr-cas in c c mouse myotube cells which are induced to differentiate. after cell harvest rt-qpcr and western blot are performed to assess mrna and protein expression, respectively. knocked out cells are also examined against wild type cells for morphological phenotype. results: slc a is a promising candidate based on: (a) muscle gwas results, (b) the expression of the gene in smooth and striated muscle tissue, (c) the lack of co-expression with other genes that have an effect on muscle; (d) mouse phenotypes associated with a mutation in the mouse ortholog slc a , (e) cell epigenetic data and (f) the topologically associated domain (tad) at chr. : , , - , , . rt-qpcr of wild type c c cells showed a fast increase in the expression of slc a 's mrna which remains constant during the entire differentiation process. conclusion: preliminary results indicate that slc a might be a promising candidate to investigate for involvement in muscle health. there is a fast and stable increase of the gene's expression during myotube formation. positive results may suggest that slc a is of importance to muscle health. to farther assess slc a role, wild type cells will be compared to knocked-out cells. this might lead to a new genetic marker for muscle health, thus extending personalized medicine in the field of sarcopenia and muscle health. jesse zanker , terri blackwell , sheena patel , kate d u c h o w n y , , s h a r o n b r e n n a n -o l s e n , s t e v e n r . cummings , , william j. evans , , eric s. orwoll , david scott , , sara vogrin , gustavo duque , peggy m. cawthon , ( ( ) background: muscle mass, strength and physical performance are independent risk factors for disability and mobility disability in older adults. it is not known how measures of body composition (muscle, lean and fat mass), strength and physical performance are interrelated or how empirical groupings of these measures relate to disability and mobility disability. objectives: to determine the relationship between measures of body composition, strength and physical performance in older men and to examine how empirical groupings of these measures relate to adverse mobility and disability outcomes. methods: muscle mass was assessed by d -creatine dilution (d cr muscle mass) in men ( . + . years) enrolled in the osteoporotic fractures in men (mros) study. participants completed anthropomorphic measures, walk speed ( m), grip strength (kg), chair stands (s), and dual x-ray absorptiometry (dxa) appendicular lean mass (alm) (adjusted for weight, body mass index or height ) and body fat percentage. factor analysis was conducted to reduce variables into smaller components. men self-reported limitations in mobility (walking - blocks, climbing steps, or carrying pounds); activities of daily living (adls); and instrumental adls at initial and follow-up visits. negative binomial models adjusted for participant characteristics were used to determine the relative risk of factors with mobility and disability outcomes. results: factor analysis reduced variables into four factors: factor , body composition, with strong loading by alm, body fat percentage, weight and muscle mass; factor , body size and lean mass, with strong loading by height, weight and alm; factor , muscle mass, strength and performance, with strong loading by walk speed, chair stands, grip strength, and muscle mass; and factor , lean mass and weight, with strong loading by alm and weight. only factor was associated with prevalent disability and background: urinary incontinence(ui) is a prevalent and costly condition that affects ~ % of older communitydwelling women.one of the contributors of ui is decreased pelvic muscle strength. objectives: to determine the effect of additional oral glutamine supplementation to kegel-exercise on pelvic floor strength and clinical parameters of ui in females. methods: it is a randomized, double-blind study. females with ui were included. digital test and a vaginal manometer were used for measuring the strength of the pelvic floor muscles. hours pad weight test was examined. participants were randomized into groups as oral glutamine gr/day and placebo. it was asked to use the supplementation and kegel-exercises to all participants for months. basic and th month measurements were compared by paired sample t -test and wilcoxon tests in each group. the progression between measurements at basic and th months was compared between the groups by using mann-whitney-u test. (clinical trials protocol id: / background: it is important to identify if middle-aged people are at risk for sarcopenia. a screening-tool identifying predictors of pre-sarcopenia early in the lifespan may inform prevention focused interventions. objectives: develop and validate a practical screening-tool to identify middle-aged adults at risk for pre-sarcopenia using data from the dunedin multidisciplinary health and development study (dmhds). methods: the dmhds is an ongoing longitudinal birth cohort study from the greater dunedin (nz) metropolitan area. the primary outcome of the screening-tool was low appendicular lean muscle index (almi) in middle-aged adults, at age . low almi was classified using prado's age-specific median cut-scores. the models were developed in % (n= ) of the cohort and cross-validated in the remaining % (n= ). possible predictors at age , were examined for associations with low almi, using univariate logistic regression. significant predictors were selected in a multivariate logistic regression to derive sex-specific prediction models. each individual in the cohort was allocated a risk-score and classified as low, medium and high risk, based on the quartile risk score. overall performance of the final models was estimated with nagelkerke r score, discrimination of the models with the area under the roc curve and calibration of the final models with hosmer-lemeshow tests. results: % of the development set and % of the validation set were female. the final models for both sexes included body mass index (b=- . , p= . ; b=- . , p= . ), vo max (b=- . , p= . , b=- . , p= . ) and grip strength (b=- . , p= . , b=- . , p= . ). the final model for females also included creatinine (b=- . , p= . ). nagelkerke's r showed that . % and . %, of the variance in low almi, is explained by the variables in the screening-tool for males and females, respectively. the area under the roc curve demonstrated good discrimination ( . ). sensitivity in the lowest quartile was . %, specificity in the highest quartile was . %. the hosmer-lemeshow p-values were respectively . and . , showing goodness of fit. conclusion: this screening-tool was able to predict the sex-specific risk of pre-sarcopenia in a large birth cohort of early middle-aged adults. clinical utility and application of this screening-tool require further investigation. background: aging-associated changes in body composition include a decrease in skeletal muscle mass, which may predispose women to physical limitations and disabilities. in women, these changes may already be accelerated during menopause, when ovarian estradiol (e ) production ceases. e , the main female sex hormone, is known to have beneficial effects on female skeletal muscle mass. objectives: the aim of this study was to investigate the effects of menopausal transition on lean body mass, lower limb muscle mass, muscle area and muscle fiber cross-sectional area in middle-aged women. methods: middle-aged women (n= ) were followed from perimenopause to postmenopause. menopausal state was defined based on repeated follicle-stimulating hormone (fsh) measurements and menstrual bleeding diaries. serum hormone levels (e and fsh; immulite ), lean body mass (lbm), right leg lean mass (dxa, n= ), and thigh muscle cross-sectional area (computed tomography (ct), n= ) were measured in peri-and postmenopause. muscle biopsies for immunohistochemistry were obtained from participants at peri-and postmenopausal phases, and muscle fiber crosssectional areas were measured. the level of physical activity (pa) from the previous months was assessed with a questionnaire (met-hours/day, n= ). statistical differences were analyzed with paired t-test and wilcoxon signed rank test. gee-modeling was used to analyze the effects of covariates during follow-up. results: the average followup time was . years (range . - . years) and there was a significant difference in e and fsh levels during the transition (p< . for both). lbm decreased . % (p= . ) and leg lean mass . % (p= . ) during the menopausal transition. no changes were found in the cross-sectional area of thigh muscles or muscle fibers. the level of pa declined during the transition (p= . ). when individual menopausal transition time and pa were controlled, only systemic e levels were positively associated with lbm (b= . , p= . ). conclusion: despite the relatively short follow-up time, significant declines were observed in lbm and leg lean mass during the menopausal transition. the decrease in lbm was associated with lower systemic e level. therefore, it seems that although pa might slow the decrease in muscle mass, estradiol loss is one key factor in whole body muscle loss during menopausal transition. hiroyuki shimada , takehiko doi , sangyoon lee , kota tsutsumimoto , seongryu bae , sho nakakubo , keitaro makino , hidenori arai (( ) department of preventive gerontology, center for gerontology and social science, national center for geriatrics and gerontology, aichi, japan;( ) national center for geriatrics and gerontology, aichi, japan)background: in , the european working group on sarcopenia in older people met again (ewgsop ) to update the original definition of sarcopenia. ewgsop uses detection of low muscle quantity and quality to confirm the sarcopenia diagnosis, and identifies poor physical performance as indicative of severe sarcopenia. however, it is not clear that the relationships between the revised definition of the sarcopenia and disability incidence in japanese older adults. objectives: to examine the associations between sarcopenia for ewgsop criteria and disability incidence among community-dwelling older japanese individuals. methods: a total of older adults participated in the study ( women; average age, . ± . years) form a japanese national cohort study called the ncgg-sgs. skeletal muscle mass was assessed using a bioimpedance analysis device and handgrip strength and walking speed were measured as physical performance. we used the cut-points of the asian working group for sarcopenia to determine the low muscle mass and low physical performances. the participants were divided into non-sarcopenia, sarcopenia, and severe sarcopenia groups. the incidence of disability was determined using data collected by the japanese longterm care insurance system over months. results: the prevalence rates of sarcopenia and severe sarcopenia were . % and . %, respectively. the participants with sarcopenia, included sarcopenia and sever sarcopenia, showed higher risk of disability incidence than those with non-sarcopenia (hazard ratio [hr]: . , % confidence interval [ % ci]: . - . ). in analysis between non-sarcopenia and sarcopenia or severe sarcopenia, although the association between disability incidence and severe sarcopenia remained significant (hr: . , % ci: . - . ), there was no significant association in sarcopenia (hr: . , % ci: . - . ). conclusion: severe sarcopenia combined low muscle mass and low physical performance could have a higher risk of disability than healthy older adults or older adults with low muscle mass alone. further studies are needed to determine whether sarcopenia without poor physical performance is associated with disability incidence.background: sarcopenia is one of the biological hallmarks of frailty that has been associated with adverse events in older adults undergoing cardiac surgery. dual x-ray absorptiometry (dxa) is a recommended modality to measure muscle mass, however, dxa may be less accurate in acute cardiac patients due to the confounding effects of peripheral edema and fluid shifts. objectives: the study aims to determine if sarcopenia as measured by a combination of dxa and timed chair rises is associated with mortality in older adults referred for cardiac surgery. methods: a convenience sample of hospitalized older adults being evaluated for cardiac surgery was prospectively enrolled at the jewish general hospital. after a questionnaire and physical performance battery, patients underwent a dxa scan (ge lunar) to measure their appendicular muscle mass (amm). patients were categorized as sarcopenic based on the european working group guidelines if they had low amm defined as < kg/m in men or < kg/m in women and low muscle strength defined as chair rises > seconds. multivariable logistic regression was used to test the ageand sex-adjusted association between sarcopenia and allcause mortality. results: the cohort consisted of patients with a mean age of . ± . years and % females. the interventions were isolated coronary bypass in %, valve surgery in %, and decision not to proceed with surgery in %. the mean amm was . ± . kg in men and . ± . kg in women. the prevalence of sarcopenia was % (n= ), similar in men and women. sarcopenia was not associated with -year mortality (or . , % ci . - . ) and, in a separate model, neither was low amm (or . , % ci . - . ). slow chair rise time was associated with higher -year mortality (or . , % ci . - . ). when patients with heart failure and reduced ejection fraction were excluded, sarcopenia appeared to be more prognostic (or . , % ci . - . ) although it did not reach statistical significance. conclusion: lower-extremity muscle strength, but not dxa-based measures of muscle mass or sarcopenia, is predictive of survival in hospitalized older adults referred for cardiac surgery. background: the "blue zone" are limited areas with a high prevalence of centenarians, with rather homogeneous characteristics, life styles and environment." this blue zone, located in the nicoya peninsula, is in the province of guanacaste. even though costa rica has this blue zone, there are no studies that characterize the prevalence sarcopenia in the centenarians of the region. objectives: the aim of this study was to determine the prevalence of sarcopenia on centenarians from nicoya, costa rica, using the ewgsop criteria. methods: this is a cross-sectional study using a population base of community-dwelling centenarians from guanacaste. antropometric measures, weight, height and strength were assessed. to assess the nutritional state, the mini nutritional assessment (mna) was used and activities of daily living (adl) scores. low muscle mass was assessed by calf circumference < cm and low strength if < kg in men or < kg in women. results: the mean age of the patients were . ± . years. from this group, ( . %) were men and ( . %) were women. patients showed comorbilities: hypertension ( . %), diabetes ( . %), copd ( . %), cancer ( %), osteoarthritis ( %) and depression ( %). mean body mass index, weight, brachial and calf circumference were . ± . kg/m , . ± . kg, . ± . cm and . ± . cm. mean handgrip strength was . ± . kg. the mean score for the mna test was . ± . and adl score . ± . . with respect to sarcopenia prevalence, a total number of ( . %) subjects were detected, ( . %) men and ( . %) women fulfilled the criteria. according to the nutritional status, patients with sarcopenia had malnourishment, were on nutritional risk and had a good nutritional state. from the sarcopenic centenarians, at least % of the subjects had dependency with adl. conclusion: we had high prevalence sarcopenia in centenarians from the "blue zone". there are few studies in centenarians, but using the ewgsop criteria, it is the first in latin america. background: sarcopenia is a geriatric syndrome characterized by low muscle mass and low muscle function and/or reduced physical performance. malnutrition is a major risk factor for sarcopenia. there is limited data on the prevalence of sarcopenia in community-dwelling older people who are at risk of malnutrition in singapore. objectives: the objectives were (i) to determine the prevalence of sarcopenia and its components i.e. low handgrip strength, low appendicular skeletal muscle mass index (asmi) and low gait speed based on the asian working group for sarcopenia consensus (chen et al., ) , (ii) to describe the characteristics and dietary intake of older adults with sarcopenia to those without sarcopenia. methods: a total of community-dwelling older adults (>= years) who were at risk of malnutrition (malnutrition universal screening tool; must score >= ) took part in this study. sarcopenia was diagnosed by low muscle mass (asmi using bioelectrical impedance analysis) plus low muscle strength (handgrip strength) and/or low physical performance ( -meter usual gait speed). anthropometric measurements, dietary intake, and short physical performance battery (sppb) were also collected. results: over % of participants had a charlson comorbidity score of . the overall prevalence of sarcopenia was %; . % had low asmi, . % had low handgrip strength and . % had low gait speed. participants with sarcopenia were significantly older, shorter, and with lower body weight and bmi, mid-upper arm circumference, calf circumference and bone mass compared to those without sarcopenia (all p< . ). they also had lower physical functions as measured using handgrip strength and endurance, leg strength, and sppb score than those without sarcopenia (all p<= . ). additionally, older adults with sarcopenia had lower total energy intake and energy-adjusted protein intake background: the prevalence of sarcopenia varies according to the diagnostic criteria used, however it is an important geriatric syndrome related to a worse functional state in the elderly. very older adults are often excluded from clinical trials. objectives: the aim of this observational prospective study is to describe the prevalence of sarcopenia in community very older adults with high comorbidity. methods: we included patients who enter the geriatric day hospital of the hospital of navarra, spain, aged more than y, underwent bioelectrical impendance analisys (bia), measurement of hand grip strength (hgs), gait speed (gs), short physical performance battery (sppb), mini-nutritional assessment (mna-sf), barthel index and cumulative illness rating scale-geriatric (cirs-g). sarcopenia were defined according to ewgsop ( ). the study begining in and it is actually ongoing. we registered variables at baseline, and at the time , and months. all-cause mortality were registered. results: we present the preliminary results of baseline value. we icluded patients ( . % men, . ± . y). sarcopenia were present in participts, vithout sex differences. sarcopenic vs no-sarcopenic patiets were older ( . ± . vs . ± . y) (p< . ) and they presented worse nutritional status (bmi . ± . vs . ± . kg/m ) (p< . ), mna-sf ( %ci - ) vs ( - ) (p< . ). sarcopenic patients presented lower barthel index ( , %ci - vs , - ) (p= . ), but we have no observed differnces nor in the sppb , %ci - in sacropenic, vs , - in no-sarcopenic participats (p= . ), neither in comorbididy index (cirs-g , - vs , - respectivelly) (p= . ). sarcopenia is significantly associated with higher mortality (hr . , %ci . - . ) (p= . ). at the present time the mean follow-up is . ± . months. at months in patients ( %) the sarcopenia reverted, and we have observed new sarcopenic cases ( %) (incident sarcopenia). conclusion: sarcopenia is highly prevalent in very older adults with high comorbidity. sarcopenia is associated with malnutrition and with higher mortality. background: disability is a multifactorial trait that contributes substantially to decline of health/wellbeing and increases steeply with age after midlife. progress in genomewide sequencing has created the potential for discovering genes influencing various health-related traits. the vast majority of such studies focus on the genetic bases of different traits assuming that they have independent mechanisms. as conceptualized by geroscience age/aging are major risk factors of geriatric traits of distinct etiologies. accordingly, the same mechanisms can predispose not to just one, but to a large fraction of geriatric conditions. objectives: identify the common genetic architecture of various traits by discovering the genetic architecture of complex multifactorial trait such as disability. methods: genome-wide association study of disability in a sample of , subjects from five studies with , disabled individuals from the women's health initiative (whi) genomics and randomized trials network, whi memory study, cardiovascular health study, framingham heart study, and health and retirement study. disability was defined as having at least one of four basic activities of daily living impairments (bathing, dressing, getting out of bed, and walking). results: we identified promising disability-associated single nucleotide polymorphisms (snps) in loci at p< - . four of them attained suggestive level of significance, p< - . in contrast, polygenic risk scores (prs) aggregating effects of minor alleles of independent snps that were adversely or beneficially associated with disability showed highly significant associations in meta-analysis, p= . × - and p= . × - , respectively, and were replicated in each study. the analysis of genetic pathways, related diseases, and biological functions supported the connections of genes for the identified snps with disabling and age-related conditions primarily through oxidative/nitrosative stress, inflammatory response, and ciliary signaling. we identified musculoskeletal system development, maintenance, and regeneration as important components of gene functions. conclusion: the discovery of adverse and beneficial prs for a multifactorial trait of distinct etiologies such as late life disability supports the concept of geroscience. the beneficial and adverse gene sets may be differently implicated in the development of musculoskeletal-related disability with the beneficial set characterized, e.g., by regulation of chondrocyte proliferation and bone formation, and the adverse set by inflammation and bone loss. key: cord- -sizuef v authors: nan title: ectes abstracts date: - - journal: eur j trauma emerg surg doi: . /s - - -y sha: doc_id: cord_uid: sizuef v nan the gertality-score: a feasible and adequate tool to predict mortality in geriatric trauma patients introduction: a large number of prediction models and subsequent outcome scores for trauma mortality have been developed over the last decades. however, feasible scoring systems for the severely injured geriatric patient are lacking. the aim of this study was to develop a new mortality prediction model for severily injured geriatric patients. materials and methods: the german trauma registry was utilized and all geriatric individuals (c years) admitted between and with an iss [ ] c were included. patient and trauma characteristics, diagnostics, therapy and outcome data were gathered. the specific odds of all variables for mortality were calculated. relevant variables were added to the novel gertality-scoring system. subsequently, this score as a sole predictor for mortality was compared with the geriatric trauma outcome score , iss, patient's age and max ais. results: a total of . trauma patients with a mean age of years were included. based on the univariable analysis, the following five variables were included in the gertality-score: age c years, pbrc-transfusion requirements from admission to ward, asa-score c , gcs b , ais c . the values of a given parameter are added to reach the total gertality-score (range - points). the auc found in the novel gertality-score was . , whereas the geriatric trauma outcome score had an auc of . . conclusions: the novel gertality-score is a simple and feasible scoring system that enables an adequate prediction of the probability of mortality in severely injured geriatric patients by using only five specific parameters. references: . champion hr, et al. the major trauma outcome study: establishing national norms for trauma care. j trauma. ; : - . . zhao fz, et al. estimating geriatric mortality after injury using age, injury severity, and performance of a transfusion: the geriatric trauma outcome score. j palliat med. ; ( ) : - . the longer the better! 'extending thawed plasma shelf life to days' introduction: major bleeding is one of the most common causes of death after severe polytrauma. one of the most recent interventions that aims for bleeding control is resuscitative balloon occlusion of the aorta (reboa). this study aims to compare macro-and microcirculatory changes of intraabdominal organs and the lower extremity during the use of reboa. materials and methods: six pigs were anesthesized and received a median laparotomy. the reboa catheter (reliant balloon, medtronic) was inserted via the inguinal artery and occluded in zones , and . the occlusion of the reboa was vizualized with fluoroscopy. the balloon was inflated for min per zone. during this time the local microcirculation was measured with oxygen to see (o c, lea). between each zone the balloon was deflated for min. blood pressure was measured at the carotis artery and the femoral artery. results: baseline values of microcirculation differ significantly among organs. the flow rate is significantly higher in intraabdominal organs (colon . a.u., stomach . a.u.) compared to the extremity ( . a.u., p \ . ). blood pressure measured at the carotic artery increased significantly after inflation of the balloon (p \ . ). this increase depends on the zone of inflation (increase of ? mmhg in zone compared to baseline). the increase of blood pressure after inflation in zone is comparable to the baseline value. the colon is most sensitive to changes of microcirculation whereas the stomach and the extremity are most robust. conclusions: reboa is a new device to control for massive bleeding. different organ systems react differently to the same occlusion of the aorta. the systemic blood pressure does not mirror the local microcirculation of the abdominal organs. during emergency resuscitation with reboa these changes should be kept in mind. none of the authors have any conflicts of interests to declare. investigation of coagulopathies and its relevance with mortality and transfusion rates using thromboelastography in trauma patients introduction: fibrinolysis shutdown after injury is a common and lethal coagulopathic phenotype. patients with polytrauma, especially those with brain hemorrhage, require delayed initiation of prophylactic or therapeutic anticoagulation despite a measurable hypercoagulable state. to understand and modulate the post-trauma coagulation milieu, we assess patients with daily thromboelastography(teg). we hypothesized that persistently high clot strength and low dissolution is associated with thrombotic adverse outcomes in severely injured patients. materials and methods: adult patients with blunt or penetrating injuries admitted to the icu of a level i urban trauma center from jan-jul were included. adverse outcomes were defined as death, ventilator-free-days (vfd) = , acute lung injury (ali), acute kidney injury (aki), and venous thromboembolic events (vte). we assessed trends of clot dissolution (fibrinolysis, ly %) and strength (maximum amplitude, ma) in the first icu days using linear mixed models to account for repeated measures and missing observations. ly % was box-cox power-transformed to approximate normality. significance for pairwise comparisons at each time was adjusted by false-discovery-rate. results: patients: median age -years, % female, iss (iqr - ), % blunt mechanism, median icu days . overall, % developed one or more of the following; %vfd = , %ali, %aki, %vte, %death. ly was persistently lower in patients with adverse outcomes compared to those without (interaction time*adverse_outcomes p = . ), with fdr-adjusted significant differences at icu days and (fig ) . conversely, ma did not differ significantly by adverse outcome status(interaction time*complications p = . , fig ) . conclusions: low clot dissolution, not clot strength, is associated with adverse outcomes in severely injured trauma patients. additional work is underway for earlier identification of sd phenotypes and strategies to mitigate impaired fibrinolysis. introduction: angioembolization (ae) is can be both diagnostic and therapeutic in management of a hemodynamically unstable trauma patient. however, patients who would benefit from ae typically require emergent surgery for their injuries. the critical decision of transferring a patient to the operating room versus the interventional radiology suite can be bypassed with the advent of intra-operative angioembolization (ioae) . while the ability to perform such an intervention was previously limited by the availability of costly rooms termed raptor (resuscitation with angiography, percutaneous techniques and open repair) suites, it has been suggested that using c-arm digital subtraction angiography (dsa) is a comparable alternative. this case series aims to establish the feasibility and safety of ioae. materials and methods: we conducted a retrospective anlaysis of all trauma patients at our level trauma centre who underwent ioae with a concomitant surgical intervention from january to april . results: a total of patients ( . % male, . ± . years, . % blunt) underwent ioae using the c-arm dsa. all but one patient underwent exploratory laparotomy, . % of which underwent an additional surgical procedure (ex. exploratory thoracotomy, orthopaedic). either gelfoam ( . %), coils ( . %), or a combination of both ( . %) were used for embolization. internal iliac embolization was performed in . % of cases ( . % bilateral) and five patients ( . %) required hepatic embolization. ae was successful in all but one case, inferior vena cava filters were placed in . % of cases, and . % of patients required a second ae. the -day mortality was . %. conclusions: our results suggest ioae is a feasible and safe management option in severe trauma patients with the advantage of concurrent operative intervention and ongoing active resuscitation with good success in hemorrhage control. introduction: partial resuscitative endovascular balloon occlusion of the aorta (reboa) is a new concept of aortic occlusion to reduce the ischemic injuries below the occlusion level. it is, however, difficult to determine when the occlusion is partial in a clinical setting. end-tidal carbon dioxide (etco ) is a product of aerobic metabolism and its production is reduced during ischemia and anaerobic metabolism. the aim of this study was to investigate if etco is a good predictor of the degree of aortic occlusion during normovolemia and hemorrhagic shock in a porcine model. methods: nine pigs, - kg, were anesthetized and surgically prepared. then, gradual zone aortic occlusion by %, % and % was induced, during first normovolemia and then controlled hemorrhagic grade iv shock. hemodynamic/respiratory variables, blood gases, aortic/mesenteric blood flow, blood pressure of common femoral artery and etco were measured continuously. oxygen consumption and carbon dioxide production were calculated for each timepoint for correlation measurement to different methods for partial occlusion determination. background: acute appendicitis is one of the most common surgical emergencies worldwide. the aim of this meta-analysis of randomized controlled trials was to compare the safety and efficacy of antibiotic treatment versus appendicectomy as the primary treatment for patients diagnosed to have acute appendicitis. methods: a systematic online search was conducted using the following databases: pubmed, scopus, cochrane database, the virtual health library, clinical trials.gov and science direct. only randomized controlled trials (rcts) that compared antibiotics treatment (a) versus surgical treatment (s) as primary treatment of appendicitis were included. results: eight rcts with . patients were included: in the antibiotics group and in appendicectomy group. higher rate of treatment success was noted in appendicectomy group . % versus only . % in the antibiotics group (p \ . ) (fig. ). follow up period for recurrence was one year in all studies and the recurrence at year was reported in . % ( / ) of patients treated with antibiotics and . % ( / ) of them underwent appendicectomy. moreover, rate of overall were . % in a group and . % in s group (odd ratio . [ . - . ], ci %, p-value: . ) (fig. ) . a longer length of hospital stay was reported among antibiotics group ( . ± . in a group versus . ± . in s group, p . ). conclusions: appendicectomy has significantly higher efficacy rate but higher complications rate when compared to antibiotics treatment. most of the studies included in this meta-analysis conveyed a high risk of bias, hence more well-designed rcts are recommended. introduction: post-operative adhesions are associated with increased risk of morbidity and mortality. up to date no effective measures has been introduced to decrease intra-abdominal adhesions following laparotomy. oxiplex-ap gel has been used in extra-abdominal surgical procedures to prevent adhesions. in the current study oxiplex-ap was tested in a mural animal model to investigate its efficacy in reducing post-surgical intra-abdominal adhesions. materials and methods: forty rats subjected to laparotomy were randomly divided into groups of . a serosa injury was made on the small intestine and three different treatments were applied: simple suture, simple suture ? oxiplexap, and oxiplex-ap only; the last group received no treatment of the injury before closure of the abdomen. all animals were kept alive for days, and a second laparotomy was done to measure the intra-abdominal adhesion by the nair classification. results: at second look laparotomy a significant difference in adhesion was noticed between the simple sutures and simple suture ? oxiplex-ap were the latter had developed less adhesions. there was also a trend towards less adhesion development between the simple sutures and oxiplex-ap only group, with less adhesions in the latter. conclusions: the use of oxiplex-ap was associated with decrease adhesion formation in the current animal model particularly without suturing. further investigations into these findings are needed. introduction: emergency abdominal surgery is known to result in high morbidity and mortality. furthermore, evidence suggests that unplanned admissions to the intensive care unit (icu) are associated with higher in-hospital mortality than those patients with planned icu admissions . the aim of the study was to describe the patient population who required an unplanned admission to icu following emergency laparotomy at the royal melbourne hospital. materials and methods: a single-centre retrospective observational study was performed using prospectively collected data between and . patients who underwent an emergency laparotomy and experienced an unplanned icu admission were included. patients who underwent a trauma laparotomy were excluded from the study. results: emergency laparotomies were performed. of these, ( . %) required an unplanned admission to icu. fourty-two patients ( %) were female, and patients ( %) were aged years and above. sixty-three ( %) were admitted due to single organ dysfunction (clavien-dindo iva). the median time to icu admission was days in patients classified to have experienced clavien-dindo iva, while it was days in patients who experience multi-organ dysfunction (clavien-dindo ivb). thirty-seven patients ( %) were admitted to icu due to complications classified as cardiopulmonary. conclusions: recognising that emergency laparotomy is a high risk procedure, with the elderly patients accounting for the majority of unplanned icu admissions, it is imperative to utilise risk stratification methods to guide optimal peri-operative management. this should result in improved utilisation of critical care resources and overall patient outcomes. introduction: the way of reconstruction following intestinal resection in the emergency settings is still controversial. the question which is better between hand-sewn and stapled anastomosis in trauma and emergency surgery occasionally arises; however, there have been few reports comparing these methods. materials and methods: a record-based retrospective study was performed to compare hand-sewn with functional end-to-end anastomoses in trauma and emergency operations from october to october in one of the largest trauma and emergency centers in japan. the patients who had intestinal resection with functional endto-end or hand-sewn anastomosis in an emergency surgery were included. the patients who had covering ileostomy or colostomy, or who underwent surgery as an elective operation were excluded. the primary outcome is the rate of complication associated with anastomosis. the statistical analyses were performed using a chi introduction: injuries are the fourth leading cause of death in europe. laparotomy is the standard treatment for penetrating abdominal wounds. because of the morbidity and the high rate of negative laparotomies, the nonoperative treatment is effectively developing. the aim of this study is to analyze the complications and the quality of life of the patients after laparotomy for this kind of wounds. materials and methods: a retrospective cohort of patients was studied between and at the laveran military teaching hospital in marseille. one hundred and eighty-six trunk gunshot or stab wound were recorded, including abdominal wounds. thirtyfour patients were managed by laparotomy and included in this study. the patients and their referring general practitioners were contacted to complete missing data and the sf- quality of life score. results: among the patients included, the average age was years and most of them were men. the indication for laparotomy was mainly based on the hemodynamic instability, then according to the results of the computed tomography in case of suspicion of specific lesions: bowel injuries, major vascular injury, mesenteric or mesocolic vascular injury, diaphragmatic injury and intraperitoneal bladder rupture. only laparotomies were negatives. eleven complications after laparotomy were found ( , %), including early (within the days) and late. no complication was found after negative or non-therapeutic laparotomies. the quality of life of the patients after one year is similar to those of the general population. conclusions: the most common indication for laparotomy for abdominal penetrating trauma is hemodynamic instability. the rate of laparotomy complications for penetrating abdominal trauma is similar to those of scheduled surgeries. the quality of life after this care remains unchanged. these results may insist on the fact that the ''gold standard'' treatment for penetrating abdominal injury remains the laparotomy objectives: splenic artery embolization (sae), a routinely used adjunct in the non-operative management (nom) of splenic injuries(si), was widely adopted in trauma about two decades ago. we examined complications that occurred with this modality at a level trauma center over a recent -year period and compared this to the prior years. methods: patients who had sae for si between - were identified. sae complications were noted. splenic abscess, splenic infarction and contrast-induced renal insufficiency were considered major complications. coil migration, fever and pleural effusions were regarded ''minor'' complications. the results were compared with data from a prior study examining similar indices at the same trauma center between and . fishers exact test was used for comparison. results: there were patients admitted with si in the recent period, of which ( %) underwent immediate splenectomy. sae was performed in ( . %) of the patients who underwent nom. of these sae patients, % had a contrast blush and . % were either aast grade or . five sae patients ( . %) had splenectomy for continued bleeding. the overall complication rate was . %. major complications occurred in patients ( . %) and minor in patients ( . %). embolization location in the splenic artery was proximal in . %, distal in . % and in both in . %. there was no association between complications and coil location by logistic regression. differences between the two periods shown in table . conclusion: sae continues to be a useful adjunct in nom of si and has seen increased utilization. complications continue to occur,although fewer minor complications were noted in the second period. no association between embolization location and complications was noted in the recent period. judicious utilization of sae is imperative given the complications that continue to be noted from this procedure. the effect of the time spent in the emergency department on the mortality rates and cause of death in patients who underwent emergent laparotomy introduction: the purpose of this study was to a) examine the effect of the time spent in the emergency department (ed) on hypotensive patients in need of emergent laparotomy and b) to determine the mortality rates and cause of death in these patients. materials and methods: between - , patients were included ( men and women, mean age . years) who underwent laparotomy less or equal to min from ed admission. of the patients, (group ) had a systolic blood pressure (sbp) greater than mmhg and had a sbp less or equal to mmhg. all patients had abdominal injuries with an injury scale score (iss) between and . the in-hospital mortality represented the primary outcome, while secondary outcomes included cause of death and time to death. results: in this study both groups spent a median of min in the ed, but the time from the ed to the operating theatre was shorter in the group ( min versus min). in total, the mortality rate was %, but in the group the mortality was %. the sbp on arrival in the ed was strongly associated with the risk of death. furthermore, we observed significant positive correlation between the probability of death and the time spent in the ed, with an increase of probability of death equal to . % per minute spent in the ed. in both groups the hemorrhage was the commonest cause of death ( %). the results of this study indicate that, in patients with abdominal injuries requiring emergent laparotomy, the probability of death is proportional to both extent of hypotension and the length of time spent in the ed. especially, in patients who were presented with a sbp inferior of equal of mmhg, this probability increased as much as % for each min. despite many advances in trauma surgery, half of hypotensive patients are going to die in the first h. introduction: injury to the pancreas may lead to significant morbidity and mortality. we studied the prevalence of pancreatic endocrine and exocrine functions and evaluated the morphological regenerations in pancreas following partial pancreatectomy. materials and methods: patients with pancreatic trauma were recruited ambispectively from january to december . endocrine functions were assessed at the time of admission and at months follow up with g oral glucose tolerance test (ogtt), serum insulin and c-peptide levels, hba c estimation and exocrine functions were assessed with faecal elastase test. pancreatic volumetry was done with imaging studies at -and -months post discharge. results: twenty patients were studied with a median age of years at the time of injury. all the patients were normoglycemic on admission; only one patient who underwent pancreatic resection developed diabetes mellitus requiring insulin on follow up. patients ( %) were found to have prediabetes by american diabetes association (ada) criteria. patients ( %) had pancreatic exocrine insufficiency. pancreatic volume increment, from mean pancreatic volume of . cm to . cm , was noted in partial pancreatectomy patients. conclusions: overt endocrine and exocrine insufficiency is rare in pancreatic trauma patients. but subsets of patients are biochemically predicted to have higher risks of endocrine dysfunction and exocrine insufficiency. hence, while dealing with pancreatic trauma patients, one should remember the possible metabolic disorders associated and the need for specific investigations. pancreatic volume increment is a new finding which opens up more opportunities for further research. hospital de santo espírito da ilha terceira, general surgery, angra do heroísmo, portugal, hospital de santo espírito da ilha terceira, orthopedics and traumatology, angra do heroísmo, portugal introduction: rope bullfights are traditional events in the azores islands, where a bull is set on the streets, arrested by a rope on its neck. around events happen every year and it is already part of the island's touristic attractions. inevitably, every year, people get injured either from direct trauma with the bull or from falls when trying to escape from the animal. the aim of this study was to characterize the type of injuries that occur in these bullfighting events, as to their incidence, mechanism of injury, anatomical affected area and severity. materials and methods: we prospectively registered all cases of injured people who suffered any type of trauma during rope bullfights and received emergency therapy in the local hospital, between and . results: patients recured to the emergency department, . % female, with mean age of . years. regarding the mechanism of injury, . % occured due to direct trauma to the animal while in the remaining . % resulted from falls during escape or handling of the rope. the most commonly affected anatomical areas were the limbs ( . %) followed by the head and neck ( . %) and thorax ( . %). in , % of the cases, patients suffered from multiple traumas. in . % of the cases the treatments performed were wound care, wound closure and/or symptomatic therapy. in total, patiens were hospitalized, patients required interventions in the operating room ( closed fracture reductions and exploratory laparotomy with splenectomy) and patients were hemodynamically unstable upon admission (hypovolemic shock due to splenic fracture and cet). conclusions: the rare articles published describe the mechanisms of injury associated with bullfights in spanish centers and injuries resulting from wild cow accidents in indian cities. this is the first local descriptive study on the prevalence of traumatic injuries associated to this specific type of rope bullfights. introduction: the two-stage splenic rupture is seldom, its risk is unpredictable and a precise diagnosis of a ct and/or mri imaging unexpectable or unexcludable. generally, and due to our experience and current literature a two-stage rupture occurs within one week after trauma. though dramatic courses after two or three weeks are known. therefore, it is suggested to perform a prophylactic angioembolization in (still) hemodynamically stable patients. materials and methods: a retrospective study in a level-one trauma centre of switzerland did analyse all patients that underwent a prophylactic angioembolisation after an explicit diagnose by ct and/or mri of a splenic parenchymal lesion after trauma between and . further inclusion criteria were hemodynamical stability (sys rr [ mmhg) and missing indication for immediate laparotomy. results: patients ( f, m) with an average age of ± years underwent preemptive angioembolisation after traumatic lesions of the spleen. the ais abdomen was in and in patients. besides a splenic injury patients did also have a kidney injury. the overall iss was ± points. patients suffered additional thoracic or head trauma. in patients the angioembolisation was performed on admission, in on the st, in at the nd and respectively in the rd and th day of. in case an uncomplicated selective embolization of a main duct of the splenic artery was performed. in patients the trouble-free proximal embolization of the splenic artery was done. the average stay was ± . days. no deaths or complications seen due to angioembolisation or splenic rupture. there were no complications or operative introduction: traumatic abdominal wall hernias (tawhs) are uncommon, and the optimal management is debated. tawhs most often result from blunt trauma and are associated with severe intraabdominal injuries. our institutional protocol mandates primary repair only if the patient undergoes laparotomy for other reasons and is without mesh. since , primary repair of lumbar hernias included bone anchors when indicated. we wanted to describe the tawh patients treated operatively during initial hospitalization focusing on injury mechanism, diagnosis, associated injuries, operative techniques, early complications and outcomes. materials and methods: we performed a retrospective, descriptive cohort-analysis of data from the institutional trauma registry from - . all operatively managed tawhs were identified based on ais codes, ncsp codes and relevant key words. results: of the identified patients, ( %) were women. median age was years (range - ). median iss was and patients had iss [ . injury mechanism was blunt except for one explosion. patients ( %) had been in a mvc, and of these ( %) had seat belt injuries. of these patients had a disruption of the muscle from the iliac crest, and one had a hernia through a fractured iliac wing. bicycle falls and fall from height had hernias in the anterior abdominal wall. two meshes were placed, with no known complications. bone anchors (twinfix Ò , mm) were used in patients. no recurring hernias were identified in the patients with routine follow-up ( - months) . conclusions: surgery for tawh is uncommon in our institution. tawh is often associated with severe torso injuries and primary repair is only done when laparotomy for other reasons is indicated. primary suture of the muscle, including use of bone anchors seems to be adequate treatment, as we have identified no recurrences. a longterm follow-up study is warranted for operated and non-operated patients with tawh. a comparison of sub-specialty operative adolescent patient outcomes in adult and pediatric trauma centers introduction: adolescent trauma victims may be treated at either an adult (atc) or pediatric trauma center (ptc). these centers have different resources, surgeon training and overnight in-house coverage. it is not known how outcomes compare with regards to the very small subset of patients that actually undergo a surgical trauma intervention. we hypothesized that presentation to a ptc would yield increased mortality when subspecialty intervention was required and that this would be most pronounced at night when in-house attending coverage is absent at all state ptcs. materials and methods: a review of the pennsylvania trauma outcome study (ptos) database was performed to capture patients aged - who underwent any non-orthopedic trauma surgery. cohorts were created for cranial, thoracic, abdominal or vascular surgery from - . trauma centers were divided as adult level (atc ), adult level (atc ) or pediatric (ptc). groups were created based on time of arrival with am- pm being dayshift and : pm- am being night shift. age, race, mechanism of injury, vital signs, gcs, iss, los and mortality were evaluated. ancova was utilized to control for iss variation. spss was used for all analyses. results: patients met initial criteria. atc s saw more minority patients and more males than other center types. atc s saw an overall older cohort ( . years vs . years in atc and . years in ptc, p \ . ). despite this age difference, presenting systolic blood pressure was lowest at the atc s ( . mmhg vs . mmhg at atc and . mmhg at ptc, p \ . ). iss and triss and overall mortality were not different and this included when grouped by day or night shift. of note, trauma thoracotomy was more likely to be performed at night in adult centers. hospital length of stay was significantly lower for atc ( . days vs . in atc vs and . in ptc). conclusions: adult and pediatric trauma centers see different patients. operative trauma cases are surprisingly low at our state's ptcs and trauma thoracotomy was more likely to be performed at night in atcs than ptcs. broader study is needed to uncover differences in operative care and outcomes. treatment of dislocation of the patella as a result of sports injuries in children. forecast and consequences in adulthood k. furmanova , o. loskutov , a. naumenko medinua clinic and lab, ortopedics, dnepr, ukraine introduction: dislocations of the patella with a rupture of the medial patellofemoral ligament (mpfl) account for - % of acute injuries of the knee joint [ , ] . inadequate therapeutic tactics of these injuries in childhood and youth, as a result of sports injuries, are fraught with complications in the form of the instability of the knee joint, residual deformities and contractures in patients in adulthood [ , ] . materials and methods: in the period from to cases of rupture of mpfl among children aged - years who were involved in sports were observed. the examination included conducting a clinical examination, axial radiography with flexion of the joint at angles of °and °, mri of the knee joint. results: in . % ( cases) the integrity of the mpfl(with a reduced number of sutures) was restored using a yamamoto suture, and in cases ( . %), the autoplasty of the mpfl was performed. excellent medium-term ( years) clinical and functional results according to the ikdc scale were noted in . % of cases, good in . %, satisfactory in . %. in patients ( . %) there was a relapse of dislocation after performing an mpfl suture during the first year after surgery mainly due to noncompliance with the recommendations. conclusions: injury to the knee joint with the patella dislocation in childhood and adolescence, associated with a sports injury is an indication for surgical treatment in order to adequately restore the integrity of the mpfl and prevent disabling complications. our yamamoto suture technique is more optimal for treating young patients with instability of the patella and is recommended for widespread use in pediatric orthopedists due to its undeniable clinical advantages. osteotomy with a defect cm placed cm below tibial plateau. types of fixation have been simulated: plate fixation of only a medial pillar, plate fixation of only a lateral pillar, plate fixation of both pillars, and locking intramedullary nailing. results: in case of plate fixation of only a medial pillar, the injury to an interosseal membrane causes an expressed valgus deformation at axial loading, leading to a reasonable ( . mpa) overload of the fixator in the osteotomy area. the use of a lateral plate leads to excessive loading on an external pillar, while the medial pillar remains unsupported. this causes overloads of the fixator in the osteotomy area ( . mpa). the double plate fixation is typical of the lowest extent of bone fragments displacements ( . mm) . this is a super-rigid type of osteosynthesis, able to cause a stress-shielding syndrome in the adjacent bone. it has been estimated that the method of im nailing is an optimal fixation method, with minimum loading of the fixator ( . mpa) and the best distribution of changed elastic strains in the bone-implant system. conclusions: the mathematical simulation demonstrates that fixation by a medial plate is possible only if support functions of the ligament system and interosseal membrane remained intact. if an injury is a high-energy one, nailing is preferable. introduction: treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. grafts are necessary to support bone healing. a well-established allograft is demineralized bone matrix (dbm) prepared from donated human bone. a recent development is a new fibrous demineralized bone matrix (f-dbm) with a high surface-to-volume ratio. in this study we examine toxicity of an innovative dbm fibers preparation. materials and methods: f-dbm was transplanted to a mm, platestabilized, femoral critical-size-bone-defect of mm in sprague-dawley (sd)-rats (n = ). healthy animals were used as control. after months histology, hematological analyses as well as serum biochemistry was performed. were measured as indicators of free radical exposure. there were no significant differences between the control group and animals receiving f-dbm. hematology as well as biochemistry did not differ between operated animals and control. histologically no evidence of damage to liver and kidney and a good bone healing could be observed in most cases. conclusions: taken together, these results provide evidence for no systemic toxicity of the bone allograft. i have received no significant financial interest, consultancy or other relationship with products, manufacturer(s) of products or providers of services or financial support related to this abstract. • i hereby confirm that my abstract is based on previously unpublished data and that i own the rights to the written summaries of research or observations presented in the abstract, or that i have obtained permission for the acknowledged sources for other excerpts taken from copyrighted works. • in submitting an abstract i hereby agree that the copyright of my abstract is transferred to the european society of trauma and emergency surgery. • i hereby confirm that i will present my abstract at the congress in case it is accepted. sponsor: german institute for cell and tissue replacement (dizg, gemeinnützige gmbh), berlin, germany. intramedullary nailing through suprapatellar approach in distal tibia fractures: a retrospective study evaluating clinical and radiographic results d. bustamante recuenco , a. gómez , j. m. pardo garcía , e. garcía , p. castillón , p. caba doussoux hospital de octubre, madrid, spain, madrid, spain, hospital mutua terrasa, orthopaedics, barcelona, spain introduction: distal tibia fractures (dtf) can be operated either by intramedullary nailing (imn) or by orif with plates. the current literature shows a higher rate of malalignment and consolidation delay with imn when compared to plates. in these studies, an infrapatellar approach for the imn is performed. recent studies show a better alignment in dtf treated with imn by suprapatellar approach, though functional and biological outcomes have not been analyzed yet. our goal is to assess the clinical and radiographic results of the treatment of dtf with imn using a suprapatellar approach. materials and methods: a two-center retrospective study was performed, collecting the cases with dtf treated with suprapatellar imn from / to / . results: a total of patients were obtained, with a mean age of . years. the average follow-up was months. % of the fractures were ao type a, presenting the remaining % intra-articular involvement. patients presented complications, corresponding in of them to superficial infections. as for clinical results, complete mobility in the knee and ankle was obtained in almost all cases. at the radiographic level, a total of % ( ) of distal malalignment cases were detected, defined as more than °deviation from normal axis in the coronal and sagittal planes. most of the fractures consolidated in a period of - months. there were cases of delayed consolidation, from which developed pseudoarthrosis. conclusions: intramedullary nailing through a suprapatellar approach for dtf offers good clinical and radiographic results, with low rates of malalignment and lack of consolidation. more studies are required to compare the results obtained with other fixation methods for these fractures. reference: avilucea fr, triantafillou k, whiting ps, perez ea, mir hr. suprapatellar intramedullary nail technique lowers rate of malalignment of distal tibia fractures. j orthop trauma. ; ( ) : - . the clinical consequences of follow-up radiographs in ankle fractures are unclear and indications for these radiographs are seldom well-defined. routine radiographic imaging in the follow up of patients with an ankle fracture adds to treatment costs, although retrospective studies dispute its usefulness. the aim of this study was to assess if a protocol with a reduced number of routine radiographs would lead to cost savings, without compromising clinical outcomes. materials and methods: a multicentre randomized controlled trial was conducted. patients were randomly assigned in a : ratio to usual-care (consisting of routine radiography at one, two, six and twelve weeks) or reduced-imaging (radiographs only obtained for a clinical indication at six and twelve weeks). functional outcome was assessed using the omas and aaos ankle questionnaires, quality of life was measured with eq- d- l and sf- questionnaires. other outcome measures included complications, pain, the number of radiographs, health perception and self-perceived recovery. costs were measured with self-reported questionnaires results: the study group consisted of participants, of which ( %) received operative treatment. patients in the reduced-imaging group received median radiographs, whilst patients in the usual care group received median radiographs (p \ . ). omas, aaos scores, quality of life, pain, health perception and self-perceived recovery did not differ between groups. we observed complications in the reduced imaging group. this did not differ significantly from the usual care group ( complications p = . ). a significant reduction in radiographic imaging costs was observed (-€ per patient, % ci - to - ). overall costs per patient were comparable ( [ % ci - to ]). conclusions: implementation of a reduced imaging protocol in the follow up of ankle fractures leads to cost savings and more importantly does not lead to worse functional outcomes. results after percutaneous and arthroscopically assisted osteosynthesis of calcaneal fractures w. grün , m. molund , f. nilsen , a. stødle oslo university hospital, orthopaedic department, ullevål, oslo, norway, Østfold hospital, orthopaedic department, grålum, norway introduction: operative treatment of calcaneal fractures using the extensile lateral approach is associated with high rates of soft tissue complications. during the last years there has been a trend towards less invasive fixation methods. percutaneous and arthroscopically assisted calcaneal osteosynthesis (paco) combines the advantages of good visualization of the posterior facet of the subtalar joint with a minimally invasive approach. materials and methods: we conducted a clinical and radiographic follow-up of patients with calcaneal fractures treated by paco with a minimum follow-up of year. there were sanders ii and sanders iii fractures. the mean follow-up period was . months (sd . ). our primary outcome was the american orthopaedic foot and ankle society (aofas) ankle-hindfoot score. secondary outcomes were the calcaneus fracture scoring system (cfss), the manchester-oxford foot questionaire (moxfq), the visual analog scale (vas) for pain and the incidence of complications. radiographs were obtained to evaluate the reduction of the fractures as well as the presence of subtalar osteoarthritis. results: the median aofas score was (range, - ), the cfss score ( - ), the moxfq score . ( - . ). the vas pain score was ( - . ) at rest and . ( - . ) during activity. the böhler angle improved from mean . degrees (sd . ) preoperatively to . degrees ( . ) postoperatively. however, the follow-up radiographs showed subsidence of the fractures and a böhler angle of . degrees ( . ). % of the operated feet showed signs of posttraumatic subtalar osteoarthritis. there were no wound healing complications. two patients were reoperated with screw removal due to prominent screws. conclusions: our results suggest that paco gives good clinical results and a reduced risk of complications in selected calcaneal fractures. prospective longterm studies will be necessary to better evaluate the potential advantages and limitations of paco. with the nascent state of microsurgical services in the region the application of negative pressure wound therapy (npwt) has proven to be very helpful. an improvised npwt has made it locally available to patients. this report aims to show how this has improved the management of open fractures of the lower limb in a resource restricted setting. materials and methods: a -month review of cases of lower limb open fractures managed at a regional trauma centre in nigeria was done. the type of wounds were classified based on region and need for soft tissue coverage. results: a total of cases were reviewed approximately % of these case were gustilo and anderson type iii. of these had npwt as part of their management. some of the benefits of observed were; reduced frequency of wound dressings, and shorter time to optimize wound for closure. conclusions: the locally improvised npwt has proven to be an affordable and cost-effective tool in the management of open lower limb fractures. it remains an invaluable alternative of care in the absence of microsurgical skills and patented device with are far from reach owing to financial constraints. references: . hussain a, singh k, singh m. cost effectiveness of vacuum assisted closure and its modifications: a review. isrn plast surg. ; : - . . isiguzo c, ogbonnaya i, uduezue a. modification of negative pressure wound therapy in the economically constrained region: a preliminary report. vol. , nigerian j plast surg. joytal printing press; . p. - . . mba u, nevo a. challenges of limb salvage in a resource limited environment: case report and review of literature. niger j plast surg. ; ( ): . . novak a, wasim sk, palmer j. the evidence-based principles of negative pressure wound therapy in trauma and orthopedics. open orthop j. ; : - . introduction: lower extremity vascular trauma may result in limb loss or mortality. this study examined outcomes of lower extremity vascular trauma (levt) and potential associations to amputation/mortality. materials and methods: a retrospective cohort study of patients (n = ; limbs) with levt between and in a single trauma center. only patients requiring a vascular procedure were included. data were extracted from the swedish vascular registry (swedvasc) and the swedish trauma registry (swetrau). results: mean age ± years; men % ( / ); trauma mechanism % ( / ) blunt and % ( / ) penetrating. % of patients underwent preoperative cta; % of patients ( / ) were transferred to hybrid operating room. arterial injury was present in / limbs ( %) and venous injury in / limbs ( %). the most frequently injured artery was popliteal artery ( / ; %) followed by superficial femoral artery ( / ; %). most common vascular operative procedure was arterial bypass/interposition graft ( / ; %). a vascular shunt was used in % of cases ( / ). fasciotomy was performed in % ( / ) of limbs. four patients were lost to follow-up after less than five days. there were eleven limbs ( / ; %) amputated within -day postoperative follow-up. all amputations were caused by blunt trauma. % ( / ) of arterial injuries below-the-knee led to amputation. thirty-day mortality rate was . % ( / ) . univariate analysis showed that fractures (p \ . ), soft tissue injury (p \ . ), multiple injuries (p = . ), and blunt mechanism (p \ . ) were associated with amputation and mortality after levt. conclusions: this study showed that amputations after levt are caused by blunt trauma. also levt combined with fractures, soft tissue injury, or multiple injuries increased the risk of amputation and mortality. multi-center study enabling multivariate analysis to adjust for potential confounding factors is imperative to confirm these findings. incidence, treatment and financial burden of tibial plateau fractures in belgium between and describe the incidence, evolution in management and financial burden of tpf in belgium between and . we compare national data with data from uz leuven (uzl), the largest university hospital in belgium. materials and methods: this study includes all tpf treated in belgium between and . we identified . tpf, of which fractures were treated in uzl. despcriptive statistics were used to analyze the data. results: the annual incidence increased from . to . / , /y. an increase in number was true for both operatively treated patients (otp) and non-operatively treated patients (notp), but was more pronounced in the latter ( % vs. % increase). the rate of surgery (ros) decreased from . % to . %. the mean ros for uzl was . %. the total financial burden in belgium increased with %, mainly driven by increasing costs in otp. hospitalisation rates for notp decreased from % to %, as day hospital admission occured more commonly. the mean hospitalisation cost was € , for otp and € , for notp. costs for uzl inpatients were € , and € , . nursing days accounted for % of the cost in otp and % in notp. the mean los was . days for otp and . days for notp. uzl patients had a mean los of . and . days. conclusions: tpf are associated with increasing hospital related healthcare costs. as nursing days determine the majority of the financial burden, measures should be taken to avoid prolonged los. introduction: rotational malalignment (rm) is a common postoperative complication after intramedullary (im) nailing of tibial shaft fractures. computed tomography (ct) is commonly used for detection of malrotation, however reliability is frequently questioned. the purpose of this study is to evaluate the intra-and inter-observer reliability of low-dose protocolled bilateral postoperative ct-assessment of rotational malalignment after im nailing of tibial shaft fractures. materials and methods: a total of patients were prospectively included with tibial shaft fractures that were treated with imn in a level-i trauma center. all patients underwent postoperative bilateral low-dose ct-assessment (effective dose of . - . mgy) as per hospital protocol. four observers performed the validated reproducible measurements of tibial torsion in degrees, based on standardized techniques. the intra-class coefficient (icc) was calculated to evaluate intra-and inter-observer reliability. the intra-and inter-observer reliability was categorized according to landis and koch. results: intra-observer reliability for quantification of rotational malalignment on postoperative ct after imn of tibial shaft fractures was excellent with . ( % ci = . - . ). the overall inter-observer reliability was . ( % ci = . - . ), also excellent according landis and koch. discussion and conclusion: first, bilateral postoperative low-dosesimilar radiation exposure as plain chest radiographs-ct assessment of tibial rotational alignment is a reliable diagnostic imaging modality to assess rotational malalignment in patients following imn of tibial shaft fractures and it allows for early revision surgery. second, it may contribute to our understanding of the incidence, predictors, and clinical relevance of postoperative tibial rotational malalignment in patients treated with imn for a tibial shaft fracture, and facilitates future studies on this topic. the trauma emergency laparotomy audit (tela) t. collaborators , m. marsden , p. vulliamy , r. carden , o. najiuba , n. tai , r. davenport tela collaboration, natric, n/a, united kingdom, queen mary university of london, centre for trauma science, london, united kingdom introduction: mortality for shocked trauma patients undergoing emergency laparotomy remains unchanged for years. the tela study aimed to describe the contemporary peri-operative management and patient outcome following abdominal injury. materials and methods: a prospective multicentre observational study of all patients undergoing emergency abdominal surgery within h of injury was performed in the uk and ireland for six months from the st january . shock was defined as the receipt of blood transfusion, with clinical or biochemical evidence of hypoperfusion. results: the study included patients from hospitals, of whom ( %) were shocked and received a median of units red blood cells. shocked patients were more likely to have a blunt mechanism of injury ( % vs %, p \ . ) and had a % mortality ( / ). half of these deaths occurred in the operating room (or). patients that died were more severely injured (injury severity score (iqr - ) vs (iqr - ), p = . ) and had a greater degree of shock at hospital arrival (base deficit . (iqr . - . ) vs . ( . - . ) , p \ . ). processes of care were equivalent or better among non-survivors, with a higher proportion of patients that died undergoing laparotomy within min of arrival in the emergency department ( % vs %, p = . ) and a lower proportion receiving crystalloid in the or ( % vs %, p \ . ). however, delays to achieving definitive haemorrhage control and delivering balanced blood transfusion ratios were observed among both survivors and non-survivors. conclusions: damage control resuscitation principles are followed most closely in patients that die. despite better processes of care, in shocked patients died in this study justifying the continued search for novel therapeutic approaches. pre-operative temporary haemorrhage control and pharmacological mitigation of the effects of shock may be productive avenues of research to improve patient outcomes. introduction: tranexamic acid (txa) has been shown to reduce mortality in bleeding trauma patients, with greater effect if administered early. normally administered intravenously, txa can also be administered intramuscularly, which could be advantageous in low resource and military settings. intramuscular use has only been tested in healthy patients, and it is likely that shock will reduce intramuscular uptake. materials and methods: in a prospective experimental study norwegian landrace pigs ( - kg) utilised in a surgical course in haemostatic emergency surgery were subjected to various abdominal and thoracic trauma. after h of surgery the pigs were injected with mg/kg txa either intravenously or intramuscularly. blood samples were drawn at , , , , , , and min. the samples were centrifuged and analysed with liquid chromatography-mass spectrometry (lc-ms/ms). results: preliminary results from animals in the intramuscular and animals in the intravenous group. mean plasma concentration with sd of txa as a function of time is shown in figure . plasma concentration in the intramuscular group was near ug/ml min after administration, and rose above ug/ml after min. conclusions: plasma concentrations reported to inhibit fibrinolysis in vitro is - . ug/ml ( , ) . if this extrapolates to the clinical situation intramuscular administration would yield plasma levels within the lower end of therapeutic range after min. in ongoing haemorrhagic shock plasma concentrations of txa after intramuscular administration were considerably lower than after intravenous administration, but within therapeutic range . introduction: fallowing laparoscopic cholecystectomy(lc), patients suffer from postoperative pain, especially in the abdomen. intraperitoneal local anesthesia (ipla) reduces pain after laparoscopic cholecystectomy(lc). acute cholecystitis(ac)-associated inflammation, increased gallbladder wall thickness, dissection difficulties, and a longer operative time are several reasons for assuming a benefit in pain scores in urgent lc with ipla application. the aim was to determine the postoperative analgesic efficacy of high-volume lowdose intraperitoneal bupivacaine in urgent lc. materials and methods: fifty-seven patients, american society of anesthesiologists(asa) physical status i or ii were randomly assigned to receive either normal saline(group a) or intraperitoneal bupivacaine(group b) at the beginning or at the end of the surgery in urgent lc. the primary outcome was the scores of postoperative pain by visual analogue scale score (vas) after surgery. results: postoperative vas scores at st and th hours were significantly lower in group b than group a (p \ . ). postoperative vrs scores at st, th and th hours were significantly lower in group b than group a (p \ . , p: . , p: . ). anelgesic use was significantly higher in group a at st postoperative hour than group b (p \ . ). shoulder pain was significantly lower in group b than in group a (p \ . ). patient satisfaction was significantly higher in group b than in group a (p \ . ). conclusions: high-volume low-concentration intraperitoneal bupivacaine instillation resulted in better postoperative pain control along with reduced incidence of shoulder pain and analgesic consumption in comparison to control group in urgent lc. introduction: in-hospital resuscitative thoracotomy is an established procedure for patients with penetrating cardiac injuries. the survival rate is dismal in patients with cardiac arrest prior to admission. prehospital resuscitative thoracotomy (prt) was introduced by the london hems with the highest published survival rate of %. we aimed to identify the number of patients who could potentially benefit from prt in our major trauma center catchment area. materials and methods: data from to were collected from the institutional trauma registry and electronic records. we included patients [ years, with penetrating cardiac injury, or penetrating chest trauma and cardiac arrest, or penetrating chest trauma and sbp \ mmhg. commonly used criteria for prt are tamponade with cardiac arrest lasting \ min at the time of ambulance arrival and with [ min remaining transportation time to hospital. results: cardiac injury was found in of included patients. of these , arrived at the hospital with signs of life and survived. of the patients who died had tamponade. criteria for prt were not met in of patients with tamponade. two patients could have been eligible for prt. one patient was found in oslo with cardiac arrest lasting min. the patient had multiple stab wounds to the chest and had several perforations of the right atrium, not technically manageable in a prehospital setting. the second patient was injured outside our primary catchment area and arrested with prehospital personnel present. prt was performed and the tamponade relieved, but compression of the aorta was necessary. the patient was declared dead shortly after hospital admission. conclusions: in years in a population of . million, two patients met london hems criteria for prt. prt was performed in one patient who was declared dead shortly after hospital admission while one patient suffered from injuries which are unmanageable in a prehospital setting. isolated tissue injury leads to fibrinolytic shutdown, tpa resistance and alterations in clot structure in a porcine model introduction: trauma-induced coagulopathy includes a spectrum of hypo-to hypercoagulable phenotypes with differing levels of fibrinolysis and tpa sensitivity. fibrinolysis shutdown is associated with increased late mortality and shown in small animal studies to be driven by tissue injury. utilizing a novel method of clot structure analysis, we hypothesize that isolated tissue injury provokes fibrinolysis shutdown, tpa resistance and is associated with altered clot structure resulting in enhanced clot stability. materials and methods: all male pigs (n = ) underwent anesthesia, intubation, femoral artery cannulation and mini-laparotomy. tissue injury (n = ), was inflicted with bilateral chest wall muscular cutdowns and bilateral femoral fractures using a captive bolt pistol. mean arterial pressure was maintained at [ mmhg. timed blood samples analyzed using tpa challenged and citrated native teg to evaluate tpa resistance and fibrinolytic shutdown respectively. after mm punch biopsy induced splenic injury, clot was collected, washed, and chemically fractioned by strong cation exchange chromatography. tandem mass spectrometry and bioinformatic analysis were used to evaluate clot structure and factor xiiia cross-linking patterns and covalently associated proteins. results: tissue injury pigs showed increased tpa resistance (change tpa-teg ly : - . % vs - . % p = . ) and a trend of fibrinolytic shutdown evidenced by teg compared to control (fig. ) . splenic clot structure analysis demonstrated altered clot structure (fig. ) and identified elevated levels of protease inhibitors such as alpha macroglobulin and alpha antiplasmin at h post tissue injury compared to baseline. conclusions: in a porcine model, isolated tissue injury provokes fibrinolysis shutdown and tpa resistance resulting in altered clot structure with an increased incorporation of anti-protease proteins resulting in enhanced clot stability. there is a high incidence of rotational malalignment after intramedullary nailing of tibial shaft fractures: a prospective cohort series of patients n. j. bleeker amsterdam medical centre, flinders university, department of orthopedics and trauma surgery, amserdam, netherlands introduction: intramedullary nailing (imn) is the treatment of choice for most tibial shaft fractures due to its minimalistic surgical approach, superior fracture healing, and rapid recovery. however, an iatrogenic pitfall is rotational malalignment (rm). the aim of this prospective cohort study was to determine the incidence of rm and to evaluate the efficacy of protocolled bilateral postoperative computed tomography (ct) assessment of rotational tibial alignment. materials and methods: between and we prospectively included patients ( male ( %)), with a mean age of years, with a unilateral tibial shaft fracture. as per hospital protocol, patients underwent a routine low-dose bilateral postoperative ct to assess rm. forty-two patients ( %) suffered open injuries; ( %) were involved in a multi-trauma sustaining more than one injury. according to the ao/ota classification, there were simple ( %), wedge ( %), and complex fractures ( %). fracture location within the tibial shaft varied with six patients ( %) being within the proximal third, ( %) middle third, and ( %) distal third. there were segmental ( %) fractures that involved more than one third of the tibia. results: fifty-five patients ( %) had post-reduction rm including patients ( %) between °- °, seven patients ( %) with a rm between °- °, and two patients ( %) with a rm greater than °w hen compared to the uninjured side. of the patients with rm, the tibia was externally malrotated in patients ( %). three patients ( % of cohort or % of those with rm) underwent revision surgery to correct the rm as detected on ct scan. conclusions: this study reveals a high incidence of rm following tibial nails ( %) with a surprisingly low revision rate ( % of those with rm). a subsequent study should aim to assess clinical relevance of rm in terms of functional outcome and gait analysis. for now ctrotational-profiling provides a platform for early recognition and correction of rm secondary to tibial imn. level of evidence: therapeutic level ii -prospective cohort study. materials and methods: the tarn database was analysed retrospectively to quantify the number of trauma team activations, patients with major trauma (mt), causes of injury, and subspecialty-specific trauma procedures. crude and risk-adjusted mortality rates, observed to expected (o/e) mortality ratio, and risk-adjusted rates of survival from mt were also calculated. results: the number of trauma team activations has risen by a factor of . the predominant injury mechanism that resulted in mt was a fall from less than m. there has been a fivefold increase in the overall number of trauma surgical procedures. orthopaedic surgeons have performed % of trauma procedures, followed by neurosurgeons, oral and maxillofacial surgeons, and visceral trauma surgeons. the rate of trauma laparotomies per consultant fluctuated between . and . per month. a fall from less than m, road traffic accident and a fall from more than m were the three leading causes of death from mt. the overall o/e mortality ratio was . . conclusions: aintree trauma profile has significantly changed since . this change highlights the potential need for a review of how mt services are offered at aintree to reduce the o/e mortality ratio. this may be achieved through more co-ordinated provision of trauma care, prevention, audit and research programmes. the role of visceral trauma surgery should be reconsidered within the context of the surgical patients' needs and demands, and fundamental requirements of the profession. inter-hospital variation in surgical intensity for trauma admissions: a multicenter cohort study l. moore , m. p. patton , i. farhat , p. a. tardif , c. gonthier , a. belcaid , f. lauzier , a. turgeon , j. clément université laval, social and preventive medicine, québec, canada, chu de québec-université-laval, québec, canada, introduction: guidelines for trauma patients are increasingly moving away from surgical management towards less invasive procedures but there is a knowledge gap on how these recommendations are influencing practice. we aimed to assess inter-hospital variation in surgical intensity for trauma patients and identify determinants of surgical intensity. materials and methods: we conducted a retrospective multicenter cohort study based on the trauma centers of an inclusive canadian provincial trauma system. we included adults admitted for major trauma between and . analyses were stratified for orthopedic (n = , ), neurological (n = , ) and thoracoabdominal surgery (n = ). surgical intensity was quantified with the number of surgical procedures during the first h. inter-hospital variation was assessed with the intra-class correlation coefficient (icc) from multilevel poisson regression models. relative risks (rr) were generated to identify determinants. results: moderate inter-hospital variation was observed for orthopedic surgery (icc = . %, % confidence interval [ci]: . - . ) whereas variation was low for thoracoabdominal surgery (icc = . %, % ci: . - . ) and neurosurgery (icc = . %, % ci: . - . ). level iv centers had similar surgical intensity for thoracoabdominal injuries (rr: . , % ci: . - . ) but lower intensity for orthopedic injuries (rr = . , % ci: . - . ) than level i/ii centers. during the study period, we observed a decrease in intensity for neurosurgery (rr for (rr for - versus . , % ci: . - . ) and thoracoabdominal surgery (rr = . , % ci: . - . ). conclusions: the observed inter-hospital variation in risk-adjusted surgical intensity suggests that there may be opportunities for quality improvement in surgical care for injury admissions. a better understanding of how surgical intensity influences clinical outcomes is needed to inform quality improvement activities. pre-hospital injury diagnosis a. easthope , m. marsden , g. grier barts and the london medical school, london, united kingdom, royal london hospital, centre for trauma science, london, united kingdom introduction: accurate pre-hospital diagnosis of a patient's injuries may improve care by facilitating effective intervention at the scene and reducing time to definitive treatment in hospital . we sought to assess the diagnostic accuracy of injuries by london's air ambulance (laa) clinicians and identify conditions in which clinical accuracy may deteriorate. materials and methods: a retrospective review was undertaken of all patients conveyed to the royal london hospital by laa from october for six-months. pre-hospital injury scores, coded using the abbreviated injury score (ais) were compared to hospital discharge ais. patient outcomes were evaluated in the case of underscored injuries. results: during the study period patients were seen and met eligibility. mean clinical sensitivity and specificity was % and % respectively. chest injury identification was most sensitive ( %) and pelvic injury least sensitive ( %). the relative risk (rr) of underscored injuries to the chest, abdomen and pelvis increased with decreasing glasgow coma scale (gcs) peaking at . (iqr . - . ). the average accuracy of injury identification was % with a negative predictive value of %. no overt patient morbidity resulted from a missed, or under-scored injury. all missed injuries were subsequently identified in the emergency department. conclusions: the pre-hospital diagnosis of injuries has reasonable sensitivity and excellent specificity. accurate pelvic injury diagnosis is more challenging than chest or abdomen. with decreasing gcs, the risk of missing injuries increases. clinicians should be aware of the potential for error when treating trauma patients with impaired conscious levels. comorbidities, injury severity and complications predict mortality in severe thoracic trauma: a retrospective analysis from the norwegian national trauma registry of epidemiology, clinical factors and risk factors for mortality of patients with thoracic injuries. materials and methods: adult patients treated for severe thoracic trauma (injury severity ais c ), between and at haukeland university hospital were included. data were extracted from ( ) the haukeland university hospital local trauma registry, and ( ) the norwegian trauma registry. additional data on comorbidities and complications was collected from patient records. the factors age, gender, comorbidities [charlson comorbidity index (cci)], anticoagulant use, injury severity [revised trauma score (rts)], [injury severity score (iss)] and complications [clavien-dindo scale (cds)] were analyzed for being predictive of in-hospital mortality. multivariate logistic regression analyses with backward selection methods were used. results: data of patients were analyzed, of which ( %) patients died. median iss was in the non-survivors (iqr , ) and (iqr , ) in survivors (p = . ). data of patients were used in the risk factor for mortality analysis. two or more comorbidities measured by cci (or: . , p = . ), injury severity measured with the rts (or: . , p = \ . ), and grade c complications on the cds (or: . , p = . ) were significant predictors for mortality. conclusions: severe comorbidities significantly decreased the chances of survival after thoracic trauma. injury severity was also found to be a significant predictor of mortality. physiological injury severity, measured by rts, appeared to be a stronger predictor of mortality than iss after thoracic trauma. finally, severe complications led to considerably higher risk of mortality following thoracic trauma. the psychosocial impact of e-bike accidents and changing values of older patients in the netherlands, a qualitative study s. berben , l. vloet , e. c. t. tan , m. edwards , , a. brants , , , g. olthuis , , , , a. oerlemans , , , , f. haverkamp , , introduction: the mechanical impact of e-bike accidents, increasingly used by older persons, has shown to be higher compared to regular bike accidents. however, the psychological impact of e-bike accidents in older trauma patients, their experiences in emergency and follow-up care, and the possible change in values and beliefs in response to the accident is still unknown. materials and methods: we used a qualitative design and included older patients ( ? years) with a variety of (severe) injuries, who were admitted to the emergency department after an e-bike accident (n = ) and their relatives (n = ). they were interviewed within one month (t ) and after three months (t ) of the date of accident. interviews were transcribed verbatim and analyzed via a thematic analysis approach using an ethical perspective. results: many patients required (in)formal care after hospital discharge. in general patients were satisfied with the provided emergency surgical care, although some patients reported limited and insufficient information on rehabilitation and homecare support. the analysis yielded impaired physical condition, anxiety, increased vulnerability and dependency of care givers as psychosocial impact. freedom impairment, shifting relational autonomy, and confrontation with vulnerability and mortality were reported changes in values. central values as mobility and freedom, vitality and health, social participation and recreation were put under pressure and needed to be negotiated again after the accident in order to decide whether to use the e-bike again. conclusions: follow-up information of surgeons and emergency physicians after initial hospital care for older trauma patients with an e-bike accident shows room for improvement, with more specific consideration for the psychological impact of trauma and changes in values after e-bike accidents. eur j trauma emerg surg. . https://doi.org/ . /s - - - . traumatic subaxial cervical fractures: functional prognostic factors and survival analysis introduction: the main goal of this study is to identify the risk factors for poor functional outcomes and to analyze the overall survival (os) and complications rate in patients with traumatic cervical spinal cord injury (sci) and subaxial cervical fracture (sacf) treated with open surgical fixation. materials and methods: the authors retrospectively reviewed sixtyfive consecutive patients from one single center with traumatic unstable sacf and associated sci treated surgically between and . we exclude cervical fractures with concomitant severe head injury, brachial plexus injury, lumbar plexus injury, superior or inferior limb fractures and patients who were lost during the followup period. statistical analysis using a chi square test, student's t-test and logist regression were used to identify factors associated with poor functional outcomes after surgical treatment. os analyses were performed using kaplan-meier curves. results: the -year survival rate was . %. four patients died in the first days after surgery and , % need a reoperation. the median time from injury to surgery was . days. the complication rate was %, being respiratory failure the most common one. preoperatively, % had an asia \ c. about % of the patients with asia between a-d had improve one or more asia grades. logistic regression analysis show that older age, sacf above c , asia \ c pre-surgery and long time from injury to surgery were related with poor prognosis. the os rate was higher in patients with neurological improvement, without signs of neurogenic shock at presentation and in sacf bellow c . conclusions: our results suggest that sacf should be treated as soon as possible in order to improve the os rates and functional outcomes. older patients, lower asia at presentation and sacf above c are related with worst functional outcomes. introduction: compression fractures of multilevel vertebral bodies are common in children. due to segmental plasticity, several adjacent vertebral bodies are compressed to a lesser degree at each body. plain ap and lateral x-ray is the first diagnostic examination in the emergency department (ed), but a proper diagnosis is often delayed or missed. materials and methods: this is a retrospective, monocentric study in children falling on their back who showed up at the orthopedic ed, between december and september . nine children ( f, m) with an average age of . years were included. trauma occurred playing games and doing sports in all cases. all children were subjected to x-ray, followed by mri scans for doubtful findings on the plain x-ray or persistent mild pain (t , t , t -stir sequences). results: cuneiform vertebral fracture or vertebral body height reduction was diagnosed with x-ray in five vertebrae while mri showed fractures in vertebrae including compression and edema of adjacent vertebrae in the t -stir sequence. therefore only . % vertebral fractures have been detected by plain x-ray. the injured vertebral bodies were so distributed: t n = , t n = , t n = , t n = , t n = , t n = , t n = , t n = , t n = , t n = , l n = , l n = , s n = , s n = . the most involved spine section was between t and t with fractures. conclusions: vertebral fractures are not always related to hyperflexion or forward hinging mechanism. mri showed vertebral compression fractures and the t -stir sequence showed edema as post-traumatic evidence that had not been detected by x-ray. in absence of a radiologically visible lesion, the persistence of pain should be investigated by performing mri scans. the middle thoracic spine level appeared to be the most involved one in pediatric vertebral fractures. introduction: occipitocervical fixation (ocf) is an effective surgical method to treat various craniovertebral junction (cvj) pathologies. a rigid fixation achieved from ocf displaces other techniques of cvj stabilization unfortunately during procedure deep and wide wound is performed. aim of this study is to share our experience in ocf and lately performed percutaneous ocfs with intraoperative ct guided navigation system. materials and methods: of patients who underwent ocf were performed percutaneously. o-arm ct scans were used to illustrate and measure radiologic parameters. screws were implanted in c lateral masses ( ) , isthmus of c ( ) and c pedicles ( ) and assessed according gertzbein robbins (gr) in modification of bredow classification from a to e. results: a total screws were implanted, of them was performed in open surgery and percutaneously. outcome in gr classification for screws implanted in open surgery was: a ( , %), b ( , %), c ( , %), d ( , %) and e ( , %) while in percutaneous: a ( , %) and b ( , %) . in open surgery one screw was revised. conclusions: percutaneous occipitocervical fusion seems to be a good option to achieve desirable effect in cervical pedicle screws implantation. during procedure whole nuchal muscles are preserved. ct guided surgery and microscope view are necessary to perform percutaneous ocf. introduction: studies have found higher risk of traumatic deaths in rural areas in norway combined with a paradoxically decreased prevalence of severe, non-fatal injuries ( ) . this study investigates the risk of fatal and non-fatal injuries among all adults in norway in the period - . materials and methods: all traumatic injuries and deaths among persons with residential address in norway from - were included. data was collected from the norwegian patient registry and the norwegian national cause of death registry. all cases were stratified according to six groups of centrality based on statistics norway's classification of centrality . mortality-and injury rates was calculated per , inhabitants per year. results: the mortality rate differed significantly according to the levels of centrality (p \ . ). the mortality rate in the most urban group ( ) was . and in the most rural group ( ) . . the lowest mortality rate was found in centrality group ( . ). there was an increased risk of death between centrality group and group with a relative risk of . (ci: . - . , p \ . ). the most common cause of death was transport injuries, self harm, fall injury and other external causes. the highest urban-rural gradient was seen in transport injuries with a relative risk of . (ci . - . , p \ . ) comparing group to group . group had the lowest risk of nonfatal injuries ( ) and group the highest ( ). the risk of nonfatal injuries increased with higher grade of rurality, comparing group and revealed a relative risk . (ki . - . , p \ . ). conclusions: the more rural the higher risk of traumatic deaths and non-fatal injuries. transport injuries had the highest urban-rural gradient. references: . bakke hk, hansen is, bendixen ab, morild i, lilleng pk, wisborg t. fatal injury as a function of rurality-a tale of introduction: virtual fracture clinics (vfcs) are an alternative to conventional fracture clinics for management of musculoskeletal injuries. they have been shown to be a safe and effective model for upper and lower limb injuries. there is limited data to support their use for specialist thoracolumbar fracture follow-up. materials and methods: lean methodology including process mapping was applied to identify a safe virtual alternative for the pathway. first cycle analysis of consecutive referrals to a traditional specialist thoracolumbar fracture clinic. second cycle analysis of consecutive referrals six months after introduction of a vfc. results: mean time to first outpatient review in first cycle was days. referrals led to booked outpatient appointments and were missed ( % non-attendance). % of referrals had or more scheduled appointments. / were ao type a - and all of these received non-operative treatment. / were ao type a or b and of these received non-operative treatment. patient received operative stabilisation (ao type b). process mapping identified two pathways-virtual review with advice letter and physiotherapy referral (outcome a-ao type a - ) or face to face review (outcome b-ao type a or b). mean time to outpatient review in second cycle was days. / received outcome a. / ( %) made a telephone call for advice and only / ( %) asked for a face to face appointment. / received outcome b and all were discharged after one visit. patients in cycle required operative stabilisation. statistically significant reduction in number of scheduled face-to-face reviews ( versus ; p \ . ) and mean time to first review ( days versus days; p \ . ). conclusion: virtual thoracolumbar fracture clinics are a safe and clinically effective alternative to traditional fracture clinic models. lean methodology can be uses to extend virtual clinic pathways to specialist trauma clinics. treatment prognosis of cases of fragility fracture of pelvis m. yoshida fujita health universityhospital, emergency, aichi, japan introduction: the number of cases of fragility fracture of pelvis in the elderly has been increasing in recent years, but there are still not enough reports of surgical treatment as a treatment method, but there is still no certainty how to treat. so we investigated prognosis of cases of fragility fracture of pelvis. materials and methods: subjects were fragility fracture of pelvis treated at a single center from april to april , males, females, average age ± . years. only cases that had ct scan were included. we examined rommens classification, the presence of injury, presence of hip implants, functional prognosis, and -year mortality. results: the breakdown of rommens classification is type ia cases, ib cases, iia cases, iib cases, iic cases there were cases of iiia, cases of iiic, case of iva, cases of ivb, and cases of ivc. surgical treatment was indicated in cases ( . %) (iic case, iiia cases, ivb cases, ivc cases) there were cases ( . %) with no injury mechanism and cases ( %) with hip implants. cases ( %) were able to follow up for more than year including telephone surveys, and . % of them did not recover to functional level before injury. the one-year mortality rate was . %. conclusions: in the cases studied here, cases ( . %) were indicated for surgery. the prognosis and mortality rate are almost the same as those reported overseas, and as with proximal femoral fractures, there is a possibility that it may be greatly involved in adl decline in the elderly. we think that further study is needed in the future. conclusions: patients with a femoral neck fracture who received a hip hemiarthroplasty and used anticoagulation had no significant longer delay to surgery and had a higher mean loss of hemoglobin points. as a clinical consequence of this, more packed cells were supplemented. also more postoperative hematomas were found in the population with anticoagulation. no differences were found in mortality rates at -days and one year. results: on all eight patients the easy-approach was applied without adverse events. in four cases the plate osteosynthesis was done completely endoscopically with excellent results for the patients regarding pain relief and scar development. in the remaining four cases the endoscopic stabilization was not performed for the following reasons: in the first overall case primarily only the endoscopic approach was planned. in the fourth overall case, ventilation showed high end-expiratory co -levels after endoscopic situs preparation, so we converted to the open plating. in the fifth overall case, the easyapproach was applied to evacuate a retrosymphyseal hematoma in a patient with a stable pubic rami fracture. in the eighth overall case, the anterior pelvic ring injury was a bilateral multifragmentary pubic rami fracture in combination with a disruption of the symphysis. after endoscopic situs preparation with clipping of the corona mortis vessel, reduction of the displaced symphysis could not be done endoscopically. conclusions: we demonstrated that the endoscopic plate osteosynthesis of the anterior pelvic ring is feasible with existing standard laparoscopic instruments. the evaluation of the easy-approach in the clinical setting is going on, while the development of suitable reduction tools is one major goal of future studies. introduction: retrograde intramedullary pubic ramus screw fixation is less invasive method and biomechanically stable compared to the plate fixation. the purpose of this study is to examine the feasibility of screw insertion using computed tomography (ct). materials and methods: we analyzed sixty ct data ( cases in male and female each). by using ct analyzing software, the virtual column with . mm diameter was inserted so that we analyzed the feasibility of the screw insertion. and the intramedullary diameter of the pubic ramus at the parasymphyseal area, base, and acetabulum were measured. results: the virtual . mm diameter screws could be inserted in % ( / ) in male and . % ( / ) in female. the cause that screws insertion was impossible was penetration to the hip joint in all cases. the screw inserting point was . mm and . mm from the medial border of the pubic symphysis and . mm and . mm from the upper border of the pubic symphysis in male and female respectively (p [ . ). the intramedullary diameter of pubic ramus was . mm, . mm and . mm at parasymphyseal area, . mm, . mm and . mm at the base of pubis, and . mm. . mm and . mm at the acetabulum in male, female who had the screw corridor and female who didn't have the screw corridor respectively. the diameter of the pubic ramus of the female who didn't have the screw corridor was significantly small compared to male and pubic ramus in three measuring points (p \ . ). , % of the screws were revised. there were no neurovascular or urologic complications. radiographic nonunion was observed in % with a minimum follow-up of months, this correlated with a peri-implant infection (p . ), operation [ months after trauma (p . ) and non-significantly with implant loosening (p . ). there was no correlation of nonunion with patient's age, the fracture mechanism or a non-excellent reduction. in total, . % of the patients were re-operated, in . % a re-osteosynthesis was conducted. conclusions: retrograde trans-pubic screws show good clinical results with lower or similar complication rates compared to alternative methods as plate fixation or external fixator. fracture union did not depend on fracture mechanism or age. hence, this minimal-invasive method is especially attractive in elderly patients with an ffp. because it is an internal fixation of the superior pubic ramus with relative stability, an anatomic open reduction is not necessary to achieve fracture union. the need for extraperitonal pelvic packing -finally confirmed to be vanishing? introduction: the presence of cerebral venous thrombosis (cvt) is increasingly recognized in traumatic brain injury (tbi), but its complication rate and effect on outcome remains undetermined. in this study, we characterize the complications and outcome-effect of cvt in tbi patients. materials and methods: in a retrospective, case-control study of patients included in the oslo university hospital trauma registry and radiology registry from - , we identified patients with cvt (cases) and without cvt (controls). groups were matched regarding abbreviated injury severity (ais) head region score - . cases were identified by ais or icd-code for cvt and a ct/mr venography confirmed to be positive for cvt, whereas controls had no ais or icd-code for cvt and a ct/mr venography confirmed to be negative for cvt. risk of mortality was assessed using multivariate logistic regression adjusting for initial gcs, iss and rotterdam score. results are also reported for subgroups according to cvt location ( fig. introduction: the aims of this prospective cohort study were (i) to identify trajectories of recovery in patients with mild traumatic brain injury (mtbi) during the first two years after trauma and (ii) assess patients and injury characteristics for these trajectories. materials and methods: all adult trauma patients with mtbi (aisseverity or and an injury severity score \ ) who were admitted to a hospital in a region of the netherlands from august to november were asked to complete questionnaires. the questionnaires could be completed at week, and , , , and months and included the euroqol- -d for health status, including a cognition dimension, the hospital anxiety depression scale (hads-d and hads-a for symptoms of depression and anxiety respectively) and the impact of event scale (ies) (for post-traumatic stress symptoms). latent class trajectory analysis was used to determine trajectories of recovery in latentgold . , patient and injury characteristics of the classes were assessed in ibm spss . . results: a total of patients ( % of total) completed at least one follow-up questionnaire. the number of classes (trajectories) ranged from for cognition to for depression. poor recovery classes of cognition and health status consisted of mostly females, patients with low education, higher age, longer length of stay at the hospital and frail patients. the class with full recovery consisted of young patients, with most recovery occurring during the first six months after injury. patients who reported poor health status before injury scored significantly lower health status after injury and showed no recovery over time. conclusions: different recovery patterns were present in patients with mild traumatic brain injury. especially frail elderly patients who reported poor health status before injury have poor outcome up to months after injury. post-concussive symptoms in children and adolescents with traumatic brain injury: a center-tbi study introduction: acute respiratory is associated with high morbidity and mortality. in addition, its etiologies are heterogeneous and the outcome depends on the underlying cause. the aim of the present study is to analyze, whether the mortality of posttraumatic ards is affected ( ) over time, ( ) attributable to geographic distribution, ( ) related to the used definition and ( ) introduction: many factors of trauma care have changed in the last decades. this review investigated the effect of these changes on overall and cause-specific mortality in polytrauma patients admitted to the intensive care unit (icu). moreover, changes in trauma mechanism over time and differences between continents were analyzed. materials and methods: a systematic review of literature on overall mortality in polytrauma patients admitted to the icu was conducted. overall and cause-specific mortality rates were extracted as well as the trauma mechanism of each patient. linear regression on changes in overall and cause-specific mortality rates was performed. results: thirty studies, which reported mortality rates for , observed patients, were included and showed a decrease of . % in overall mortality per year ( fig. ). brain-related death has become more common over the years, whereas multiple organ dysfunction syndrome (mods), acute respiratory distress syndrome and sepsis became less prevalent (fig. ) . mods was the most common cause of death in north america and brain-related death was the most common in asia, south america and europe (fig. a) . penetrating trauma was most often reported in north and south america and asia (fig. b) . conclusions: overall mortality in polytrauma patients admitted to the icu has been decreasing as a result of the improvements in trauma care. a shift from mods to brain-related death could be observed. more research on preventative measures for the latter is required to ensure a further decline in mortality. moreover, we have shown geographical differences in cause-specific mortality, which may provide learning possibilities between similar trauma centers resulting in improvement of trauma care introduction: aim of the current study was to assess an association between trauma patient volume of the intensive care unit and inhospital mortality. materials and methods: from data of the japan trauma databank, this retrospective cohort study selected adult (c y) trauma patients hospitalized in the intensive care unit with the injury severity score of c . after applying a multiple imputation on all the study variables, a logistic regression generalized estimating equation after adjustment for age, sex, mechanism of trauma, and the injury severity score as covariates and hospitals as a cluster assessed an association between quartile of patient volume in intensive care unit and hospital mortality. introduction: quality and content of early fracture hematoma (fh) dictate the healing process in long bone fractures. different reaming protocols for intramedullary nailing (imn) are available. however, the impact of reaming strategies on immune cell characteristics of early fracture hematoma is unclear. we hypothesized that the application of reaming irrigation and aspiration (ria) techniques optimizes cellular content of fracture hematoma. materials and methods: twenty-four pigs underwent standardized femur fracturing. then, animals were exposed to different protocols of imn. group a underwent no reaming prior to imn. group b was treated with conventional reaming plus imn and group c composed of animals treated with ria and subsequent nailing. fracture hematoma was collected h after reaming. fh-immune cells were isolated and studied by flowcytometry. cell viability was tested by annexin-v-labelling. neutrophil activation was determined by mac- /cd bcell surface expression levels, whereas fcyriii/cd -receptor expression was utilized to investigate neutrophil maturation. results: all animals survived the observation period. propertions of white blood cell subtypes in fh did not differ between conditions. however, the percentage of viable fracture hematoma immune cells was significantly higher in the ria-group, compared with conventional reaming (respectively mean . % vs. . %, p = . ). additionally, both neutrophil cd -expression (- %) and cd bexpression (- %) were significantly lower in those animals treated with ria compared with the conventional reaming condition. conclusions: this experimental study reveals that reamed irrigationaspiration (ria) prior to imn is associated with increased immune cell viability and less neutrophil senescence/activation in early fracture hematoma. this underlines the important role of imn in optimizing local cellular immune homeostasis during the formationphase of early fracture hematoma. introduction: the study and determination of the traumatic pattern in bicyclists-delivery employees. the recording of personal protective equipment and evaluation of the selection criteria of their self protection. materials and methods: a total of patients ( men and woman) with mean age of . years ( - years) were included over a study period from january to march . twenty-one patients admitted to the hospital with a total of injuries treated operatively, whereas injuries were treated conservatively. we recorded and evaluated the use of adequate personal protective equipment of these delivery employees. results: the mean hospitalization time was . days ( - days) . a total of thoracic injuries, traumatic brain injuries, spine injuries, lower extremity injuries and upper extremity injuries were recorded. surgical treatment concerned patients with upper extremities and patients with lower extremities injuries and the anatomic regions involved were the distal radius ( ), pelvic ring injury ( ), femoral fractures ( ), tibial plateau fractures ( ), patella fractures ( ), diaphyseal tibial fractures ( ), and ankle fractures ( ) . conclusions: the lack of an adequate personal protective equipment due to their low financial status in combination with the absence of driving professional education among workers in this category of delivery employees results in lower extremity injuries with the majority requiring hospitalization and surgery. further investigation is needed, as well as constant training and setting right criteria for the pursuit of such employment. results: a total of nine rct's ( patients) and the sixteen observational studies ( patients) were included. the pooled nonunion rate did not differ significantly between both treatment groups (risk difference: %; or . , % ci . - . ). more patients treated with nailing required re-intervention (risk difference: %; or . , % ci . - . ) with shoulder impingement being the most predominant indication. more patients treated with pate fixation developed radial nerve palsy compared to nailing (or . , % ci . - . ). notably the absolute risk difference is small ( %) and during follow-up the palsy resolved spontaneously in the majority of patients. nailing lead to a faster time to union (mean difference: . week, % ci . - . ), lower infection rate (risk difference: %, or . , % ci . - . ) and shorter operation duration (mean difference: min, % ci . - . ). functional scores were comparable in both groups (standardised mean difference: - . , % ci - . to . ). there was no difference between effect estimates form observational studies and rct's. conclusion: there appears to be no difference between plate fixation and nailing for humeral shaft fractures with regard to non-union rate and functional outcome. patients treated with plate fixation have a higher risk for infection and radial nerve palsy, but lower risk for reintervention. the absolute differences, however, are small. nailing does differ significantly from plate fixation in terms of shorter operation duration and time to union. the pooled estimates from randomised clinical trials did not differ significantly from estimates obtained from observational studies. post-traumatic complications are more often after medial clavicle injuries compared to lateral clavicle injuries introduction: medial clavicle injuries (mci) are widely unexplored, especially in contrast to lateral clavicle injuries (lci). current research concerning mci assumes a higher severity of mci, e.g. concerning concomitant injuries. our aim is to evaluate by big data analysis if these rare injuries would also lead to a higher number of post-traumatic complications. materials and methods: we focused on the mci subgroup consisting of medial clavicle fracture and sternoclavicular joint dislocation. the lateral clavicle fracture and the acromioclavicular joint dislocation were summarized to the subgroup of lci. the midshaft clavicle fracture was analyzed for comparison. the data are based on icd- codes of all german hospitals as provided by the german federal statistical office. anonymized patient data from to were evaluated. the retrospective analysis addresses the fracture healing in dislocation, delayed union and non-union. results: the proportion of all patients suffering from complications was . %, which were attributed to one of the three post-traumatic complications. each complication rate for the single injury and the single complication was rather low with a maximum of %. mci were more likely to be affected by post-traumatic complications than lci with a ratio of . to . times (p \ . ). the midshaft clavicle fracture was similarly frequently affected by complications with . % of all complications as the mci ( . %). the lci accounted for the smallest proportion at . %. conclusions: we proved that mci are more often associated with post-traumatic complications than injuries of the other parts of the clavicle. this is another hint that mci appear to be more complex than lci. this could be due to a missing standard procedure and the higher number of concomitant injuries in mci. further representative clinical studies are required since miscoding is a frequent issue in research concerning clavicle injuries, especially in a big data analysis. quantification of trauma center accessibility using gis-based technology introduction: there is no generally accepted methodology to asses trauma system access and optimal geographical trauma center distribution. the goal of this study is to determine the influence of trauma center(tc) distribution during high and low traffic density using geographical-information-system(gis)-technology. methods: using arcgis-pro, we calculated differences in transport time (tt) and population coverage in seven scenarios with , , or tcs during rush [r]-and low traffic [l] hours in a densely-populated region with tcs in the netherlands (fig. ) . results: in the seven scenarios, the population that could reach the nearest tc within (\) min, varied between - % ( fig. ) in the three-tc-scenario, roughly % of the population could reach the nearest tc \ min in [r] and [l] . the hypothetical scenarios with two geographically well-spread tcs showed similar results as the current three-tc-scenario. in the one-tc-scenarios, the population reaching the nearest tc \ min decreased by - % in both [r] and [l] compared to the three-tc-scenario. in the three-tcscenario the average tt increased with about . min to almost min in [r] , in comparison to min during [l] (fig. ) . similar results were seen in the scenarios with two geographically well-spread tcs. in the one-tc-scenarios and the geographically close two-tcscenario the average tt increased by - min [l] and - min [r] in comparison to the three-tc-scenario. conclusion: this study shows that a gis-model for trauma center access offers a quantifiable and objective method to evaluate trauma system configuration in areas with different geography and demography. applying this technology to one of the most densely populated areas in the netherlands shows that the transport time from accident to trauma center would remain acceptable if the current situation with three trauma centers would be changed to a scenario with two geographically well-spread centers. classifying posttraumatic stress disorder courses in physical trauma patients: an observational prospective cohort study introduction: the aim was to identify different courses of posttraumatic stress disorder (ptsd) in physical trauma patients. then, to examine whether these classes could be characterized by sociodemographic, clinical, psychological, and personality outcomes. methods: patients completed the impact of event scale-revised (ies-r), m.i.n.i.-plus after inclusion, , , , and months after injury to examine different courses. the hospital anxiety and depression scale, neo-five factor inventory, state-trait anxiety inventory-trait, and the whoqol-bref were completed after inclusion only. latent class analysis, chi square tests, and anova were performed to analyze the aims. results: in total, patients were included. the mean age was . (sd = . ) and % were male patients. the ies-r (see figure ) and the m.i.n.i-plus had five classes ( : moderately, : little bit, : worse, : none, : quite a bit of ptsd symptoms). patients in class are diagnosed with ptsd (cut-off score c ). on both questionnaires, patients (proportion & %) in class or , scored higher on anxiety, depressive symptoms, neuroticism, and trait anxiety compared to the other classes over months after trauma. lower scores on all domains, except for social domain on the ies-r, were found compared to the other classes (ies-r; physical domain: class vs. (mean ± sd): . ± . vs. . ± . , p-value = \ . ). psychological and personality outcomes were significantly different on all courses. also, patients in class or were younger compared to the other classes (ies-r; class vs. : . ± . vs. . ± . , p-value = \ . ). no medical outcomes for ptsd were found. conclusions: about % suffer from ptsd symptoms months after trauma. different courses were defined by sociodemographic, psychological, and personality characteristics. professionals can, short after trauma, recognize patients at risk for ptsd when they focus on these characteristics. then, an intervention can be offered. six meter, the criterion for severe adult trauma to falls from heights in cdc field triage needs to be lowered introduction: trauma is one of major public health care issue which is costly to society. differences vary from region to region, but blunt trauma accounts for a large part of the total trauma, and the rates of the falls from heights among the blunt trauma is getting higher. it is serious that falls from heights is often accompanied by severe multiple trauma. therefore, authors studied the relationship between the height of the fall/other related factors and outcomes including hospital stay/mortality. materials and methods: retrospective cohort study of the adult falls-from-heights patients visited a regional trauma center for years (from . . to . . ). results: of total patients, the number of d.o.a patients were . the height from falls of the deceased patients was statistically significantly higher than that of the survived patients. ( . ± . m vs. . ± . , p \ . ) the auc of the roc curve of the height from fall to mortality was . . (figure) the sensitivity of . m was . % and . m was . %, respectively. the traumatic brain injury, pelvis fracture, visceral organ injury, age, and the height from fall were statistically significant risk factors in multivariate analysis for mortality (p = \ . , . , , , . , and . respectively). conclusions: the height from the fall is closely related with mortality. we think the current height for the severe fall injury in cdc field triage for trauma is high and needs to be lower to . introduction: operative management of severe trauma is a team effort, requiring excellent communication skills. surgeons, anesthesiologists and nurses need to coordinate effectively in order to ensure an excellent clinical outcome. the definitive surgical trauma care (dstc), definitive anesthesia trauma care (datc) and definitive perioperative nurses trauma care (dpntc) courses provide an excellent opportunity to train efficient teamwork. we aimed to study the impact of the joint dstc-datc-dpntc courses in candidates' perceptions and skills in perioperative communication. materials and methods: study population of candidates ( surgeons, anesthesiologists and nurses) participating in a joint dstc-datc-dpntc course in coimbra, portugal. median age of years (range - ). female gender in ( %) of cases. all participants attended joint lectures, case discussions and surgical skills session, emphasizing intraoperative communication. postcourse survey on several aspects of peri-operative communication, with responses on a likert scale. participants were also asked which aspects of intraoperative communication they valued the most. statistical analysis with spps, . (wilcoxon signed rank test, significance with p-value \ . ). results: all participants responded to the survey. results displayed an increase in the self-assessed importance of team briefing and intraoperative communication, particularly routine periodic communication, rather than only at critical moments (p \ . ). postoperative team debriefing was also valued as highly relevant. closed-loop and direct, by-name communication were highly rated (p \ . ). self-reported communication skills improved significantly during the course (p \ . ). conclusions: joint training in the dstc-datc-dpntc courses provides a unique opportunity to improve candidates' self-awareness and skills in intraoperative communication. a public health approach to knife related trauma in liverpool: a geospatial study r. shellien , n. misra , , j. germain , m. whitfield aintree university hospital, emergency general surgery and trauma unit, liverpool, united kingdom, liverpool john moores university, public health institute, liverpool, united kingdom introduction: liverpool is a city that has undergone recent rapic socioeconomic change. despite reductions in overall deprivation, incidents of stabbings have increased by % in the last years. this study will describe the trend in knife crime, drawing on governmental data and policies to conclude the reasons behind the trend. materials and methods: a retrospective cohort study of patients presenting to north-west ambulance service (nwas) with a penetrating injury in liverpool between and . data collected included patient demographics, geography and timing of incidents and correlation to datasets of multiple indices of deprivation and knife crime prevention outreach education programmes. results: incidents of stabbings have increased by % between and . victims were more likely to be males ( %) between the ages of and ( %). the peak rate was between : - : ( . %) and trough between : - : ( . %). there is a spike in incidents of stabbings of - year olds from : to : , correlating with school closure. there appears to be statistically poor correlation between deprivation of lower super output areas and stabbings (r = . , . and . for , and respectively). however, when the data is split into larger areas, middle super output areas (msoas), deprivation appears to be a further risk factor. this study has identified certain geographical areas as high risk. conclusions: this study allows for targeted public health interventions at populations most at risk of knife trauma, including geographical mapping of high-risk areas, so that interventions can be distributed appropriately. references: ministry of housing, communities and local government ( government ( , government ( , introduction: trauma teams treat complex patients with injuries posing significant resuscitative and management challenges. effective teamwork is essential to optimise patient outcomes and improve survival, with failure contributing to adverse events [ ] . the role of multidisciplinary (mdt) trauma training has been demonstrated by the military operational surgical training course (most) [ ] . it is imperative that civilian trauma training adopts similar methodology to optimise team work. materials and methods: the three-day multidisciplinary trauma course comprised cadaveric-based skills teaching supplemented by lectures and real-life scenario discussion. delegates were senior surgical and anaesthetic registrars and consultants, alongside trauma team leaders (ttl), scrub staff and operating department practitioners (odp). pre-and post-course questionnaires assessed perceptions of multidisciplinary trauma simulation and confidence in specialty specific skills. results: all delegates reported mdt simulation clarified each role, including their own, in the trauma team. post-course, scrub staff and odps felt confident gaining intraosseous access (p \ . ), surgical delegates had improved confidence performing all skills (p \ . ), with anaesthetists and ttls more confident in haemorrhage control and performing resuscitative thoracotomy (p \ . ). conclusions: mdt trauma training improves team understanding of role and effectively teaches skills. mdt courses with experienced faculty are one way of improving mdt trauma team function. further careful evaluation is required to assess performance of trauma teams in real scenarios. introduction: despite a dramatic rise in youth knife crime, the factors associated with it remain underexplored, especially in the critical pre-college years, which hinders effective counter-knife carrying interventions. the current research is the first to addresses this deficit. materials and methods: british male school students (mean age = . , sd = . ) coming from four different schools completed a short -min survey. they indicated their standing on a number of dimensions (school-adapted and shortened-scale-based predictors) derived from theories of violence, developmental psychology and related research (i.e. violence acceptance, need for respect, belief in self-defence, belief in a just world, narcissism, psychopathy, impulsivity, sensation seeking, and need for closure). results: for perceived knife harmfulness (i.e., the knife's assumed value in inflicting injury and death)-the total variance explained by the model was . %, r = . ; f( , ) = . . the only statistically significant predictors were: right-wing authoritariamism (b = . , p = . ) and need for respect (b = . , p = . ). the other factors were not statistically significant. for the perceived value of knife defence (i.e., its assumed defensive worth in violent confrontations) -the total variance explained by the model was . %, r = . ; f( , ) = . , pviolence acceptance (b = . , p = . ), followed by need for closure (b = . , p = . ), narcissism (b = . , p = . ) and psychopathy (b = . , p = . ). conclusions: this study provides evidence for future knife-carrying prevention interventions, such as talks in schools or social media videos, to focus more on how to increase self-esteem, stimulate empathy for and better understanding of other people, and approach problems from multiple (rather than just two) perspectives, emphasizing the ultimate superiority of the human intellect over brute force. introduction: the physician's response unit (pru) is a novel service that operates from the royal gwent hospital's emergency department (ed), in newport, south wales. it involves an emergency medicine consultant and a paramedic responding to calls in a rapid response vehicle. their aim is to treat and, hopefully, discharge patients at the scene, reducing ed admissions. the pru can also refer patients on to other departments, e.g. the medical assessment unit, allowing patients to bypass the ed. methods: the author spent six weeks out in the pru and in the ed to observe and speak to patients. to assess whether ed admissions were reduced, the dispositions of patients seen by the pru were recorded on a daily log sheet. the service users' satisfaction with the pru was evaluated using simple questionnaires. this included both patients and paramedics, who can request the pru for support with a patient. results: the pru saw patients during the project's timeframe. % (n = ) of these patients were discharged at scene, while % (n = ) were sent to the ed. % (n = ) of patients asked described the care they received from the pru as equal to or better than care they have received previously. % (n = ) of patients rated their overall satisfaction with the pru as / . conclusions: the pru is very well received by both patients and paramedics and has been shown to reduce the number of patients attending the ed. this system excellently implements the principles of prudent healthcare introduction: in germany reducing alcohol related harms in youth is still a priority, because adolescents and young adults still have the highest accident risk in road traffic. therefore, the p.a.r.t.y.-project aim to increase awareness of alcohol and risk-related issues. the purpose of this study was to analyse the risk behaviour of adolescents before and after a prevention project in two different hospitals in germany. materials and methods: during a one-day prevention project, young people within the age of to years got an overview of the route an accident victim go through from the ambulance until the rehabilitation. before and after the prevention day, a structured written survey was completed by the adolescents. results: students participated in the p.a.r.t.y. program between and . the gender distribution of the participating students were balanced. the average age of the adolescent was years. according to the program, the risk assessment and risk behaviour improved through the project significantly (\ . ). the evaluation of the students' satisfaction was rated as good. the majority of students prefer to repeat the project day after years. conclusions: the prevention program shows that the program increase for short-term the awareness for risk related trauma in youth. nevertheless, long-term studies are necessary to receive data regarding the long-lasting effect. references: the present study is funded by the ministry for energy, infrastructure and digitization of the country mecklenburg-vorpommern, germany. development of a claims-based risk adjustment model for trauma introduction: duodenal injury is rare. the diagnosis requires a high index of suspicion which might result in delayed treatment. there is limited data on the delayed diagnosis group, especially high grade duodenal injuries. the purpose of this study is to determine the characteristics and outcomes of delayed high grade duodenal injuries. materials and methods: charts of all patients from - who had history of small bowel injuries are reviewed. the inclusion criteria were age between - years old, diagnosis with duodenal injuries at least grade with delayed operation at least h after injuries. baseline characteristics and postoperative outcomes were recorded. results: of the small bowel injuries, ( %) were duodenal injuries. the overall mortality was %. delayed diagnosis more than h with at least grade of duodenal injuries were cases. the overall in-hospital mortality rate of the delayed group was . % ( / ) who had concomittent hemorrhagic shock and low initial systolic blood pressure. cases ( . %) were diagnosed within h and had better outcomes without leakage. they could step diet within days and had shorter length of hospital stay (mean = days). patients ( . %) presented with delayed diagnosis more than h (the maximum was h after injuries). all these patients had anastomosis leakage and need reoperation. they had initial low level of serum albumin (mean . mg/dl), high white blood cell count, low serum bicarbonate and presented with preoperative acute kidney injury. conclusions: delayed diagnosis and surgical treatment of high grade duodenal injuries lead to poor outcome. low initial blood pressure associated with mortality and delayed treatment more than h had higher morbidity. references: gary sa, frederick am, charles sc, et al. delayed diagnosis of blunt duodenal injury: an avoidable complication. acs meeting. ; ( ) : - . routine follow-up imaging has no advantage in the non-operative management of blunt splenic injury in adult patients modality. the aim of this study was to investigate the incidence and time to failure of nom as well as to evaluate the relevance of follow-up imaging. materials and methods: all adult patients with bsi admitted to our level i trauma center, including two associated hospitals, between / / and / / were retrospectively analyzed. demographic data, injury severity score, splenic injury grade, modality, results and consequences of follow-up imaging were retrospectively analyzed. results: a total of patients with a mean age of . ± . years ( - years) met inclusion criteria. patients ( . %) underwent immediate intervention. patients ( . %) were treated by nom. failure of nom occurred in patients ( . %). failure was significantly associated with active bleeding (or . , % ci . , . , p = . ) , and liver cirrhosis (or , % ci . , . , p = . ) . patients ( . %) in the nom-group received followup imaging by ultrasound (us, n = ) or computed tomography (ct, n = ). in cases, routine imaging examinations were conducted ( us and ct scans) without prior clinical deterioration. ( . %) of these imaging results revealed no new significant findings. every failure of nom was detected following clinical deterioration. conclusions: to our knowledge this study includes the largest monocentric patient cohort undergoing ultrasound as first-line followup imaging modality in the nom setting of bsi in adult patients. the results indicate that a routine follow-up imaging, regardless of the modality, has no therapeutic advantage. indication for radiological follow-up should be based on clinical findings. if indicated, a ct scan should be used as preferred imaging modality. the association between bmi and mortality of renal injuries in adult trauma patients introduction: the role of body mass index (bmi) on solid organ injuries remains debatable. while some studies have shown no association between bmi and hepatic or splenic injuries, others have reported that severe hepatic injuries were more common in pediatric patients with bmi [ . the aim of this study is to examine the association of bmi and mortality, as well as any significant differences between operative vs. non-operative management. materials and methods: this was a retrospective study using the american college of surgeons-trauma quality improvement program database to identify all adult patients (ages to \ ) with traumatic renal injuries. the primary analysis showed a different pattern of mortality between patients with bmi \ and those with bmi c kg/m . then, the study population was divided into patients with bmi \ and those with bmi c kg/m . multivariable logistic regression was conducted to assess any association of mortality with age, gender, bmi, and injury severity score (iss). results: adult trauma patients were identified. a greater proportion of males ( . %) and females ( . %) had bmi \ kg/m (p = . ). the average age of patients with bmi \ kg/m was . (sd = . ) years which was significantly younger than that in patients with bmi c kg/m , . (sd = . ) years (p = . ). patients with bmi \ kg/m were found to have a significantly higher mortality rate of . % vs. . % in patients with bmi c kg/m (p = . ). however, there was no significant difference in type of operative or nonoperative management between patients with bmi \ vs. bmi c kg/m . after multivariable logistic regression, mortality was associated with age, bmi and iss. no effect modification of sex was observed in the relationship of mortality and bmi. conclusions: adult patients with renal injuries and bmi \ kg/m have significantly higher rates of mortality compared with adult patients with renal injuries and bmi c kg/m . introduction: trauma is an ever-evolving surgical discipline. trauma remains a major source of global mortality. the operative and non-operative options for trauma patients has steadily increased. the development of trauma protocols, advancement in transport to trauma centres and radiological techniques has seen a shift in trauma surgery caseload. observing and understanding this shift from operative management to an increasing non-operative management of trauma cases will better prepare the acute medical team in this setting. materials and methods: prospective trauma registry data was collected and analysed retrospectively. patients presenting to a tertiary referral hospital between jan to dec with an injury severity score of [ were reviewed. patients who were transferred to another facility for management were excluded. the demographic data and surgical outcome data were collected and analysed. trend analysis of the operative cases performed for each specialty. results: major trauma patients presented to the john hunter hospital between january to dec . there was a non-statistically significant increase in the number of presentations ( pt in vs in , p = . ). there was a decreasing rate of operations performed for trauma patients ( % in vs % in , p \ . ). there was an increasing rate of orthopaedic surgery cases and operative time compared to other specialties ( in vs in , p \ . ). general surgical major trauma operating cases noted a significant decline over the study time ( in vs in , p \ . ). conclusions: there is a sizeable shift in the caseload of different surgical specialties in regard to major trauma patients over the course of years from to . orthopaedics has seen a significant increase in operative caseload and surgical time required to adequately manage major trauma presentations. the workload and experience of general surgical teams will likely be affected by these changes. the distribution of resources needs to be reflected in the changing work demands of each surgical subspecialty. traumatic internal hernia with delayed small bowel strangulation after pelvic ring injury hospitalization, follow up abdomen ct checked. there was no other specific change than increased thigh hematoma. eight days after hospitalization, ct was re-examined due to abdominal pain with abdominal distraction. an ct showed peritonitis with pneumoperitoneum and small amount of ascites. small bowel herniation through right pubic bone fracture site with ischemic change also noted. diagnosis: diagnosis was traumatic pelvic hernia with delayed small bowel strangulation. therapy and progressions: an emergency operation was performed. ileal loop was hernitated and perforation was found. emphysematouns change and fluid collection was exsited at perineal area and left high. after small bowel loop segmental resection, wound vac was applied at thigh area. comments: traumatic pelvic hernia is rare. diagnosis is challenging in the acute setting and often delayed due to lack of awareness. when diagnosed, efforts should be made to look for other serious injuries as traumatic pelvic hernia usually associated with concomitant intraabdominal injuries. the optimal management of traumatic hernia should be individualised based on the mechanism and severity of injury, presence of concomitant injuries, size of defect, and presence of incarceration. delayed treatment may read to fatal outcomes. careful inspection of the patient is important. references: vincent k, cheah sd. traumatic abdominal wall hernia-a case of handlebar hernia. med j malaysia. ; ( ): - . angio-embolization in pediatric trauma patients with blunt splenic injury: a systematicreview t. nijdam , r. spijkerman , l. hesselink , t. hardcastle , l. leenen , f. hietbrink umc utrecht, traumasurgery, utrecht, netherlands, inkosi albert luthuli central hospital, trauma, durban, south africa introduction: non-operative management (nom) for children with blunt splenic injury (bsi) is nowadays a commonly used treatment in pediatric trauma departments. in adult trauma departments the addition of splenic angio-embolization (sae) is suggested to decrease the failure rate of nom in high grade splenic injuries. however, the use of sae in pediatric trauma departments is very uncommon and it is unknown if sae is of additional value in pediatric trauma patients. therefore, the aim was to analyze the available literature on sae in pediatric trauma patients with bsi. materials and methods: a literature search was performed to find eligible studies that analyzed sae in pediatric patients with bsi. the primary outcome was failure of treatment in these patients. secondary outcomes were the success rate of sae, length of stay and mortality. the relative risk (rr) was calculated to compare primary outcome between study groups. results: in total studies were identified through the search, a total of studies matched our inclusion criteria and were selected for this review. studies included a total of . pediatric patients, of whom underwent sae. patient age ranged from &lt; year to years, mean age was . years. both injury severity score and spleen injury grade were higher in the sae group compared to the nom group. failure rate of sae was %. no spleen related morality was observed in the sae group. conclusions: the literature suggests that sae might be of added value in a very selective group of pediatric trauma patients with high grade splenic injures. however, since limited evidence is available concerning the use of sae in pediatric trauma patients with bsi, no firm conclusions can be drawn about safety and effectiveness. introduction: the management algorithms for trauma have changed with the development of specialised trauma centres. the aim of this study was to review the management and outcomes of patients with traumatic small bowel (sb) and colonic injuries. material and methods: patients treated for sb and colonic injuries between - at aintree university hospital (liverpool) were identified using the prospective trauma audit and research network database. the management and outcomes of the patients included were analysed. results: patients sustained sb and colonic injuries. there were ( . %) sb injuries and ( . %) colonic injuries ( patients had a sb and colonic injury). patients ( . %) of injuries were due to knife stabbing wounds, ( . %) patients were due to gunshot wounds, and ( . %) patients were due to road traffic accidents/ blunt blows. damage control surgery was performed in ( . %) patients. colonic injuries included ( . %) haematomas and ( . %) perforations. a resection and stoma (rs) procedure was performed in patients ( . %), primary repair (pr) in patients ( . %) and resection with anastomosis (ra) in patients ( . %). sb injuries included ( . %) haematomas and ( . %) perforations. pr was performed in ( . %) cases and ra in ( . %) cases. the overall complication rate after sb and colonic injury was % ( patients) with a significant complication rate ( patients, p value = . ) for patients undergoing rs in colonic trauma. the -day mortality rate was . % ( patient). conclusions: pr in sb and colonic injuries appears safe. in our dataset, rs appeared to have a higher complication rate. our study highlights that such injuries are uncommon with a high complication rate. surgeons need to provide individualised treatment. introduction: nowadays, patients with high grade bsi are preferably treated using spleen preserving treatments (spt). it is assumed that patients with low grade bsi treated with spt have a good splenic function after recovery. however, there is no consensus on splenic function after high grade bsi. in several institutions, asplenic/hyposplenic infection prevention protocol will be executed in all patients who had spt after high grade bsi, where other institutions evaluate splenic function first. scintigraphy is believed to be the best flow/activity test to approximate splenic functionality. the aim of the study was to analyze whether spleen injury grade is associated with diminished splenic function. secondarily, we aimed to evaluate whether splenic function testing is necessary in pediatric patients after bsi. material and methods: a retrospective study was performed from january to january . in our institution patients with bsi grade iv of v are assumed hyposplenic and will receive a splenic function test. we included all patients with a minimum follow-up test period of days. all tests were analyzed by the radiology specialist. for each patient we furthermore collected clinical data, including the date of trauma, gender, age, mechanism of injury, ais of splenic injury and iss. results: patients consisted of male and female, with a median (iqr) age of . ( . - . ) . median iss was . ( - . ) and the median spleen ais was ( ) ( ) . nom was used in patients, sae in five patients and two patients were treated with surgical mesh technique. the median follow-up time of all performed tests was ( - ) days. a total of patients ( %) had a grade iv or v splenic injury. scintigraphy was utilized to test most patients. a total of out of patients had an adequate splenic function, including all sae patients. conclusions: even high grade splenic injuries show adequate splenic function in the follow-up of pediatric trauma patients after bsi. therefore routine diagnostic follow-up by scintigraphy is not necessary in this specific patient group. evaluation of abdominal injuries treated at stavanger university hospital: occurrence, severity and mortality j. w. larsen , k. søreide , , j. a. søreide , , k. tjosevik , k. material and methods: retrospective evaluation of data recorded prospectively in the hospital's trauma registry between january and december . patients with abbreviated injury scale (ais) code for abdominal injury were included. descriptive analyzes are presented for demographic data, injury type, mechanism, and severity, as well as -days mortality. results: a total of patients with abdominal injuries were included ( . % of all trauma patients). % where men. median age was . the injury mechanism was blunt in %. transport accidents were the most frequent cause of injury ( %). median iss was , and median niss . overall -days mortality was . %, with a median trauma injury severity score (triss) of , . multiple abdominal injuries were recorded in % of the patients. % had associated injuries in other body regions, most frequently in the thoracic region ( . %). solid organ injury occurred in % of the patients, with liver injury ( %), splenic injury ( %), and kidney injury ( %) encountered most frequently. an ais score c was found in % of liver injuries, % of splenic injuries, and in % of patients with kidney injuries. hollow viscus injuries were found in % of the patients. injuries to the small intestine ( %) and colon ( %) were most frequent. abdominal vessel injuries were encountered in %, and % of these had an ais score c . conclusions: abdominal injuries are dominated by solid organ injuries following blunt injury mechanism and are often associated with concomitant thoracic injury. patients who dies within days from admission are characterized by a low probability of survival shown by triss. pancreatic trauma management in a third level centre a. gonzález-costa , r. gracia-roman , s. montmany-vioque , a. campos-serra , r. lobato-gil , c. zerpa-martin , f. j. garcía-borobia , p. rebasa-cladera , s. navarro-soto management. the aim of the study is to review the management and describe the most frequent complications of pancreatic trauma in our centre. material and methods: observational study with prospective collection of data, from march to march . inclusion criteria: trauma patients older than admitted to the emergency department who were admitted to icu or died before admission. demographic data has been collected, also vital signs, iss, mechanism of action, mortality, complications, and lesions. results: between and , polytraumatic patients were registered. only had pancreatic trauma ( . %). the male: female ratio was : ; with an average age of . years (sd . ) . mean iss of . (sd . ), mean ais of . (sd . ) and mortality of . % ( patients). the most frequent pancreatic lesion was at the head of the pancreas ( patients; . %), followed by body-tail ( patients; . %) and two patients with full section ( . %). . % of patients were treated with non-operative management. five patients required urgent surgery ( %), requiring corporocaudal pancreatectomy in cases and drainage in patients. an embolization of a gastroduodenal artery aneurysm was performed in patient. respiratory complications were the most frequent. patients developed a pancreatic fistula ( . %), although in surgical patients this complication was much higher ( % in our series). one of them required puestow pancreaticojejunostomy and patient developed necrotizing pancreatitis ( . %). conclusions: pancreatic trauma is very uncommon. its management can be difficult, depending on the degree of injury (aast), with a high rate of complications. therefore, combined management and monitoring by the surgery and intensive care team will be very important. introduction: the aim of this retrospective study was to evaluate and compare the clinical outcomes of conservative versus surgical treatment in a series of patients with liver injury. material and methods: between - , there were included patients. according the treatment chosen, the patients were subdivided in two groups. non-operative management was considered in hemodynamically stable patients. the failure of conservative treatment was defined as need to resort to operative management after a period of strict monitoring when the reason was related to the liver or associated injuries or need for late angioembolization. all hemodynamically unstable patients were subjected surgical treatment. results: conservative treatment was selected for patients and only in of them was failed due to associated delayed bleeding and small bowel injury. patients underwent emergent surgery which included packing, lobectomy and splenectomy. operative findings revealed grade iii liver injuries in % and grade iv in %. pneumonia, sepsis and ards were the most frequently associated complications. the overall mortality rate was . %. in patients of conservative group, non-surgical treatment failed with surgery being required. the mortality in the group of patients who underwent emergent laparotomy on admission was of patients. conclusions: conservative treatment of blunt traumatic hepatic injuries is applicable in patients presenting hemodynamic stability with mild hepatic injuries and it could be successful even in high graded injuries with low morbidity and mortality. surgical treatment is indicated in grade v injuries. nevertheless, failure of conservative treatment does not necessarily lead to an increase in the incidence of complications or mortality. with the trend towards more conservative management strategies, surgeons' exposure to laparotomies for blunt injuries in rtas has decreased. the aim of this study was to examine surgeons' exposure to laparotomies following blunt trauma which remains important to maintain low patient morbidity and mortality rates. material and methods: data was collected for adult patients admitted to mater dei hospital (malta) following rtas with ctproven intrabdominal injuries between january and january . results: patients ( ( . %) males vs. ( . %) female (p value \ . ), mean age = . years) were included in the study. patients ( . %) were car occupants whilst patients ( . %) were pedestrians. ( . %) patients had single intraabdominal organ injury, whilst ( . %) had multiple intraabdominal organ injuries. the -day mortality rate was . % ( patients). liver injuries occurred in ( . %) patients, splenic injuries occurred in ( . %) patients, kidney injuries in ( . %) patients and other organs were injured in ( . %) patients. conservative management was followed in ( . %) patients, angioembolisation was utilised in ( . %) patients and operative management was performed in ( . %) patients during the -year period. this resulted in trauma laparotomies following rtas per year. conclusions: only a minority of patients require operative management after rtas. surgeons in small countries have limited exposure to complex rta's. in view of the low exposure to emergency laparotomies following rtas, changes to our local training programme was done. trauma courses, lectures and fellowships in eu have been implemented to maintain surgical skills to an optimal level. references: european commission, annual accident report. european commission, directorate general for transport june . case history: a year old female presented to the accident and emergency department h post colonoscopy with complaints of left sided abdominal pain. this colonoscopy was requested under a -week wait for a history of chronic diarrhoea. this was a complete and uneventful examination ath the time, with random colonic and ileal biopsies taken. she attended a ? e with left sided abdominal pain increasing in severity. clinical findings: she was found to have an exquisitely tender abdomen, experienced more in the left upper quadrant. she was clinically shocked with a marked hypotension and tachycardia. investigation/results: a ct of her abdomen and pelvis showed free fluid within the abdomen and pelvis, with active bleeding and large haematoma adjacent to the spleen. the grade of splenic injury however was not commented upon by the reporting radiologist. interventional radiological embolism was considered but unfeasible as patient not stable haemodynamically. diagnosis: she was diagnosed with a splenic injury post-colonoscopy, with internal bleeding and haemodynamic instability. therapy and progressions: she underwent an emergency splenectomy overnight and was transferred to the intensive care unit for postoperative care. she recovered well, was stepped down to ward level care and was discharged with post splenectomy protocols, including all necessary vaccinations. comments: splenic rupture post-colonoscopy is a very rare event, with less than cases reported worldwide since . however, it still should be considered as a cause of a ? e presentation in patients with upper abdominal pain and haemodynamic instability after recent colonoscopy. we wanted to present this rare case to the international audience of estes congress to raise awareness of this rare complication. clinical findings: hemorrhagic shock and consciousness disorder were observed. her abdomen was distended, and she was intubated in the emergency room. investigation/results: ct revealed massive intra-abdominal bleeding. diagnosis: massive intra-abdominal bleeding due to hepatic laceration. therapy and progression: damage control surgery (dcs) and transcatheter arterial embolization (tae) were performed. she was transported to a hybrid operating room. she experienced cardiac arrest before operation. cardiopulmonary resuscitation was immediately initiated, resulting in the return of spontaneous circulation. laparotomy with perihepatic packing (php) was performed, but she experienced two more episodes of cardiac arrest during operation. then, tae was performed for right hepatic artery extravasation. after physiological function restoration, including rewarming, coagulopathy correction and hemodynamic stabilization in the intensive care unit. she gradually became hemodynamically stable. however, incomplete hemostasis was obtained at second-look laparotomy h later. because of bleeding, we repeated php. we performed cholecystectomy and abdominal closure after confirming complete hemostasis ( h post-accident). she was discharged ambulatory without neurological deficit (day ). comments: prognosis of traumatic cardiac arrest is generally poor, and survival without considerable neurological deficit is very rare. we reported a surviving patient with severe hepatic laceration. sharing of strategies and tactics, such as blood transfusion, tae, trauma team approach to surgery, early decision of dcs improves outcome of patients with severe abdominal trauma. references: resuscitation. ; : - . introduction: the spleen is the most commonly injured organ after blunt trauma. non operative treatment (nom) of splenic injuries has gained wide acceptance. transcatheter embolization of the splenic artery is considered a useful adjunct in aast lesions c without active bleeding. we report a retrospective review of all patients admitted to a level trauma center with blunt splenic injury from to and compare their treatment and outcome with a previous series from to , when angioembolization was performed only in case of contrast blush at ct scan. patients and results: from to june , patients with blunt splenic injuries were admitted to the ed of a level university hospital in milan, italy. men to female ratio was : ,the mean age . ± years (range - ), and the iss ± . (range - ). eight patients ( . %) underwent emergent splenectomy due to hemodynamic instability. of the stable patients treated with nom, those with aast lesions c (n = ) were submitted also to angiography and to embolization of the spleen ( %), either proximally ( ) or distally ( ). two nom failed, and the patients were submitted to splenectomy or distal embolization. the median hospital stay was . ± . days. the total spleen salvage rate was %. no associated abdominal injuries were missed in the nom group. in the previous series of patients (mean age . ± . years, range - , #:$ = : , iss ± , range - ), underwent emergency splenectomy ( %), and ( %) were treated conservatively, with only embolization ( , %) in case of aast c at ct scan. failure of nom were , and the spleen salvage rate . %. liver injury following multiple cardiopulmonary resuscitations case history: this is a case of a year old woman who presented to the emergency department (ed) due to worsening dyspnea complicated by two lengthy cardiac arrests. after the first resuscitation and return to spontaneous circulation (rosc), echocardiography was done and showed severely dilated right ventricle with strain, suggestive of massive pulmonary embolism, for which rtpa was given. arrest occurred again, and post rosc, heparin was started and the patient was transferred to the icu. extracorporeal membrane oxygenation (ecmo) was initiated but complicated by severe hemodynamic instability and a third cardiac arrest, so cardiopulmonary resuscitation (cpr) was performed till rosc and massive transfusion protocol was started for suspected intraperitoneal bleeding. clinical findings: after ecmo cannulation, abdominal distention was noted with a severe drop in hemoglobin and an increased intraabdominal pressure ( mmhg). abdominal bedside ultrasound showed significant amount of dense free fluid. the decision for an urgent exploratory laparotomy was made and the patient was taken to the operating room. therapy and progressions: deep liver laceration over the right hepatic dome with rupture of the capsule and an estimated hemoperitoneum of l were found intra-op. controlling the bleeding was difficult due to the laceration site and the patients coagulopathic status, so packing was done and the patient was transferred to icu for correction of the coagulopathy and re-evaluation in h. the liver was unpacked after h, bleeding sites were cauterized and sutured and the liver was wrapped with a mesh with an attempt for a tamponade effect. the patient's stay in icu was complicated with kidney injury requiring chronic dialysis but otherwise recovered well. comments: liver injury is a rare but serious complication after cpr that should be considered in case of persistent hemodynamic instability along with bedside findings. this case is intriguing due to the right sided liver injury with no overlying rib fractures. blunt renal trauma after electrical injury: a series of curious events. a. nixon , e. falidas , d. davris , a. botou , g. sofos chalkida general hospital, department of surgery, chalkida, greece case history: a yr old patient was referred to the emergency department (ed) of our hospital from a primary health center after sustaining an electrical injury ( v ac). the patient experienced loss of consciousness (loc) and promptly fell to the ground in a supine position. the patient arrived approximately h after the incident. clinical findings: vital signs: bp: / mmhg, hr: bpm. the patient's major complaint was left flank and abdominal pain. no obvious thermal injuries were observed or any other signs of external trauma. a left abdominal mass developed which was evident on physical examination. in addition, examination of urine revealed gross hematuria. investigation/results: ekg monitoring documented sinus tachycardia without evidence of cardiac arrhythmias. fast indicated the presence of a massive retroperitoneal hematoma. the fast exam indicated the left kidney as the probable source of hemorrhage. the initial hematocrit (hct) from the primary health facility was % while results from the ed recorded a hct of %. diagnosis: grade v renal trauma. therapy and progressions: a massive transfusion protocol was initiated. the patient underwent an emergency laparotomy and a left nephrectomy was performed. subsequent imaging did not reveal other injures. comments: the history of electrical injury could have misdirected investigation efforts towards cardiogenic shock. this case suggests that even in the absence of a high energy impact, sustained hemodynamic instability should always be attributed to hemorrhagic shock until disproven. in addition, the management of grade v renal trauma in blunt injury remains a controversial topic, however we believe that in cases of class iv shock, surgical management is imperative. case history: y.o. female with a history of chagas' disease of years duration and esophageal involvement in the last few months. she's admitted for a first endoscopic balloon dilatation due to dysphagia, which is performed according to protocol, and a tear of the mucosa layer is observed during it. clinical findings: she's stable for the first h but with continuous thoracic pain of moderate intensity according to the gi specialist. on the second day there's a general worsening of the patient's condition, with dyspnea, fever, desaturation and tachycardia. results and diagnosis: she develops leukopenia and elevations of acute phase reactants, and a ct scan reports a distal esophageal perforation with free extravasation of contrast in the mediastinum and bilateral pleural effusions. therapy and progressions: emergency surgery is performed through a midline supraumbilical laparotomy which shows peritonitis around the epigastric area. after opening the hiatus, a very long transmural esophageal tear with devitalized tissues and severe contamination are observed. a trans-hiatal esophagectomy was decided and, given the hemodynamic stability, a gastroplasty is performed and brought up to the neck without anastomosis, along with a terminal cervical esophagostomy and feeding jejunostomy. the patient did well in the postop period. we were able to do the esophagogastric anastomosis in the neck days later, during the same admission. comments: the surgical technique in esophageal perforation depends mainly on the time elapsed since the perforation, and on the condition of the patient. esophagectomy is sometimes unavoidable, and a gastroplasty can be brought up to the neck at the same time in selected cases, with reconstruction of the upper gi tract during the same admission. introduction: the spleen is one of the most frequently injured abdominal organ. the anatomy of the lesion defines the degree according to aast, ranging from grade i to v in increasing complexity. the diagnosis of splenic trauma may be difficult, as % of patients may show no signs or symptoms at primary survey. the approach involves two main strategies: conservative or surgical. the strategy should take into account four aspects: hemodynamic status, anatomy of the lesion, associated injuries and organizational structures of the evaluation site. this study aims to evaluate the type of approach performed on different degrees of splenic trauma during years in a portuguese trauma center. material and methods: we conducted a retrospective study including all patients diagnosed with splenic trauma during a period of seven years. by consulting the patient's clinical files we evaluated and compared: demographic data, trauma kinetics, degree of splenic injury and the approach taken as well as morbidity and mortality. results: of the patients studied, most were male with blunt trauma. in patients the inicial approach was surgery and in the option was conservative treatment. in grade iii or iv lesions conservative treatment failed in % of patients. patients in whom the surgical approach was first chosen had predominantly grade iv lesions, with total splenectomy being the preferred approach. in grade iii lesions, the option was mainly conservative surgery of the spleen. conclusions: the initial approach of splenic trauma results essentially of the experience of emergency teams and support structures for surveillance and intervention (intervention radiology and -h operating room availability). the attempt to try conservative strategy is increasing over time. introduction: for decades, helicopter emergency medical services (hems) contribute greatly to prehospital trauma patient's care by performing advanced medical interventions on scene. unnecessary dispatches, resulting in cancellations, cause these vital resources to be temporarily unavailable. these cancellations contribute to overtriage and provide additional costs to society. an earlier study showed a cancellation rate of % in our trauma region. however, little empirical knowledge exists about reasons for cancellations for different mechanisms of injury (moi) and type of dispatch. this study aims to examine the current cancellation rate in our trauma region over a -year period. additionally, insights in cancellation reasons for different moi and type of dispatch are evaluated. methods: a retrospective study was performed, using data derived from the hems database of trauma region north west netherlands, between april st and april st . information regarding patient's characteristics, date and time of day, moi, type of dispatch, and cancellation reason were compared. results: in total, , patients were included. hems was cancelled in . % of dispatches. the majority of dispatches ( . %) were cancelled because the patient was physiologic-and neurologically stable. dispatches simultaneously activated with ems were cancelled . % of times, compared to . % when hems assistance was additionally requested by ems on scene. no differences were found between dayand night-time dispatches. trauma related dispatches were cancelled more frequently compared to non-trauma related dispatches. conclusions: this study found a considerable-and increased cancellation rate compared to previous research. an explanation for this finding could be better adherence to dispatch protocols. furthermore, a great variety in cancellation rates was found among different moi's. therefore, continuous critical evaluation of hems triage is important and dispatch criteria should be adjusted if necessary. case history: two separate cases of high speed road traffic collision. the first is years old female without significant past medical history. the second is years old male who had short extremitis due to history of spastic quadriplegic cerebral palsy alongside congenital kyphosis and postural scoliosis. clinical findings: on examination the first patient was hemodynamically stable with soft abdomen and bruising over the left pelvic area. the second patient had left side neck and right side chest bruises; furthermore, he was tachycardic with normal blood pressure, but he was generally pale, getting clammy and significantly sweaty. investigation/results: fast scan for both patients showed free fluid in the abdomen and ct scan was uncertain of the source in the first patient. in the second, a large mesenteric haematoma was evident on ct with contrast extravasation with corresponding significant drop in hemoglobin and raised lactate levels. diagnosis: case : hemodynamically stable blunt abdominal trauma. case : hemodynamically unstable blunt abdominal trauma. therapy and progressions: the first patient was managed conservatively initially but worsened overnight with a drop in haemoglobin and increase in lactate mandating emergency laparotomy. hemoperitoneum and cm of ischaemic bowel with tear in the mesentery was found. she had an uneventful recovery after resection and primary anastomosis. the second patient underwent immediate emergency laparotomy. there was evidence of hemoperitoneum ( l) and similar mesenteric tear with ischemia involving cm of the terminal ileum. resection with end to end anastomosis was done. patient was then transferred to itu; however, he developed chest infection which prolonged hospital stay. comments: hemodynamic instability is a major factor in mandating urgent exploratory laparotomy in bat and bucket-handle injury is not uncommon following road traffic accidents. introduction: incisional hernias are one of the most common complications post-abdominal surgery, affecting between - % of patients undergoing a laparotomy. a number of risk factors are associated with their development such as age, bmi, type of surgery and co-morbidities. these risk factors also affect their levels of recurrence which is why the technique undertaken to repair these is of such interest. the primary purpose of this meta-analysis was to examine which repair technique is associated with the lowest level of recurrence whilst a secondary aim was to examine whether the frequency of common complications was dependent on the type of repair utilised. material and methods: this systematic review and meta-analysis was conducted by both co-authors. the following information sources were utilised; cochrane/embase/google scholar/pubmed/scopus. in relation to the eligibility criteria-papers that were published from onwards and in the english language were included with any length of follow-up. study selection was as per the inclusion/exclusion criteria below and only cohort studies/rcts/systematic reviews/ meta-analyses and case control studies were included. inclusion criteria: abdominal incisional hernias, all types of repairmesh/open/laparoscopic/sutured repair/primary repair etc. in terms of the exclusion criteria-any hernia repair that was not incisional was excluded. results and conclusions: in terms of the primary question posed by this repair, meta-analysis shows that there is a significant difference between open vs laparoscopic technique and recurrence rates in relation to the primary question posed by this paper whilst the use of mesh impacts negatively on post-operative wound infection rates. this invites an interesting debate on the merits of each technique whilst demonstrating the need for a multicentre randomised controlled trial. laparoscopic approach in penetrating abdominal trauma: case study and review of the literature b. vieira , v. taranu , a. silva , d. galvão , a. soares hospital de santo espírito da ilha terceira, general surgery, angra do heroísmo, portugal introduction: laparoscopy(ls) has greatly improved surgical outcomes in many elective abdominal procedures. the use of ls in acute care is becoming widely accepted. however, a number of safety issues have limited its application in abdominal trauma. notwithstanding with the reports and studies of the past decade proving its safety and accuracy, ls is slowly replacing the need for exploratory laparotomies. case report: a yo male sustained with penetrating stab wound on the left flank. he was hemodynamically stable. ct confirmed intraperitoneal positioning of the knife, without free fluid or air nor any evidence of organ injury. an exploratory ls was performed and confirmed the intraperitoneal positioning of the knife. abdominal exploration revealed a jejunal transfixating lesion about m from treiz's angle that was manually closed. the patient maintained a favorable po evolution and was discharged on the thpo day. discussion/conclusion: a number of concerns have limited the use of ls in abdominal penetrating trauma. initially, it resulted in high rates of missed injury, mainly of the small bowel, generating considerable criticism. the development of systematic abdominal explorations in ls, as described by choi and kawahara, resulted in a rate of missed injuries close to zero. moreover, direct visualization using ls has shown superior specificity and sensitivity in identifying peritoneal penetration, hollow viscus injuries and diaphragmatic lesions when compared to ct. in the case reported here, ct didn't show any image suspected of perfuration such as free air or fluid, and yet ls showed a small bowell injury. besides its advantages as a diagnostic tool avoiding negative laparotomies in more than % of the cases, thanks to evolving techniques and improved practice, it may also be therapeutic and allow safe definitive treatment for many types of injuries as described here. method: this is a monocentric retrospective study from a database entered prospectively. all patients admitted to the university hospital in nice with splenic trauma between / / and / / were included. the primary endpoint was performing splenectomy as a failure of a nom. results: patients were included in our study. the majority of splenic lesions were severe grades, that is to say greater than . in total, splenectomies were performed urgently, i.e. % of patients; angio-embolizations were performed, i.e. % of patients with a success rate greater than %; . % of patients who had not anterior angio-embolization required secondary splenectomy; . % of the patients who had anterior angio-embolization required secondary splenectomy. in the patient group with successful angio-embolization, the mean age was years vs . years in the nom failure group (p = . ). a decrease in hemoglobin between admission and h after admission was found in the nom failure group compared with the successful embolization group (p = . ). conclusion: hemoglobin monitoring in the hours following admission of a patient with splenic trauma may be an important factor in the surveillance of hemodynamically stable patients. prospective studies could confirm these results. missed ureteric injuries in gunshot injuries of the abdomen: how to avoid? introduction: traumatic ureteral injuries are uncommon. penetrating rather than blunt trauma is the most common cause of ureteral injuries. the aim of this study is to make a strategy to avoid missing ureteric injuries in gunshot injuries of the abdomen. material and methods: patients were operated in our hospital in years period. all patients were managed according to atls guidelines. for stable patients, full radiological work up was done, while hemodynamically unstable patients were shifted to or immediately for laparotomy and exploration. all patients demographic and clinical data were recorded these include :patient age, sex, mechanism of injury, hemodynamic state on arrival to the rr, anatomical site of gunshot injury, associated injuries, ureteric injuries detected early or late, early repair, delayed presentation and morbidly associated with delayed discovery. results: ureteric injuries were found in patients out of patients who underwent laparotomy for gunshot injuries had ureteric injury in an incidence of . %. ureteric injuries were missed in the first laparotomy in patients. associated injuries of other abdominal viscera include; colon injuries affecting ascending and descending colon in all the patients. conclusions: ct and pyelogram are the modalities of choice in stable patient but in unstable patients the early recognition of ureteric injuries depends on high index of suspicion leading to surgical exploration of the ureter along its course. case history: we present a case of a year old man, who was injured by his agricultural machine in the abdomen. clinical findings: he was transferred in the emergency department and he was hemodynamically stable. he had several traumas in his abdominal wall. from the largest one, in the left iliac fossa, omentum, transverse colon and loops of the small intestine were protruded out of the abdominal wall. the small bowel was ischemic and ruptured. investigation/results: computed tomography investigation, revealed small amounts of liquid and air in the abdominal cavity. diagnosis: the patient was immediately operated. the destroyed loop of the small bowel was resected with the use of a stapler and the field was washout. then with a midline incision the abdomen was opened. there were no other injuries inside the abdomen cavity. there was an extensive injury with a creation of a large gap in the anterolateral abdominal wall. it was impossible to identify the left rectus abdominis muscle as also the lateral muscles (external and internal oblique and transversus abdominis). therapy and progressions: a side to side entero-enteric anastomosis was created and a meticulous observation and washout of the abdomen were performed. for the closure of the abdominal wall a double-sided mesh from polypropylene coated with silicone on one side ( cm) was placed and the operation was completed. all the other wounds of the abdominal wall were closed with loop nylon stitches no . a closed suction drain was placed above the mesh. the patient had a very good postoperative course. he was dismissed from the hospital after days in a very good condition. comments: the usage of mesh was very useful for the reconstruction of the abdominal wall. there is no conflict of interest. strategy shift from damage control surgery to primary radical surgery improve the outcome of blunt hepatic injury involving inferior vena cava introduction: the diagnosis of abdominal trauma is a real challenge even for surgeons experienced in trauma. clinical findings are usually unreliable, and abdominal examination is made up of various factors. diagnostic tools that help the attending physician make critical decisions, such as the need for laparotomy or conservative treatment, are mandatory if we propose a favorable outcome. material and methods: the study was performed in the clinic i surgery, the county clinical emergency hospital craiova, between - and analyzed a number of abdominal traumas hospitalized, investigated and treated in the clinic. the methods of paraclinical diagnosis are evaluated comparatively, the study analyzing the evolution and the tendencies during the studied period, from , to . results: the study allowed an evaluation of the diagnosis and treatment methods compared to the data in the literature. conclusions: thus ct scan remains the standard criterion for detecting solid organic lesions. in addition, a ct scan of the abdomen may reveal other associated lesions. fast ultrasound is an important and valuable alternative for diagnosing abdominal trauma, especially for patients who are hemodynamically unstable and cannot be mobilized. there is a tendency in the treatment of abdominal trauma, as evidenced by the literature data on the use of conservative versus surgical treatment for a larger number of cases introduction: antiplatelet agents and anticoagulant drugs are widely used in prevention of cardiovascular incidents, which poses a challenge in surgical emergencies. the drafting of a multidisciplinary protocol for the treatment of pharmacological induced coagulopathy in patients who require urgent surgery standardizes management and increases patients' perioperative safety. material and methods: aims of the study were to describe the results from the protocol implementation. a retrospective study was conducted by examining reports of every patient presenting pharmacological induced coagulopathy and undergoing emergent surgery, recorded in our center from to inclusive. different algorithms used were explained and data such as need of transfusion, reintervention rate and perioperative complications were analyzed. results: data from patients were analyzed, median age of , ( %) men. patients ( %) used anticoagulant drugs. fresh frozen plasma transfusion and/or prothrombin complex concentrates were used according to the guideline. ( %) patients used antiplatelet agents. % of them underwent a delayed h surgery directly. tirofiban therapy was established in patients on dual therapy due to medium-high risk of cardiovascular event. regarding surgical approach, ( %) were laparoscopic, ( %) open and conversion occurred in ( %) cases, but only of them due to intraoperative hemorrhagic complication. only cases of postoperative hemorrhagic complications led up to reintervention and only one isolated case of thrombotic complication was reported. finally, ( %) mortality cases were reported, but none was caused by hemorrhagic nor thrombotic complications. conclusions: establishment of a guideline on management of pharmacological induced coagulopathy in emergent surgery is crucial in all surgical emergency units and has proven to be effective and safe. introduction: digestive haemorrhage is a frequent pathology. most of the episodes are self-limited, but in some cases massive haemorrhage occurs, leading to a % mortality rate. severe problems occurs when endoscopic treatment is not effective, requiring emergent surgery with poor prognosis. the aim of this study is to evaluate the implementation of interventional radiology techniques on short-term results. methods: a retrospective descriptive study was performed reviewing patients who underwent radiological embolization after failure of endoscopic conventional treatment between - in our hospital. a total of patients were included. results: patients were male. cases were from lower gi track and were from the upper gi with a similar death rate between them, with a higher rebleeding rate in upper gi ( . % vs . %). % of the arteriographies did not show any bleeding site, of them developed a new bleeding episode. overall patients who undergo embolization, urgent surgery was avoided in of the patients diagnosed as upper gi haemorrhage and in of the patients diagnosed as lower gi haemorrhage. patients died, those death occurred later on the recovery of the acute bleeding episode and embolization, all of them related to patients comorbidities. conclusions: arterial embolization has become an important tool in order to treat massive haemorrhages of the gastrointestinal tract. it seems to decrease the mortality and morbidity rate, but some complications can be associated such as rebleeding or bowel ischaemia. massive transfusion protocol with early administration of platelet and fresh-frozen plasma along with packed red cells in the initial phase of resuscitation is associated with improved outcomes introduction: massive transfusion (mt) in a ratio of : : (prbc:platelet:ffp) is the standard of care in hemorrhaging trauma patients. the aim of our study was to compare the outcomes of patients who receive near balanced resuscitation (nbr) compared to unbalanced resuscitation (ubr) during the initial phase of resuscitation. material and methods: we performed a -year analysis of the acs-tqip. all adult patients (age [ ) who received mt (defined as transfusion of prbc c units in -h) were included. patients were stratified into two groups: nbr defined as prbc:platelets:ffp in : [ . : [ . and ubr ( : \ . : \ . ) in the first h of resuscitation. primary outcome measure was mortality. secondary outcome measures were complications, and hospital length of stay. propensity matching was performed to match the two groups. results: a total of , patients received mt. mean age was ± years, median iss was [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . overall h mortality was . %. only % patients received nbr while % received ubr in the first -h. using propensity score matching, patients were matched for demographics, ed vitals, iss, ais and injury parameters. patients who received nbr in the early resuscitation phase had lower mortality ( % vs. %, p = . ), lower overall complications ( % vs. %, p = . ), with no difference in hospital length of stay ( days vs. days, p = . ) compared to the ubr group. conclusions: only one-third of patient receiving massive transfusion receive prbc, ffp and platelet in a ratio closer to : : in the initial -h and they have lower mortality and complications compared to patients with unbalanced resuscitation. material and methods: the goal is to assess mtp strategies in level- trauma centres in the netherlands and compare these with each other and (inter) national guidelines. a trauma surgeon or anaesthesiologist involved in compiling the mtp in each level- trauma centre in the netherlands and dutch ministry of defence was approached to share their mtp and comment on their protocol in a survey or oral follow-up interview. results: all eleven level- trauma centres responded. content of the packages and transfusion ratio (red blood cells/plasma/platelets) was : : , : : , : : , : : , : : , : : , : : and : : . tranexamic acid was used in all centres and an additional dose was administered in eight centres. fibrinogen was given directly (n = ), with persistent bleeding (n = ), based on clauss fibrinogen (n = ) or rotem Ò (n = ). standard coagulation monitoring are used in all centres, but most hospitals use also rotational thromboelastometry (rotem Ò ) (n = ), thromboelastography (teg Ò ) (n = ) or both (n = ). all centres used additional medication for patients using anticoagulants, but its use was ambiguous. conclusions: mtps in dutch level trauma centres differs from (inter) national guidelines in transfusion ratio and additional medication, which could be explained by misinterpretation of the : : ratio, changes in components and following an outdated dutch national guideline. whether these differences in mtps actually leads to different patient outcomes will follow from data that is currently being collected. this study is sponsored by the dutch ministry of defence. anastomotic bleeding after colorectal surgery: incidence, management and complications introduction: postoperative anastomotic bleeding (pab) is a frequent minor complication ( - %) that usually resolves by a conservative approach. hemodynamic instability and anemization may develop requiring urgent management. the aim of our study is to describe pab and its treatment. material and methods: observational retrospective cohort study of patients with pab collected between july and september . pab was defined as an episode of lower gi bleeding after colorectal surgery with at least one anastomosis. characteristics of patients, surgery, length of hospital stay, morbidity and mortality, and management of pab were reviewed. results: a total of ( . %) patients with pab was collected. median age was of years (iqr - ), with a median estimated asa grade of . the most common procedure was a right hemicolectomy ( %), followed by sigmoidectomy ( %). % of surgeries were laparoscopic. only cases were converted to an open approach. % of patients had the first episode of pab during the first h after surgery, while % after the third postoperative day. pab was treated conservatively in % of the cases. the remaining % required urgent endoscopic management identifying the bleeding through the anastomosis line, using clips in patients and hemospray in patient to control it. no complications were recorded after endoscopic treatment. just case required surgical reintervention. a total of ( %) patients required blood transfusion with a median of (iqr - . ) units. length of hospital stay was . days. no mortality related to pab was registered. conclusions: pab is a mild complication after colorectal surgery. most of the patients respond to conservative management. urgent endoscopic treatment seems to be effective and safe to control pab even during the first postoperative day. introduction: hemorrhagic shock and associated reperfusion injuries are davastating situations during the treatment of polytrauma patients. the aim of this study was to analyze and compare alterations of the local circulatory changes of various body regions during hemorrhagic shock and after fluid resuscitation. material and methods: this study was conducted on male pigs. they suffered a standardized polytrauma including femoral fracture, blunt thoracic trauma and liver laceration. further, the suffered a hemorrhagic shock for h (aimed map mmhg). fluid resuscitation with three times drawn blood volume after hemorrhagic shock. retrograde nailing for femoral fracture and chest tube in case of pneumothorax liver packing. measuring circulation at liver, colon, stomach, and extremity. results: inclusion of animals. local circulation at the extremity decreased significantly compared to baseline values during hemorrhagic shock ( . a.u. versus . a.u., p \ . ). after resuscitation the flow rate at the extremity was comparable to baseline values. the stomach was least sensitive to hemorrhagic shock, whereas the oxygen delivery rate at the colon decreased during shock phase and remained decreased during fluid resuscitation (p \ . ). conclusions: different body regions react differently to hemorrhagic shock. the colon appears to be most vulnerable to changes based on hemorrhage. the delayed improvement of circulation in liver, colon, and extremities may represent a trigger for systemic hyperinflammation and subsequent sirs and sepsis. none of the authors have any conflicts of interest to declare. massive transfusion in penetrating trauma: the search for a specific prediction system introduction: prediction systems of massive transfusion (mt) were developed from cohorts with a small proportion of penetrating trauma. some of them required laboratory tests. we aimed to evaluate abc score and to identify independent predictors of mt in a cohort of torso penetrating trauma (tpt) material and methods: adults with tpt, managed in a level-i trauma center, who received one or more packed red blood cells (prbc), were included. variables obtained during the evaluation in the trauma bay were registered prospectively. the ability to predict mt was evaluated with simple, multiple logistic regressions and roc curves. results: we included patients; . % were male, and . % received fire-arm wounds. twenty-one ( %) received mt. mt patients were intubated more frequently in the pre-hospital, had lower sbp, higher hr, lower gcs, and received more frequently vasopressors (p \ . ) when compared with the no-mt patients. trauma mechanism, number or localization of the wounds, and positive fast could not discriminate mt (p [ . ). hypotension, tachycardia, and alteration of the glasgow coma scale or its motor response behaved as independent predictors of mt. models created with these variables showed better discriminative ability than abc score, with adequate goodness to fit. conclusions: prediction models of mt, based on heart rate, systolic blood pressure, and neurologic alteration outperformed abc score in a tpt cohort. introduction: rectus sheath hematoma presents with abdominal pain and anterior abdominal wall mass. it can be followed conservatively and rarely causes mortality ( ) . in this study we aimed to review rectus sheath hematoma cases consulted to our department and to present our management. material and methods: the data of patients admitted with rectus sheath hematoma between and was collected using hospital database. treatment modalities, demographic data and complications were reviewed retrospectively. results: all the cases presented with abdominal pain and/or with a palpable abdominal mass. . % of the patients (n = ) were receiving anticoagulant therapy at the time of admission. the mean inr value was . . patients were followed up with es&ffp transfusion and conservative treatment. patients not eligible for conservative care underwent inferior epigastric artery embolization and hematomas in patients were evacuated via a percutaneous drainage catheter. patient went through laparotomy for an infected hematoma and one patient underwent laparotomy plus packing. the patient who had laparotomy plus packing died due to intraabdominal hematoma and sepsis. conclusions: rectus sheath heamatoma is a rare cause of acute abdominal pain. the patients diagnosed early and have suitable indications can be treated conservatively ( ) . rectus sheath hematoma should be considered in the differential when a patient with a history of anticoagulant drug use presents with acute abdominal pain in order to prevent unnecessary surgery and complications. introduction: an early delivery of blood products when massive transfusion protocols (mtp) are triggered is mandatory to improve trauma patients survival. scores predicting massive transfusion (mt) have already been described ( ) . the aim of our study is to compare scores for predicting mt and identify the best trigger for mtp. material and methods: multicentric retrospective study from the trauma registry of the spanish surgeons' association. severe trauma patients (injury severity score [iss] c ), admitted to different level trauma centers, from january to september were included. demographic and clinical information was recorded, and predictive scores for mt were assessed. results: patients were included. medium age was . ± . years, ( . %) were male. median iss was (iqr ). in % of the patients a mt (defined as c units of packed rbc) was necessary, while a mtp was triggered in . %. surgery was performed in . %. the overall mortality was of . %. predictive scores for mt were compared: gap (glasgow coma scale, age, systolic blood pressure), shock index (si), assessment of blood consumption (abc) and mabc (modified abc). auroc for gap was . ± . , si . ± . , abc . ± . and mabc . ± . , showing differences between gap (the worst score) and the others, p \ . . no differences were found between si, ab and mabc. best cut-off points were calculated. si c . better predicts mt with a sensitivity %, specificity . %, positive and negative predictive values . % and %. conclusions: si, abc and mabc are all good scores for predicting mt in our population. appealing by its simplicity, we recommend si as the best trigger for mtp. protocols should be standardized to improve the accuracy of mtp activation for trauma patients. introduction: the prevalence of knife-related offences is rising in the uk. successful management of trauma patients requires the co-ordinated response of specialist services, including transfusion. we aimed to assess the impact of knife-crime on transfusion support within a uk adult major trauma centre (mtc). material and methods: retrospective review of patients admitted to a uk mtc following knife injuries resulting from interpersonal violence during a three-year period (may -april ). source material included electronic patient records, tarn database and massive transfusion protocol (mtp) logbook. patient characteristics, resource utilisation including transfusion, mtp activation and outcome were collated. results: patients were identified, ( %) were male. median age was years. ( %) were under the age of . patients ( %) presented with circulatory compromise (sbp \ ). patients ( %) had attended our hospital previously for violencerelated trauma. % arrived at hospital between h to h. ( %) required one or more surgical procedures. median length of stay was days. patients ( %) received blood transfusion. median units transfused were prbc, ffp, platelets (atd). mean component use was pbrc (range - ), . ffp ( - ), platelets . ( - ), cryoprecipitate . ( - ). annual mtp activations increased from to during the study period (total ). stabbings accounted for . % of these ( patients), of which ( %) were transfused. conclusions: knife crime presents a burden to blood transfusion, accounting for a quarter of mtp activations. patients typically present out of hours with implications for service planning and delivery. patient profile together with repeat healthcare attendance and surgery requiring transfusion has implications for red cell allo-immunisation. we recommend timely baseline blood grouping and triage to optimise the safe use of rhd positive cellular components. introduction: spontaneous intramural small bowel hematoma is a very rare complication of anticoagulant therapy. nowadays, the prevalence is increasing due to the widespread use of computerized tomography and the increasing number of patients receiving anticoagulant therapy. material and methods: patients admitted to our center between january and june and treated with the diagnosis of intramural hematoma were retrospectively evaluated. results: the median age of the patients was years ( - ) and ( %) were male. at the time of appeal, warfarin intoxication was present in cases ( %) and the median inr was . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one patient had known factor deficiency. diagnosis was made by computerized tomography in all cases. one intramural hematoma was localized in the duodenum ( . %), nine in the jejunum ( %), and five in the ileum ( . %) six patients ( %) had ileus findings. all patients underwent fresh frozen plasma replacement due to high inr levels and bleeding. median tdp transfusion was units ( - ). only patients ( %) required erythrocyte suspension replacement. all cases were followed up conservatively and there was no need for intensive care. the median hospital stay was ( - ) days. conclusions: due to the limited number of studies in the literature with a large number of cases, retrospective evaluation of singlecenter cases may be helpful. spontaneous intramural small bowel hematoma should be considered in the elderly population under warfarin therapy who present with abdominal pain, especially if inr values are above therapeutic limits spontaneous regression is seen in the majority of cases. non operative management and correction of coagulopathy with fresh frozen plasma replacement is the preferred approach. references: abbas ma, et al. spontaneous intramural small-bowel hematoma: clinical presentation and long-term outcome. arch surg. ; ( ) : - . pre-hospital decision-making: identifying the challenges assessing and managing traumatic haemorrhage and coagulopathy m. marsden , r. bagga , k. gillies , r. lyon , s. kellett , r. davenport , n. tai expert pre-hospital clinicians in making decisions about the diagnosis and treatment of patients with major haemorrhage and suspected tic. methods: semi-structured interviews were conducted with senior pre-hospital consultants from london's air ambulance and air ambulance kent, surrey and sussex. interviews probed clinicians on how they make decisions relating to the pre-hospital assessment of major haemorrhage and tic and subsequent blood product transfusion. the interviews were analysed using descriptive thematic analysis. results: all clinicians agreed that identifying and treating major haemorrhage was vital. half of the clinicians reported making no conscious assessment for tic and six reported tic should be managed in a hospital setting. four broad themes were identified: collation of information, weighing utility of different approaches, influence of experience and evaluation of unknowns. collating information from multiple sources drove clinical decision-making. decisions on blood product transfusion were made after weighing potential benefits (e.g. improve microvascular perfusion) against harms. clinical experience was reported as key to nuance clinical assessment, detect subtle signs and identify patterns. uncertainty complicated clinical decision-making in two domains; incomplete knowledge of a patient's injury and uncertainty of best clinical practice. conclusion: the pre-hospital identification and treatment of major haemorrhage was recognised as challenging and fundamental. necessity of pre-hospital tic diagnosis and treatment divided opinion. identifying these four themes allows for a greater understanding of the factors involved in making these decisions and will guide the creation of more accurate decision support tools to aid pre-hospital clinicians. nothing to declare. introduction: massive transfusion (mt) is defined as the administration of c packed red blood cells (prbc) in h. alternative definitions have been proposed; however, there is little understanding about the discriminative ability of different mt definitions with regards to mortality and multiorgan failure (mof). we aim to assess and compare the discriminative ability of different definitions of mt concerning mortality and mof. material and methods: we included patients who arrived to the emergency department and required trauma team activation at a level i trauma center in the city of cali, colombia between - . demographics and trauma characteristics were evaluated. the following mt definitions were measured: units of blood products in h (t ), u prbc in (t - ), u prbc in h (t - ), prbc in h (t - ), the combination of t - and t (t-combi), prbc in h (t - ), prbc in h (t - ) and units of prbcs in min. the operative characteristics were calculated for each definition. mof was defined as a sofa score of c points. results: we included subjects, . % male. trauma mechanism was penetrating in . %. the median and interquartile range (iqr) of age was years iqr ( - ) and of iss ( - ). lesions were located in the torso in . % of patients, and . % had a positive abc score. a total of ( %) received at least unit of prbc. tables and presents the operative characteristics of definitions of mt with respect to mortality and mof, respectively. conclusions: although all definitions showed an association with higher odds with the outcomes of interest, none of them showed an accurate diagnostic capacity regarding mof and mortality. thus, we advise caution when relying on the classical definition of mt ([ rbc units in h) to guide the flow of care of severely injured patients. trauma and coagulation: trends in coagulation factors in the severely injured trauma patient introduction: trauma-induced coagulopathy (tic), affects about - % of the major trauma patients. in the past, tic was considered as a consequence of the coagulation factors' dilution after a highvolume colloid administration. today tic is seen as a phenomenon that can arise after trauma; the first event is the c-protein activation by the tissue damage and hypoperfusion, resulting in the subversion of the hemostatic process. material and methods: the patients of the pilot study ''trauma and coagulation'' run in irccs san raffaele scientific institute have been reviewed and analyzed using a suite of experimental coagulation factors including rotem parameters, activated protein c (apc), thrombomodulin, endothelial protein c receptor, thrombin-antithrombin complex (tat), plasminogen activator inhibitor (pai- ), seselectin, interleukin- (il- ), interleukin- (il- ), d-dimer (xdp), antithrombin iii (atiii), and prothrombin fragment f ? (f ? ). new patients have been enrolled to validate the results of the pilot study. results: there is a statistically significative correlation between clinical scores of severity of trauma and risk of massive transfusion (iss, abc and tash) and some of the experimental coagulation factors analyzed. case history: to evaluate the role of negative pressure wound-care systems applied to the pleural cavity in case of severe acute empyemas and frail patients not amenable to conventional surgery. clinical findings: we report the case of a yrs old male critically ill patient suffering from complications of cardiac surgeries who developed a severe right empyema with broncho-pleural fistula through the site of a previous pulmonary hernia. investigation/results: we review the actual indications of negative pressure therapy in thoracic surgical emergencies especially in septic patients unfit for surgery. in our case the repeated application of negative pressure with dedicated dressings through the initial thoracotomy was the chosen damage control approach because of the sepsis and poor conditions. diagnosis, therapy and progressions: air leaks were later found to originate from a subsegmentary branch of middle lobe bronchus. subsequent video-assisted debridement procedures followed by negative pressure therapy managed to ( ) control the infection, ( ) reduce the thoracotomy incision into a thoracoscopic access and ( ) heal the pleural cavity, restoring eventually better general conditions of the patient. the closure of the bronchial fistula required further procedures after the acute phase when sepsis was overcome. comments: negative pressure systems can be applied to the pleural cavity with many advantages in selected critically ill patients. they allow to contain, treat and resolve infections both of chest wall and pleural cavity in case of severe empyemas reducing also wound pain and eliminating the need of chest drains. air leaks may also be managed by negative pressure therapy with adequate indications and particular attention to its settings. references: sziklavari z. mini-open vacuum-assisted closure therapy with instillation for debilitated and septic patients with pleural empyema. eur j cardiothorac surg. . flail chest: the renaissance of rib osteosynthesis c. leite , a. oliveira , a. lemos , b. barbosa , c. casimiro centro hospitalar tondela-viseu, general surgery, viseu, portugal case history: we present the clinical case of a male patient of years old. injury mechanism: fall from his own height over the right hemithorax. clinical findings: rib fractures with flail chest and significant displacement of bone edges. symptoms: intense thoracic pain. diagnosis: rib fractures with flail chest. therapy and progressions: multimodal analgesia. on the th day, he presented a tension pneumothorax. after adequate intercostal drainage, the pneumothorax relapsed. on the th day, he underwent a right posterolateral thoracotomy, open reduction and internal fixation of ribs with plates and screws and intercostal drainage. evolution: he received respiratory kinesiotherapy and was discharged on the th pos op day. follow-up at st and th months, without functional impairment and with preservation of quality of life. comments: rib fracture is the most common injury in the setting of thoracic trauma and is associated with a higher morbimortality. in the last years, positive pressure mechanical ventilation was the first line treatment of respiratory insufficiency caused by rib fractures. however, severe complications associated with prolonged mechanical ventilation, have elicited the rising implementation of open rib reduction and internal fixation techniques. the most consensual indications are: flail chest with fracture of at least ribs, significant displacement of bone edges or uncontrolled pain. rib osteosynthesis is a simple method but requires clinical experience in thoracic approaches and handling of specific instruments and material. its implementation in non-ventilated patients reduces the need for mechanical ventilation, pain, length of stay and allows preservation of quality of life. yokohama city university medical center, advanced critical care and emergency center, yokohama, japan, saiseikai yokohama-shi nanbu hospital, department of surgery, yokohama, japan, yokohama city university, department of general surgery, yokohama, japan, yokohama city university, department of emergency medicine, yokohama, japan introduction: although americans and europeans report emergency room thoracotomy (ert) is of value in penetrating trauma patients, most of ert is performed for blunt trauma in japan. after the establishment of the local government-directed major trauma center in the city of yokohama, the unexpected trauma survivor rate increased in the single center study. we report our experience in ert and surveyed the effect of the establishment. material and methods: patient characteristics (backgrounds, mechanism of injury, indication for ert, anatomic injuries, interventions and survival) of those who underwent emergency thoracotomy compliant with the guideline of western trauma association, between october and september were analyzed. results: fifty-eight patients ( males) underwent emergency thoracotomy. median age was . ( - ) years. fifty-seven were performed for blunt trauma ( %) and only for penetrating injuries. twenty-three patients presented with cardiac arrest on arrival, while thirty-five had deep and refractory hypotension. overall, survival rate improved from ( / ) to % ( / ) (p = . ) after the establishment of the trauma center. of patients presenting with cardiac arrest, only one survived. conclusions: the establishment of major trauma center seemed to affect the survival rate of the patient edt was performed. introduction: more than % of polytrauma events involve chest injuries. one third of these patients sustain thoracic instability due to serial rib fractures. thanks to numerous innovations in implant development several approaches currently exist for surgical rib stabilization (srs). however, no consensus exists regarding patient selection for srs to date. material and methods: retrospective single center cohort analysis in trauma patients. serial rib fracture was defined as three consecutive ribs confirmed by chest ct. cohort includes patients that were treated conservatively and patients that underwent srs by plate osteosynthesis. demographic patient data, trauma mechanism, injury pattern, injury severity score (iss), glasgow coma scale (gcs) and hospital course were analyzed. two matched pair analyses stratified for iss ( pairs) and gcs ( pairs) were performed to minimize selection bias. results: the majority of patients was male ( %) and aged ± years. serial rib fractures were located left/right/bilateral in %/ %/ % of cases. other thoracic bone injury included sternum ( %), scapula ( %) and clavicula ( %). visceral injury consisted of pneumothorax ( %), lung contusion ( %) and diaphragmatic rupture ( %). average iss was ± . . overall hospital stay was . and icu stay . days. in hospital mortality was %. srs did not improve hospital course or postoperative complications in the complete study cohort. however, patients undergoing srs had significantly reduced gcs ( . ± . vs . ± , ; p = . ). matched pair analysis stratified for gcs showed a reduced need for blood substitution and shorter icu stays ( vs days; p = . ) including shorter respirator time ( vs h; p = . ) and reduced in hospital mortality ( vs %). conclusions: patients with serial rib fractures and simultaneous severe cerebral injury benefit from surgical rib stabilization. tracheal and bilateral recurrent laryngeal nerve disruption injury secondary to accidental strangulation by dupatta case history: year old female brought to trauma emergency with a/h/o accidental strangulation injury with dhupatta at farm field while working with thresher machine after h of injury. patient had severe dyspnoea, dysphagia, paining neck clinical findings: primary survey revealed threatened airway with extensive surgical emphysema, rr- /min, spo - % on high flow oxygen mask, hemodynamically stable, and had no neurological deficits. patient was immediately intubated, however ventilation could not be maintained and surgical emphysema worsened hence immediate tracheostomy was established. investigation/results: computed tomography (ct) head and ct angiography of neck with venous phase study of neck and chest with ct esophagogram revealed complete disruption of cricotracheal junction with extensive cervical and upper thoracic surgical emphysema and no other injuries. diagnosis: disruption of trachea from cricoid cartilage with crushed trachea with loss of approximately cm, cricoid and thyroid cartilage fracture, complete avulsion of bilateral recurrent laryngeal nerves and serosal tear of esophagus. therapy and progressions: neck exploration with debridement of tracheal margins and anastomosis between trachea and cricoid cartilage with repair of cricoid, laryngeal cartilage and esophageal serosal repair was performed. comments: post-operatively patient underwent fibreoptic bronchoscopy and revealed paramedian location of vocal cords. at present patient is with tracheostomy tube in situ undergoing speech therapy and is able to generate comprehensible sounds. further laryngeal framework surgery is being planned. introduction: emergency resuscitative thoracotomy (ert) is a lifesaving procedure in selected patients and it is often considered a controversial ''last chance'' method of resuscitation. objectives of ert are to resolve pericardial tamponade, to repair heart injuries, to perform an open cardiac massage, to cross-clamp the aorta to redistribute blood flow to the myocardium and brain, to control intrathoracic bleeding and air embolism in the bronchial venous system. outcome mostly in blunt trauma is believed to be poor. material and methods: we retrospective reviewed patients c years who underwent ert at san camillo-forlanini hospital (rome, italy) between january and september with traumatic arrest for blunt or penetrating injuries. results: of ert, ( . %) were for blunt trauma, ( . %) were for penetrating trauma. . % of patients were male. the collectively reported overall survival was % (n = ). when including erts designated as done in the emergency department for blunt mechanism, only patient survived ( . %). survival after erts for penetrating trauma was % ( of ). conclusions: our experience suggests that ert is a technique that should be utilized for patients with critical penetrating injuries. the reported outcome after ert in european civilian trauma populations is favorable with an overall survival of %. multicenter, prospective, observational data are needed to validate the modern role of ert in blunt or penetrating trauma. references: narvestad jk, et al. emergency resuscitative thoracotomy performed in european civilian trauma patients with blunt or penetrating injuries: a systematic review. eur j trauma emerg surg. ; ( ) case history: an -year-old male driving a car collided with a wall at a speed of km/h and was brought to a hospital near the scene. he was diagnosed with right multiple rib fractures and hemopneumothorax, and transferred to our emergency center for definitive care. clinical findings: the patient's consciousness was clear and his heart rate, blood pressure, respiratory rate, and o saturation (room air) on arrival were /min, / mmhg, /min, and %, respectively. subcutaneous emphysema was identified on the right side of his chest and his right breathing sound decreased on auscultation. there was no tenderness and rebound on abdominal examination. investigation/results: an enhanced whole-body computed tomography scan revealed a small disruption on the right diaphragm behind the sternum and free air in the abdomen. diagnosis: the diagnosis was right traumatic diaphragmatic injury, sternum fracture, and right multiple rib fractures with pneumohemothorax. there was free air in the abdomen but without evidence of perforation of the digestive tract as there was no finding of peritonitis on physical examination. thus, pneumoperitoneum from the thorax was strongly suspected. therapy and progressions: laparoscopic observation revealed a . cm-length of disruption on the diaphragm in the right sternocostal triangle. this was covered with falciform ligament using extracorporeal knot tying method because there was little seam allowance in front of the disruption on the sternum side, and direct suture was not possible. prognosis was good following surgery, and the chest drain was removed on postoperative day and the patient was discharged on postoperative day . comments: laparoscopic repair of the diaphragm using extracorporeal knot tying method is often used for retrosternal (morgagni) hernias. however, the method was also useful in this case because the diaphragmatic injury occurred in the sternocostal triangle. rib fractures associated with pneumo-and/or hemothorax; does everyone need a chest tube? v. snartland , p. a. naess , c. gaarder , m. hestnes , p. majak , , faculty of medicine, university of oslo, oslo, norway, oslo university hospital, department of traumatology, oslo, norway, oslo university hospital, trauma registry, oslo, norway, oslo university hospital, department of cardiothoracic surgery, oslo, norway introduction: pneumo-and/or hemothorax are often seen in trauma patients with rib fractures (rfs). standard treatment for pneumothorax (ptx), hemothorax (htx) and hemopneumothorax (hptx) is tube thoracostomy (tt). however, a non-operative approach can be applied in selected patients. we wanted to assess our practice in patients with rib fractures and associated ptx, htx or hptx. material and methods: all adult patients (c years) with rf, admitted by a trauma team at oslo university hospital in were identified retrospectively and those with associated ptx, htx or hptx were then included in the study. patients who underwent tt prior to arrival and those who died were excluded. spss v was used for statistical analysis. results: of the patients with rfs, a total of patients had ptx, htx or hptx. fifty-one percent ( / ) of these patients were treated with tt and % ( / ) of the patients underwent tt within h after arrival. the presence of opacification (p \ . ), chest wall deformity (p \ . ) and pneumothorax size (p \ . ) were significantly higher on chest x-ray in the tt group compared to the nonoperative group. intubation at arrival was also significantly more common in patients treated with tt (p \ . ). there was no difference in the presence of subcutaneous emphysema between the groups. the tt group was sicker than the non-operative group (had a significantly lower systolic blood pressure, a lower gcs and a higher lactate on arrival). oxygen saturation, heart rate, respiratory rate, ph and hemoglobin did not differ significantly between the groups. conclusions: in trauma patients with rf concurrent ptx, htx or hptx should be suspected. in our study only half of these patients were treated with tt, and % of tubes were inserted within h after admission. size of the ptx, radiological presence of opacification and deformity of the chest wall should be addressed when choosing treatment strategy. introduction: emergency department thoracotomy (edt) is a potentially life-saving surgical procedure performed in the emergency department (ed) in patients presenting with cardiac arrest following penetrating thoracic trauma. however, it is not clear if all surgeons are prepared or motivated to perform this procedure. furthermore, not all institutions are equipped, either in terms of logistics or team training, to perform edt. our purpose was to perform a pilot study in a cohort of polish surgeons of various specializations, in order to ascertain who would and who would not (and why) perform edt in their departments. material and methods: study population of surgeons ( specialists, residents) from various hospitals in poland, mean age: - years, . % men, . % women. study respondents were asked to fill in a questionnaire on the indications and motivation to perform edt in their clinical practice. results: most respondents (n = , %) correctly recognized the indications to perform edt. however, only ( %) declared they would perform it. the reasons for not performing edt were: lack of team training ( . %); lack of equipment ( %); lack of motivation among ed personnel ( . %); the ed is not prepared ( . %); the respondent is not prepared ( %). only participants ( . %) declared that their institutions had the edt protocol. conclusions: this survey demonstrates that, although most surgeons agree on the indications for edt, the level of preparedness in its execution is lacking. the main reasons are the lack of team training, the lack of equipment and the lack of motivation among ed personnel. other relevant reasons were the lack of preparation of either a surgeon or a department. these results demonstrate that improvements in institutional logistics as well as in team and individual training can translate into improved care. we strongly advise the performance of a pan-european survey on edt to address other unrecognized issues. mediastinum widening: how to manage it? a. gonzález-costa , r. gracia-roman , s. montmany-vioque , m. s. santos-espi , r. lobato-gil , m. pascua-solé , a. campos-serra , a. luna-aufroy , p. rebasa-cladera , s. navarro-soto parc tauli hospital universitari, trauma and emergency general surgery department, sabadell, spain, parc tauli hospital universitari, esofagogastric general surgery department, sabadell, spain, parc tauli hospital universitari, angiology and vascular surgery, sabadell, spain case history: a -year old male was admitted to our emergency department as a polytrauma code, because of a gunshot wound in the neck. clinical findings: his airway was compromised with expansive cervical hematoma. intubation was difficult. he was hemodynamically unstable with cervical bleeding, in which manual compression was applied. results: chest x-ray showed mediastinal widening without pneumo or hemothorax. diagnosis: urgent sternotomy while maintaining manual compression on the cervical bleeding, followed by left antero-lateral cervicotomy. injuries: section of left jugular vein and left carotid artery, lesions of unnamed vein. free cervical chylous fluid. left pleura and pericardium were opened without identifying major injuries. therapy and progressions: jugular vein was repaired with continuous suture and carotid artery with patch sutured. unnamed vein was sectioned between ligatures. thoracic duct was ligated. after surgery, ct scan showed cervical and mediastinal hematomas without signs of active bleeding, and correct permeability of the vessels, with no cranial lesions. the patient was admitted to the intensive care unit. tracheostomy was performed. fibrobronchoscopy, fibrogastroscopy and esophagogastricoduodenal discarded airway and esophageal lesions. he presented the following complications: • small mediastinal collection • right diaphragmatic paralysis. • paralysis of vi left cranial nerve (mononeuritis of vascular origin). the patient was discharged on the th postoperative day. comments: in this kind of trauma is essential the airway management with intubation when necessary. it is important that mediastinal widening visualized in the chest x-ray in a traumatic patient, should be an indication of surgery. in our case, it was essential to start it with sternotomy while maintaining manual neck compression, and in a second time, perform the cervical approach since that prevented the patient from suffering a greater blood loss. background: clavicular fracture is very common in childhood. otherwise, the medial third of the clavicle is the less affected. the current report describes a new pattern of clavicular injury, in which a medial third clavicular fracture and posterior sternoclavicular joint (scj) dislocation occur together in a skeletally immature patient. clinical findings: an -year-old boy sustained a direct impact to his left shoulder resulting from the fall of a sofa. at admission, he complained of severe pain in the clavicular and shoulder associated with functional limb impotence. physical examination revealed deformity of the proximal third clavicle, with swelling and tenderness to palpation along the medial left clavicle. no signs of skin pression or neurovascular impairment were found. the anteroposterior radiograph of the left clavicle showed a fracture of the proximal third shaft and an asymmetry of the scj. computed tomography confirmed the association of a greenstick fracture of the proximal third clavicular shaft, accompanied by a mild posterior scj dislocation. therapy and progressions: the left limb was immobilized with a sling during weeks, after which physical therapy was initiated to improve range of motion using active and gentle active-assisted exercises. at the months medical consultation, he presented asymptomatic, with good bone healing, full range of motion of the shoulder and absence of relevant aesthetic deformity. comments: in the immature skeleton, scj dislocation and epiphyseal fracture of the proximal clavicle are very rare entities due to the multiple strong ligaments that stabilize the scj. trauma in the proximal third of the clavicle typically results in fractures in the region of the physis and only more rarely culminate in dislocations of the scj. these injuries warrant a high index of suspicion, and early ct scanning is recommended. although treatment may be conservative, in situations of major displacement, surgery should be considered. use of rib fracture scoring systems in a uk major trauma unit: a retrospective audit and lessons learnt introduction: rib fractures are detected in % of trauma patients [ ] . significant morbidity and admission to intensive care units (itu) is common [ ] . rib fracture scores do not have strong validity as a predictor, but are a useful screening tool to identify patients at higher risk, of morbidity. the aim of this study was to audit the use of rib fixation scores in a single major trauma centre. material and methods: a retrospective audit of trauma patients with rib fractures presenting to a single major trauma centre over a -year period subsequently admitted to itu was performed. demographics, length of itu stay, rib fracture score (rfs) and ribscore were recorded and comparisons made between patients who had surgical rib fixation and those who did not. results: patients with traumatic rib fractures were admitted to itu over -year, of whom had rib fixation. mean age of patients undergoing surgery was compared to in the non-surgical cohort. average rfs was higher in the surgical cohort ( vs ; p = \ . ), as was average ribscore ( vs ; p = \ . ). incidence of flail segment was higher in surgical cohort ( % vs %; p = \ . ), as was number of rib fractures ( vs ; p = \ . ) and incidence of st rib fracture ( % vs %, p = \ . ). rib fractures treated surgically had a longer itu stay ( . days vs . ; p = \ . ). conclusions: surgical rib fixation patients were older and had longer itu stay. higher rib fracture scores correlated with need for surgical intervention. this highlights the need for careful patient selection for rib fixation, as they appear to fall in a more vulnerable patient demographic. there is a need for a score combining ribscore and rfs, ensuring the nature of fractures and presence of flail segments are interpreted in the context of patient age, to ensure this vulnerable patient group undergoes surgical fixation only when necessary. jichi medical university, shimotsuke tochigi, japan case history: an -year-old female individual hurt her back while walking during a hospital rehabilitation program after experiencing a brain stroke. her hemoglobin level gradually decreased to . g/dl on the th day after injury. a non-enhanced abdominal ct scan revealed a burst fracture of the lumbar spine. the patient was brought to our emergency center for a thorough examination. clinical findings: her vital signs on arrival were gcs: e v m , hr: , bp: / , rr: , and bt: . . her back presented a severe kyphotic spine. the palpebral conjunctiva was anemic and there were no injuries on her surface. no abnormalities were detected upon auscultation of the thorax and no tenderness and rebound was detected upon physical examination of the patient's abdomen. investigation/results: hemoglobin level was . g/dl and lactate . mmol/l on arrival. an enhanced chest and abdominal ct scan revealed a burst fracture of the th lumbar spine, a large hematoma around it, and a pseudoaneurysm of the lumbar artery. diagnosis: a pseudoaneurysm of the lumbar artery and a burst fracture of the th lumbar spine was diagnosed. therapy and progressions: the angioembolization of the lumbar artery was abandoned because the distance between the abdominal aorta and the aneurysm was \ mm. endovascular aneurysm repair (evar) was finally performed. after the successful completion of the surgery, the patient was discharged on the th day after evar. comments: slight injury caused the fracture of the lumbar spine, possibly yielding pseudoaneurysm of the lumbar artery. such pseudoaneurysms are rare and employing evar for its treatment is equally rare. blunt lumbar artery injury may be a differential diagnosis for the elderly patients who present burst spine fractures with extreme anemia or shock, even if it results from a minimal injury. case history: a year old co-driver was hit by another car on her side. air rescue found the patient with gcs and right tension pneumothorax. oral intubation, decompression with chest tube and transportation to the nearest level one trauma center was undertaken. clinical findings/investigation/results: on presentation in the emergency room the patient was hemodynamically instable with free fluid in efast-sonography and a haemoglobin of . g/dl. she was immediately taken to the operation room where laparotomy was performed. liver rupture and right diaphragm rupture was found. diagnosis: right hilar bronchial disruption. therapy and progressions: despite packing of the liver the patient remained instable. due to continuous bleeding from diaphragm rupture side right anterolateral thoracotomy was performed. bronchial disruption close to the hilus was detected leading to total pneumonectomy. after surgery the patient recovered under intensive care. six weeks after initial trauma the patient presented with ileus. a gastric tube was placed without complications. chest x-ray was performed showing intrathoracal displacement of the gastric tube. in an emergency operation the insufficient bronchus trunk was covered with an intercostal muscle flap. comments: this case shows the rare necessity of total pneumonectomy after blunt chest trauma and its typical complication with insufficiency of the bronchial trunk. after total pneumonectomy surgery covering the bronchial trunk should be performed as soon as possible to prevent insufficiency. in these patients gastric tubes should only be placed under endoscopic vision. because of the high complication rate total pneumonectomy should only be performed as a last resort procedure in the context of damage-control surgery. introduction: multiple rib fractures continue to be a challenging problem as the associated pain leads to a compromise in respiration. proper analgesia is required for physiotherapy, and to prevent development of respiratory failure. ultrasound-guided serratus plane block (spb) has recently been described as a regional anesthetic technique to provide analgesia to a hemithorax by blocking the lateral branches of the intercostal nerves. material and methods: from sept we applied the serratus plane block for pain control in patients with multiple rib fractures. we administered . - . % bupivacaine solution with easypump for - days, the infusion rate was ml/h. after admission we measured pulmonary function of patients and recorded the forced vital capacity (fvc). we repeated the test after the catheter insertion on the - - days. in our control group ( patients introduction: rib fractures are the most frequent injury after blunt thoracic trauma. it is very important to choose the most appropriate interventions to minimize or prevent complications. but who will benefit most of those interventions remains a challenge. material and methods: a retrospective study with a prospective data collection from march to december . there have been included all traumatic patients older than years old, that were admitted to the icu or who were died before the admission and had a plain chest radiograph (cxr) and thoracic or thoraco-abdominal scan (ct scan) in the first h. demographic data has been collected, vital signs, iss, mechanism of action, need of ventilation or intubation, lesions, complications, cause of death. a total of cxr were reevaluated by one general surgeon (one of the authors) and one radiologist, who were blinded to the results of the subsequent chest ct scan, the written radiology report and the patient's outcome. rib fractures, pneumothorax, hemothorax, pulmonary contusion, laceration and atelectasis were described. results: attending to the number of fractures, the kappa between the radiologist, the surgeon and the ct report is very low: surgeon-ct k = . , radiologist-ct k = . , and radiologist-surgeon k = . . both radiologist and surgeon under-diagnosed rib fractures. we tried to predict respiratory failure and pneumonia using the number of fractures, and scores (chest trauma score, ribscore and rib fracture score). results are shown on the table. conclusions: plain radiography seems not to be a good diagnostic method for rib fractures. both radiologists and surgeons under-diagnosed rib fractures. scores based on radiography seem un-useful given that this under-diagnoses rib fractures; but with a precision of % by the surgeon evaluating cxr and using a score like rfs perhaps it is enough to decide which patients require a ct scan or more specific treatment in the icu. surgical experience of traumatic diaphragm injury in a single regional trauma center for years introduction: this study is a retrospective review of the experience with the management of traumatic diaphragm injury in our trauma center from to . material and methods: we identified a total of patients with the traumatic diaphragm injury coded from the institutional trauma registry. we reviewed the radiographic finding of radiologists and the electronic medical record (emr). results: the mean of injury severity score (iss) was . ± . . except for case, the plain chest x-ray was evaluated in the patients before surgery, only patients were revealed positive finding for diaphragm injury (n = / , %). the computed tomography (ct) was performed for patients, the positive finding was . % (n = / ). according to the clinician impression before surgery, the diagnosis for diaphragm injury was showed . % (n = / ). approaches were laparotomy in patients ( . %), thoracotomy in ( . %), thoracoscopy in ( . %), laparoscopy in ( . %), open conversion after thoracoscopic or laparoscopic exploration in ( . %), median sternotomy in ( . %). the occurrence of herniation was ( . %). the mean of the calculated rupture size in the operation field was . ± . cm. in our study, the herniated peritoneal organ was observed in more than cm size rupture of the diaphragm. patients were performed surgical management of diaphragm rupture after h. conclusions: without herniation of organs, the radiologic evaluation was difficult to detect diaphragm injury. and, detect of diaphragm injury with herniation of organ, the injury of the diaphragm was predicted a larger than cm. case history and clinical findings: a -year-old man presented to the emergency room with a single self-inflicted left chest gunshot wound at the level of nd rib. on arrival patient was conscious, with systolic blood pressure mmhg and heart rate bpm. extremities were pale, cold. jugular veins distended. investigation/results: fast scan was negative. chest radiograph revealed a metal foreign body with the size of mm at the projection of heart. a ct scan of chest and abdomen demonstrated bullet inside the dorsal wall of the left ventricle and blood in pericardium and left pleural cavity (figs. , ) . therapy and progression: patient was taken to the operation room for median sternotomy. due to severe deterioration of patient's condition, ml of blood was aspirated from the pericardium prior to sternotomy. during subsequent pericardiotomy ml of blood was evacuated. main pulmonary artery wall gunshot injury was detected above the pulmonary valve. the wound was sutured, after which the hemodynamics stabilized. cardiac surgeon was consulted about the air gun bullet inside the myocardium. it was decided that removal of the bullet is not indicated. the patient was observed in the icu for the next h, later transferred to the thoracic surgery ward. the postoperative course was uneventful. an echocardiogram demonstrated a perforation of the anterior leaflet of mitral valve with a mild to moderate regurgitation, otherwise no abnormalities. patient was discharged on day . patient has been followed up on an annual basis for the last years. patient's exercise tolerance and cardiac function according to repeated echocardiography remains unchanged with no evidence of dyskinesia or other abnormalities. bullet is retained in the same location (fig. ) . comments: this case illustrates a successful management of usually lethal injury of main pulmonary artery and reflects that retained myocardial foreign body does not necessarily cause any complications. profile of penetrating chest injuries in hostile environment: a three year study introduction: penetrating chest injuries are one of the leading causes of death and major morbidity in operations involving high energy weapon systems. this study aimed at assess the profile of penetrating chest injuries suffered during armed combat operations in a hostile environment over a three year period. material and methods: a retrospective and prospective, non-randomized study designed to assess the profile of chest injuries in armed combat operations over years. all patients with penetrating chest injuries were included in the study. results: there were trauma cases out of which patients suffered penetrating chest injuries. the age range of patients was - years and all were male. a total of casualties were brought dead ( . %). there were lung injuries and two diaphragmatic injuries. thoracotomy was required in patients ( . %) and intercostal chest drainage (icd) in patients ( . %). average blood loss was ml and duration of hospital stay ranged from to days. conclusions: ballistic injuries to the chest are frequently fatal due to injuries to the heart, major vessels and tracheobronchial tree. prompt and efficient pre hospital treatment, expedient evacuation to a surgical facility and swift management by critical care specialists and surgeons can be instrumental in reducing mortality and morbidity. the cornerstone of management is bedside intercostal chest drain insertion as a formal thoracotomy is seldom needed. penetrating chest injuries can be managed by general surgeons with training in thoracotomy and repair of intra-thoracic structures does the number of a-or low symptomatic but intervention requiring complications justify regularly chest x-ray controls after less than rib fractures? c. deininger , , f. wichlas , , s. deininger , v. hofmann , university hospital of salzburg, orthopedics and traumatology, salzburg, austria, universitätsklinikum salzburg, klinik für orthopädie und traumatologie, salzburg, austria, universitätsklinikum salzburg, universitätsklinik für urologie und andrologie, salzburg, austria introduction: fractures of less than ribs may still cause delayed complications ( ) . the aim of this retrospective study is to determine whether standardized control imaging in a-or low symptomatic patients reveals a significant number of intervention requiring complications and therefor should be recommended. material and methods: all patients with less than rib fractures presenting in our emergency department after any trauma mechanism in the study period of years ( - ) and available for follow up were included retrospectively in the study. results: we included patients in this study, ( . %) of which were male, female ( . %), with a median age of . ± . years. in patients ( . %) rib was affected, in patients ( . %) , the fractured ribs being true ribs ( - ) in cases ( . %), false ribs ( - ) in cases ( . %) and both in cases ( . %). the affected thorax half was the left side in cases ( . %), the right side in cases ( . %) and both thorax halves in cases ( . %). the trauma mechanisms were falls at home, traffic accidents, sporting accidents, work accidents, fighting related and minor trauma in ( . %), ( . %), ( . %), ( . %), ( . %) and ( . %) cases, respectively. the median follow up time was ± days. patients ( . %) required delayed intervention: case of hemopneumothorax and cases of pneumothorax all treated with chest tube. conclusions: planned chest x-ray controls seem not to be necessary. symptom triggered reappearance for patients after rib fractures in hospitals seems to be sufficient and more economical compared to regularly re-imaging ( ) is computed tomography a first line modality in stable blunt chest trauma elderly patients? a. becker , , y. berlin , , d. hershko , emek medical center, department of surgery a, afula, israel, technion-israel institute of technology, haifa, israel, emek medical center, surgery, afula, israel introduction: adult older, patients aged [ years, represent up to - % of all trauma patients admitted to the trauma centers. chest trauma in older patients have been recognized to strongly influence mortality. the estimated of % mortality and pneumonia rate for these patients was observed ( , ) . based on low diagnostic accuracy of cxr, interpretation difficulties due to aging chest wall deformities, we hypothesized that ct chest should be the first imaging modality in stable elderly blunt chest trauma patients. patients and methods a retrospective analysis of all blunt trauma admissions at emek medical center between - years was performed in order to identify patients with blunt chest trauma. only stable trauma patients with abbreviated injury score (ais). results: among patients that met inclusion criteria, there were ( %) patients aged - years old and ( %) patients aged c . in the first group of patients ( - ), had ct chest on arrival. in the second group of patients (aged c ), there were ( . %) patients with missed injuries. in this group, patients who had ct chest on arrival, of ( . %) patients had missed injuries. eleven of ( %) patients who had no ct chest on arrival, diagnosed with missed injuries (p- . ). readmission rate in the first group of patients ( - ) was of ( %) who had ct chest on arrival, and of ( %) who had cxr on arrival only (p- . ). in the second group (c ), readmission rate was of ( . %) patients with ct chest on arrival, and of who had cxr on arrival only ( %) (p- . ). conclusions: based on our study result we conclude that ct chest should be a first imaging tool in stable elderly patients with blunt chest trauma. no disclosures. efficacy and safety of small-bored tube thoracotomy for chest trauma: large-bored chest tubes will no longer be needed introduction: tube thoracostomy drainage is an important treatment for traumatic pneumothorax and hemothorax. traditionally, largebored chest tubes have been recommended for successful drainage and prevention for clogging by clots. however, there is little evidence that large-bored tubes are more effective than smaller ones. in consideration of invasiveness, in our emergency room (er), we use fr chest tube for all trauma patients when chest thoracotomy is indicated. the aim of our study is to investigate the efficacy and safety of small-bored tubes for chest trauma patients. material and methods: we conducted a retrospective observational study. we included the adult patients ([ years old) who had undergone tube thoracostomy with fr chest tubes for chest trauma during the years from october to september in our er. the patients with cardiopulmonary arrest on contact or on arrival were excluded. we evaluated tube-size related complications defined as obstruction and worsening of pneumothorax/hemothorax due to ineffective drainage. results: there were eligible patients, % were male, mean age was . and the average injury severity score was . (± . ). sixty-six tube thoracostomies were performed by emergency physicians and were performed by thoracic surgeons. the average duration of tube placement was . days (± . ). there were not any tube-size related complications nor any patients who required additional tube insertion. case history, clinical findings: different stable hemodynamic cases with thoracoabdominal penetrating trauma and negative fast evaluation were enrolled in study. subsequent hemo/pneumothorax was managed initially by tube thoracostomy. investigation/results: hence laparoscopic investigation is an effective method for evaluation of diaphragmatic injuries in thoracoabdominal penetrating trauma, patients underwent diagnostic laparoscopy. in case , classic approach was done by open technique mm port insertion in sub umbilical. two mm ports inserted in lower abdomen at the level of midclavicular line. then mm port was added in subxiphoid area and by introducing zero-degree camera through it a better exposure was obtained. in case , mm sub umbilical port, mm port in subxiphoid and another mm working port at the level of umbilicus and right midclavicular line were applied. a -degree camera used. exposure, working space and exploration maneuvers were much easier to perform in compare with case . in case , port placement was identical to case but zerodegree camera was used. due to poor exposure, subxiphoid port was replaced by a mm one and used for camera insertion, then an acceptable exposure was obtained. in case , port placement of case was used by using -degree camera which resulted in a great exposure. diagnosis, therapy, progressions: patients tolerated the operation well and underwent appropriate management according to their intra operation findings; post-op courses passed without any complications. comments: in patients with suspicious diaphragmatic injury and according to available facilities in our centers, in unilateral injuries we suggest that a mm port in subxiphoid area can be used instead of contralateral midclavicular mm port. in bilateral injuries, if enough exposure doesn't achieve, a mm port in subxiphoid can be added. in absence of degree cameras, mm port use in subxiphoid can give surgeons better exposure. hemodynamic instability in patients with extremity injuries: motor vehicle accidents and shot wounds vs. explosions a. mahamid , i. ashkenazi hillel yaffe medical center, hadera, israel introduction: we previously reported that hemorrhagic instability (hs) was a complication of extremity injuries in as many as of of patients treated in one medical center following explosions. the objective of this study was to evaluate whether the prevalence of hs in patients with other high energetic injuries such as motor vehicle accidents and shot wounds (mva/sw) is different or not. material and methods: victims following mva/sw with extremity injuries and hs treated in one medical center during were identified with the aid of the national trauma registry and the center's blood bank. hs was defined as tachycardia (pulse [ /min) and/or hypotension (systolic pressure \ mmhg) in need of blood transfusions to reverse instability. patients in whom hs could be attributed to injuries other than the extremity injury were excluded. these were compared to patients treated following bomb explosions ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) conclusions: the proportion of patients in need of blood transfusion is much higher in patients whose extremity injury was caused by an explosion. the relative risk for hs is almost times higher in these patients. new technologies in soft tissue wound management limit reconstruction complexity and enhance recovery introduction: large soft tissue losses are associated with infection, increased morbidity and mortality, increased costs and poor outcome functionality. the purpose of this study was to evaluate the efficacy of a combination treatment of combined topclosure Ò tension relief system (trs) and administration of regulated oxygen and antibiotic irrigation negative pressure-assisted wound therapy (roi-npt) in the treatment of patients suffering from significant soft tissue loss. patients with open abdomen, large infected wounds, and extensive soft tissue loss treated with trs and roi-npt. results: full wound closure was achieved in [ patients treated without skin grafts or flaps. primary failure was successfully followed with secondary closure with the same system. the trs system allowed early postoperative physiotherapy with good to excellent functional results. limitations and complications will be discussed. . trs is a novel device for stretching, and securing wound closure, applying stress relaxation and mechanical creep for primary closure of large skin defects that otherwise would have required closure by skin grafts, flaps or tissue expanders. . irrigation may accelerate the evacuation of infectious material from the wound and may provide a novel method for antibiotic administration. . supplemental oxygen to the wound reverses reduced o levels in the wound's atmosphere inherent to the conventional negative pressure-assisted wound therapy restricting vacuum use in anaerobic contamination. moris topaz is the inventor and patent holder of the topclosure Ò and vcarea Ò . attendees' perceptions about tourniquet safety use aboard, easiness of application, and preference among four devices tested assessed. material and methods: the descriptive study design assessed employing a post-seminar survey, participants' perceptions of tourniquet safety use, application easiness, and preference among the four devices tested (cat, sam-xt, swat-t, and rats). the first two variables measured on a one-to-ten scale (being ten the easiest or safest, and one the least easy or least safe), while preference was measured by frequency count, with only one device to select as the preferred. frequencies and percentages for categorical variables and averages calculated and compared using the anova test (p \ . ). results: a total of sailors, ( %) females, and ( %) males, aged between and , participated in the workshop and completed the survey. the mean for the perception of safety regarding onboard usage was . . as for application easiness, cat and sam-xt ranked equally high ( . ), followed by swat ( . ) and rats ( . ), and the only statistical difference found was for rats (p \ . ). cat was reported as preferred by participants ( %), followed by sam-xt ( %), swat-t ( %), and rats ( %). conclusions: jse crewmembers (non-medical personnel) considered safe the use of tourniquets on board. of the four devices assessed, cat and sam-xt were regarded as equally easy to use and rats the least of all. cat was reported as preferred by almost three out of every four respondents. introduction: surge capacity is the ability to manage the increased influx of critically ill or injured patients during suddenly onset crisis, like a mass-casualty incident (mci) or disaster. during such an event all ordinary resources are activated and used in a systematic, structured and planned way. there are, however, situations where conventional healthcare means are insufficient and additional resources must be summoned. this study investigates the possibility of using community resources such as primary health care centers, nonmedical professionals and non-standardized facilities together with educational initiatives to increase surge capacity in a flexible manner. purpose: to investigate the possibility of an increased and flexible surge capacity during a crisis, disaster or mass casualty incident (mci) by examining the main components of surge capacity (sc) (staff, stuff, structure, and system) in the västragötaland region of sweden. method: this thesis uses a mixed methods research approach with an explanatory sequential design. a literature search was performed by using standard search engines utilizing relevant keywords, questionnaires and semistructured interviews were used for data collection from primary health care centers, dental and veterinary clinics, schools, hotels and sports facilities to determine capabilities, barriers, limitations and interest to be included in a flexible surge capacity system. results: preliminary findings indicate that there is interest, capacity and capability in the investigated municipalities to partake in a fscplan: primary healthcare centers can be toned up with drills and exercises, civilians can be educated in advanced first aid procedures (immediate responders) and focused leadership (scene management), schools, hotels and sports facilities can be prepared with advanced first aids kits and be used as alternative care facilities. these alternatives together represent the concept of flexible surge capacity. conclusion: flexible surge capacity can be a possible approach to create extra resources in disaster situations, mci's, or whenever supporting infrastructure is not intact. new educational initiatives, drills and exercises, laymen empowerment and organizational and legal changes might be needed to realize a flexible surge capacity. introduction: a hospital may need partial or total evacuation because of internal or external incidents, such as in natural disasters and or armed conflicts. an evacuation aims either to transport a large number of patients to other medical facilities or to prepare enough space to receive a large number of victims. despite many publications and reports on successful and unsuccessful evacuations, and lessons learned, there is still no standardized guide for such an evacuation, and many hospitals lack the proper preparedness. we aimed to analyze the preparedness of hospitals for a total evacuation by looking into some key parameters necessary for a successful performance. material and methods: a literature search was performed by using the standard search motors in the related fields, and by using relevant keywords. eleven questions were sent to representatives from euand non-eu countries. results: our findings indicate that there is neither a full preparedness nor a standard guideline for evacuation within the eu or other non-eu countries included in this study. some countries did not respond to our questions due to the lack of relevant guidelines, instructions, or time. conclusions: hospitals are exposed to internal and external incidents and require an adequate evacuation plan. there is a need for a multinational collaboration, specifically within the eu, to establish a standardized evacuation plan. references: nero c, Ö rtenwall p, khorram-manesh a. hospital evacuation; planning, assessment, performance and evaluation. j acute dis. ; ( ): - . introduction: the importance of and the need for medical management during any armed conflict is a fact. many medical achievements have been accomplished due to wars and armed conflicts. the world is, however, divided into countries with and without related military healthcare services. there is a need for joint structure with the civilian in the former, while in the latter the civilian healthcare is responsible for offering services to the military. this study aims to identify the needs of military healthcare system and military medicine as an independent specialty. material and methods: a literature search was performed by using the standard search motors in the related fields, and by using relevant keywords. relevant professionals were asked about the pros and cons of having established military healthcare. the data was collected and analyzed. results: although our findings indicate a need for military medicine/ healthcare as a professional specialty, the organizational divisions between military and civilian healthcare systems seems to be changing. the current security issues worldwide, the pattern of injuries and resource scarcity indicates a need for improved collaboration and maybe a fusion between these entities. conclusions: new security threats, modern technology, the pattern of medical injuries, and the lack of adequate surge capacity may indicate a very close collaboration between military and civilian healthcare systems. such a close collaboration may develop to fusion and a total defense healthcare system that can act both in peace and during conflicts. references: ringel js. the elasticity of demand for health care. a review of the literature and its application to the military health system. https://apps.dtic.mil/docs/citations/ada khorram-manesh, a. facilitators and constrainers of civilian-military collaboration: the swedish perspectives. eur j trauma emerg surg. . https://doi.org/ . /s - - - . alternative methods of mandibular comminuted fracture fixation in severe maxillofacial injured patients introduction: severe maxillofacial injuries refer to significant facial trauma with communitive bony fractures and soft tissue loss. they result in violent trauma as firearm injuries (wartimes injuries, terrorist attack, suicide attempt) and high velocity motor vehicle accidents. the initial management consist of fighting hemorrhage, fighting asphyxia, wounds debridement and suture, and fractures stabilization, especially mandibular fracture stabilization. our study aims to share thoughts on the alternative methods of comminuted mandibular fracture fixation within the context: kind of injury, multitrauma patients, mass-casualty situation, precarious situation or hostile environment. material and methods: based on our experience (clinical cases), on senior surgeons questioning and on medical literature data, we sought to identify, to evaluate and to compare the different available methods to stabilize comminuted mandibular fractures in severe facial injured patients. results: open reduction and stable internal fixation (using macro plate), external pin fixation and closed reduction with maxillomandibular fixation are the methods of treatment which are the most classically used and described. however, some methods using kirschner wires are reported: in cross extrafocal pinning ( fig. ) , external fixation and handmade splints. all these methods differ in their complexity of use, in their availability, and in their possibilities to treat one kind of mandibular fracture or another. conclusions: the stabilization method of comminuted mandibular fracture will be choose depending on material availability, on surgeon's abilities, on the time available (mass-casualty situation) and on the patient's overall condition. even if stabilization methods using wires are less commonly used, they appear to us to be useful in the initial management of the severe maxillofacial injured patient with comminuted mandibular fracture, especially in austere conditions. causes of combat casualties' death at medical treatment facilities (mtf) in modern conflicts: russian experience i. samokhvalov , v. badalov , k. golovko , t. suprun , v. chupriaev material and methods: data including mechanism of injury, physiologic and laboratory variables, staged surgical treatment and cause of death were obtained from the combat trauma registry of the kirov military medical academy war surgery department. the combat trauma registry includes russian wounded in military conflicts over the past decades, of them ( . %) dead of wounds (dow) at the mtf. results: . % of the total dow number died at the role ii field medical units, . % died at the forward military role iii hospitals, and . % died at the role iv hospitals. the causes of dow patients delivered to the mtf were nonsurvivable traumatic brain wound ( . %), life-threatening consequences of injuries-mainly massive blood loss due to external and internal bleeding and acute respiratory failure ( . %), as well as the late septic complications ( . %). terms of death depended on the cause of dow. so for nonsurvivable traumatic brain injuries, they amounted to . ± . days, for lifethreatening consequences of wounds- . ± . days, and in the development of complications- . ± . days. conclusions: there is a high mortality rate among the combat casualties delivered to mtf in modern asymmetric warfare ( . %). moreover, half of these patients ( . %) die at role ii field medical units mostly from nonsurvivable injuries and from acute irreversible blood loss that occurred at the prehospital stage. the main cause of hospital combat mortality is severe septic complications of combat trauma. in consideration of the present counterterrorism practices, prevention and initial treatment for primary blast injury by shock waves constitute a particularly urgent subject because blast injuries and gunshot wounds account for the majority of terrorism deaths. in japan, due to strict ethical standards in animal experiments, there is no appropriate animal model of blast injury. we established an original small animal model of blast injury using a laser-induced shock wave at the national defense medical college (ndmc). however, since the experiments were conducted using only small animals, such as mice and rats, it was necessary to establish a medium-sized animal model aimed to test the applicability in human patients in the long term. correspondingly, we established a blast tube, which was authorized globally as a shock wave-generating device that causes blast injury based on air pressure differences, in the ndmc research institute using the budget of advanced research on military medicine of japan in . this allowed us to conduct scientific studies on blast injury using mediumsized animals. in this presentation, we will introduce the structure and function of the blast tube installed in the ndmc and present some of the results of our research thus far. this research is financially unfortunately, even if hospital and their staff are an essential key for successful response to mcis, the plan are seldom well-known and, above all, exercises are quite neglected at local and national levels. due to mci rarity, simulation exercises are the only way to achieve proficiency in mci response. therefore, we tested an original mci training system (macsim Ò ) adapted to the pemaf of a large university hospital in milan (italy). material and methods: the original mci training format called macsim-pemaf (emergency plan for massive influx of casualties)was developed for the italian society for trauma and emergency surgery (sicut) in . it uses macsim Ò , a simulation tool scientifically validated for training and assessment of healthcare professionals in mci management. between and the course was held for the emergency department staff of a single university hospital of milan (italy) (foundation cà granda-ospedale maggiore policlinico). macsim Ò was used to reproduce the hospital resources, with different mci scenarios. during the simulation the participants had the opportunity to test the local pemaf, in adjunct to their knowledge and skills. course effectiveness was evaluated by a pre-and post-course self-assessment questionnaire. results: macsim-pemaf was tested in seven courses, for a total of participants. pre-and post-test questionnaires showed a significant improvement in hospital staff self-perception of knowledge and skills in mci management. on a - scale, the improvement value was from . ± . to . ± . (p \ . ). conclusions: macsim-pemaf is a useful tool to test single hospital pemaf. it is versatile enough to adapt to specific realties, mimicking different traumatic scenarios. participants, acting in their usual professional roles, can increase their self-perception to be able to respond to a mci with in-hospital resources. introduction: emt are field health facilities, specifically structured to operate in case of disaster, where local healthcare resources are insufficient. there are types of emt. ''emt regione piemonte'' is the first italian emt to be certificated by who. it's a type , meaning that more than triage and stabilization of emergency cases it's provided with an icu, a / working operation room, a test lab, radiological and ultrasound devices. it can admit up to inpatients. cyclone idai made landfall on / / in the district of dondo in mozambique. it brought torrential rains and strong winds and had heavy impacts on the city of beira and surrounding areas resulting in loss of communication and access. in addition important damage and destruction to shelter, settlements, health and wash facilities occurred. on / italian government approved the aid mission, from march st to th three italian military aircraft transported the medical staff and the boxes containing the hospital to maputo and then in beira. on / , the hospital began working, treating an average of patients and performing - surgeries per day, involving mozambican staff who immediately well integrated with the italian colleagues. results: days of activities. surgeries ( orthopaedic, general surgery, gynaecology, plastic surgery). . % of the cases related to cyclone. mean tiss: ( - ). mean age ( - ) females, males. types of anaesthesia: % locoregional, % general, % analgosedation. conclusions: our first experience in a mass casualties' scenario showed how important is to refresh team skills through periodic drills. the leadership is of paramount importance to keep the team united and to support collaboration with other nations' teams and with the local population. adaptability and open-mindedness are fundamental. emts do not arrive in loco immediately so that longer periods of mission and integration with local medical staffs should be programmed. introduction: in utrecht, the netherlands, a worldwide unique major incident hospital is continuously standby to receive multiple victims during mass casualty events. each year, different types of mass casualty events are simulated with a varying number of victims, to train command and control under extreme circumstances. in utrecht, on march th , a terrorist opened gunshot fire in crowded public transport. the aim of the study is to compare our experiences in simulation versus reality. material and methods: an internal evaluation was performed by questionnaires completed by participants and an external evaluation was performed by interviews. results: all five victims were brought to the major incident hospital, of whom two were dead on arrival, one died seven days after due to multiple organ failure and two survived after multiple surgical procedures. all victims arrived within min after the major incident hospital was activated. a sufficient number of medical staff was alarmed for these five victims, however, since the event occurred during office hours, at least a double amount of staff showed up. among some medical staff on commanding key positions fear arose about their own safety and of relatives outside the hospital. this was exaggerated by incomplete and incorrect provided information from the scene. although medical care of the victims was not affected at all, occasionally the anxiety negatively influenced the command and control structure. conclusions: the combination of anxiety and a surplus of awaiting and benevolent curious medical staff resulted in occasional insufficient performance of the existing command and control structure, despite proper training. however, simulation of fear in a training is very difficult. nowadays, with the increasing threat of terror attacks, one should be aware of the influence of fear and anxiety on personnel, even with low numbers of victims. ethic and law issues during mass casualties management operations in foreign countries introduction: mass casualties incidents occur even more frequently during the last years globally. international help in order to manage them, when needed and asked, has to take into consideration special aspects of ethics and local law status in order to successfully fulfill its expectation. purpose: to demonstrate the ethic and law issues that arise during mass casualties management operations in foreign countries. material and method: literature review from recent management operations in syria, iran and sub saharan africa. results: during such operations a lot of ethical and law issues arise. the knowledge of ethics and laws in the country that these take place is essential and critical for the successful result of them. special care must be taken for the management of women, children and dead people. traditions and religion status of the local populations also must be taken into consideration and actions must take place in accordance to respect of the local authorities and social conditions. conclusions: mass casualties management operations in foreign countries is a challenging mission. ethic and law issues arise and must be taken into consideration for the success of the mission. western surgical experience is one thing, but surgical practice in countries in conflict zones is another. the pathologies are different, the thermal conditions are often difficult and the follow-up of the patients is fundamentally modified. humanitarian surgery is becoming more professional and most organizations are setting up a training program for new surgeons embarking on the humanitarian adventure. international committee of the red cross (icrc) has implemented an onboarding-surgeon experience, before to become a fully icrc surgeon. i hereby present my personal onboarding experience in south sudan: how to learn a new type of surgery, how to come with an helicopter to collect patients in the bush and then, how much you learn about yourself. conflict of interest: i only represent my own experience and i do not represent icrc. surgical clinical reasoning during the war in the period between and , i was the head of operating rooms and icu at the clinic for orthopedic surgery and traumatology, in sarajevo. working in the operating room whose walls are shaking because of the sniping and shelling was not remembered by any other generation of surgeons. there were around traumatized citizens of sarajevo. thousands of injured, dying patients were seeking for help from a small number of surgeons. the duty of a surgeon working in the war conditions, without water, electricity, medicines, or heat, is not easy at all, and there were a lot of difficult situations. for example, one day, operated children were again wounded by direct shelling on the walls of pediatric department of our clinic. after we re-operated the children, we also operated the injured nurses. th may, , th february, , and th august, were the most painful experiences in the surgical treatment of disaster in the center of sarajevo, with a large number of massively traumatized patients. while you were helping one casualty, others were pulling our arms or legs. while you were helping one patient, others were dying in the cramp of pain. during the war, a series of traumatic events happened. above many thousands of them, i admitted a -year-old girl, severely injured, with traumatic lower leg amputation of the leg, and severe injuries of the thigh, pelvis, and neck. we operated on her through the night. during the surgery, she received whole blood transfusions. following the surgery, she was stabilized on pediatric department of our clinic. one day, i saw her mother brought her a gift, immensely valuable in those days, a small canister of pure water. in the , one girl approached me, and asked me if i remembered her. i remembered the canister of pure water. she was happy to show me how she can walk now, and told me she lives in canada and works as a university assistant. i was more than happy to see her walk proudly, as she was leaving. she injury pattern of earthquake in athens, greece: the panic-effect introduction: earthquakes are devastating events. greece is known to be in the first place of seismicity in europe and sixth worldwide. lately, a . richter earthquake shook the greek capital, and fortunately no substantial construction damage was sustained. the aim of the study is to evaluate the classification and severity of all injuries, as well as the type of orthopedic surgical procedures performed, in addition to the role that panic plays on the occurrence of these kind of trauma material and methods: prospective case-series study, conducted in the emergency department of our hospital after the july th, earthquake. the study included patients treated by our department, who sustained injuries in their attempt to run away from the scene. age range was from to years old (mean . y.o), were female and were male. results: a total of injuries reviewed. upper extremities were involved in of all cases, lower extremities in and one patient suffered minor head trauma. four patients required hospitalization and all of them underwent surgical treatment. open reduction and internal fixation performed in patients ( calcaneus fracture and olecranon fracture), patient underwent intramedullary nail fixation (tibial shaft fracture) and external fixation was applied to another (distal tibia fracture). six patients were conclusions: panic is an independent contributing factor in natural disaster associated trauma. prior education, preparedness and combined team effort are clearly needed, in order to reduce the incidence of these injuries. regardless of age, panic may result in various types of fractures, even in cases there are no substantial construction damages after an earthquake. digital and analogue record system for mass casualty incidences at sea: results, reliability and validity introduction: mistriage may have serious consequences for patients in mass causality incidences (mci) at sea. therefore, an exercise was conducted to compare the reliability and validity of an analogue and tablet based recording system for triage of sample patients. material and methods: volunteers were asked to triage with the start-algorithm (black, red, yellow and green) patients in a given time using an analogue and tablet based system. triage score distribution and agreement between the two triage methods and a predefined standard were reported. the present study assessed the triage results as well as the reliability through cronbachs alpha and kappa. for testing of validity and internal consistency, the sensitivity, specificity and predictive value was measured. results: forty-eight participants completed a total of triages. while the number of triaged patients in the given time was significantly higher with the analogue system compared to the digital system (p-value . , t-test), the validity measured with the cronbachs alpha and unweighted cohens kappa was higher with the digital system. for each triage category, higher values were gained with the digital system. the sensitivity, specificity and predictive value for the digital system was higher than for the analogue system. conclusions: this study gives reliable and valid results comparing a digital versus an analogue triage system for a mci at sea. significant differences could be found for the number of triages and the number of under triage. the results of the study show that the used digital system has a slightly higher reliability and validity than the analogue triage system. references: the present work is part of the project improved emergency treatment and organization in the event of a mass casualty of casualties at sea (venomas), planned within the framework of the research network ''kompetenz und organisation für den massenanfall von patienten in der seeschifffahrt'' (kompass) and funded by the federal ministry of education and research (grant number: n ). predicting outcome for extremity wounds in pediatric casualties of war introduction: during the early s, the international committee of the red cross (icrc) implemented the red cross wound classification (rcwc) for penetrating wounds. wound grades of , and describe the amount of kinetic energy transferred to the tissue (low, high and massive, respectively). currently, this classification system mostly serves as a descriptive tool, but it is hypothesized it could also support clinical decision making. the aim of this study is to assess whether the wound grade of a pediatric patient's extremity wound correlates with patient outcomes. material and methods: this study included pediatric patients (age \ years), who have been treated by the icrc for conflictrelated extremity injuries between and . the correlation of the following variables with the wound grade were analyzed: number of surgeries required, length of stay, and in-hospital mortality. results: the study cohort consisted of pediatric patients. the higher the wound grade, the more surgeries were performed per patient (p \ . ), with a mean of surgeries per patient if they had a wound grading of . there were no significant differences in mortality rates between any of the wound grades, which were . % ( / ), . % ( / ) and . % ( / ) for wound grade , and respectively. pediatric patients with wound grade were hospitalized for the longest period (mean . days), followed by wound grade (mean . days) and wound grade (mean . days; all with p \ . ). conclusions: the wound grade of pediatric patients' extremity wounds appears to correlate with some patient outcomes, but not with mortality. grading of extremity wounds according to the rcws could support clinical decision making in pediatric patients. introduction: during the last few decades, french armed forces have regularly deployed in asymmetric conflicts. surgical support for casualties of these conflicts occurs in nato role and medical treatment facilities (mtf); definitive surgical care occurs in france following a strategic medical evacuation. the aim of this study was to describe the combat injury profile of these soldiers who presented with either non-exclusively orthopedic and/or non-exclusively brain injuries. material and methods: this descriptive study is a retrospective analysis of the surgical management of french casualties performed in role or mtf in afghanistan, mali, niger, djibouti and the central african republic between january and december . results: one hundred patients were included. forty had fragment wounds. the most severe lesions were of the head, neck or thorax. the average injury severity score (iss) was . (ic % . - ). damage control procedures were performed. thirty patients died with a mean iss of (ic % - ); deaths were considered as preventable deaths. the most frequent surgical procedures in the mtf were digestive (n = ) and thoracic surgery (n = ). thirty patients needed second-look surgery in france; eleven had severe complications. no patient died following medical evacuation to france. conclusions: results from this study indicate that the mortality following non-exclusively brain or orthopedic injuries remains high in modern asymmetric conflicts. introduction: telemedicine has been applied to disasters and extreme environments for more than years, however, despite the many lessons learned so far, telemedicine is still not a common part of the immediate disaster response. for this reason, a review of the literature was conducted to investigate whether telemedicine technology can be used to address medical and non-medical needs in extreme environments. material and methods: this systematic review included studies published in the period - , originating from literature search bases medline, scopus, cinahl and pubmed. the case of neemo project were studied so to evaluate the diagnostical and surgical care of the patients regarding the emergency response in a remote and constricted area, with limited human medical resources and using the telecommunications and telerobotic technologies. results: the majority of the included studies have highlighted the importance of telemedicine interventions in extreme environments, stressing that it is a viable solution to health care provision. in addition, it has been found that telemedicinal technology provides the possibility of virtual collaboration between healthcare professionals with various specializations. projects neemo , , engaged to eliminate the challenges of telesurgery. conclusions: future studies such as large multicentre randomized trials will have to be conducted that will lead to safe conclusions on the usefulness and efficiency of telemedicine applications in extreme environments. introduction: tourniquets are a critical tool in the immediate response to life-threatening extremity hemorrhage. the optimal tourniquet type and effectiveness of non-commercial devices is unclear, and the aim of this study. material and methods: this prospective observational cadaverbased study was performed using a perfused cadaver model with a standardized superficial femoral artery injury bleeding at ml/ min. five devices were tested: cat (combat application tourniquet), rats (rapid application tourniquet system), swat-t (stretch, wrap, and tuck tourniquet), a triangle bandage and a stick and a leather belt. volunteer medical students with no prior clinical tourniquet experience participated. each student underwent a practical hands-on demonstration of each of the tourniquets, prior to the test. using a random number generator, they then placed all tourniquets in random order. outcomes measures included time to hemostasis, total time to secure devices, estimated blood loss (ebl) and difficulty rating. a one-way anova repeated measures was used to compare efficacy between the tourniquets in achieving the outcomes. results: participants' mean age was ± . years and ( %) were male. all participants were able to stop the bleeding with of the tourniquets. with the rats there was a % failure rate. among the five types of tourniquets, time to hemostasis and ebl were not statistically significantly different (p [ . ). the swat-t required the longest time to be secured ( . ± . ), while the belt was the fastest ( . ± . ; p \ . ). conclusions: all five tourniquets, including the non-commercial devices, were effective in achieving hemostasis. a standard leather belt was the fastest to place and able to stop the bleeding. however, it required continuous pressure to maintain hemostasis. nevertheless, in an emergency setting where commercial devices are not available, improvised tourniquets may be an affective lifesaving bridge to definitive care. hospital preparedness for mass gathering events and mass casualty incidents in matera, european capital of culture for introduction: mass casualty incidents (mci) may occur during mass gathering events (mge). lack of preparedness of health system increases mortality. education and training are crucial. hospital mci plans are mandatory in italy, but they are poorly known. on , matera was declared italian host of european capital of culture for : the local hospital decided to revise the hospital plan for massive influx of injured (pemaf) and to start a program to train the staff. material and methods: the pemaf was reviewed through simulations that involved all the staff. a partnership with mrmi-italia (italian chapter of the international association medical response to major incident and disaster-mrmi&d) leaded to the support of experts and to the organization of residential courses based on the macsim Ò (mass casualty simulation) simulation tool. educational capacity of the residential events was tested through a self-assessment tool. results: alert, coordination and command sequences were defined. all the available resources were recorded and the functional areas identified. the communication network was improved. documentation and registration system was prepared. standard operational procedures (action cards) were created for the key positions. residential educational events of macsim-pemaf were organized. the educational capacity was tested through self-evaluation: knowledge of participants resulted improved. conclusions: mge are a great opportunity for the hosting community but they also represent an increased risk of mci. preparedness is mandatory for health system. the format macsim-pemaf seems to be adequate to review the existing plans and transfer skills to attendants. introduction: the cruise industry is facing a constantly growth of infectious diseases. some of them are reaching the extent of mass casualty incidences (mci), which are overwhelming the capacity of the local rescue system. our aim was to improve the ability to act in a mci due to an infectious emergency regarding the situation at sea/in the port. hamburg, as one of the largest ports in europe, was chosen for analysis. material and methods: the collaborative project ''adaptive resilience management in the port'' (armihn) is funded by the german federal ministry of education and research. scenarios due to an infectiological emergency were developed together with the university central department of occupational medicine and maritime medicine and the hamburg port health center in hamburg, germany. these scenarios were specified with all key stakeholders in the port. the organizational structure of the current emergency management was analyzed and a new concept was developed. results: for the ship and the port, emergency strategies dealing with mass casualties of injured persons are available. nevertheless, current concepts regarding this special situation of an infectiological mci were missing. we developed a new concept, which based on the models concerning mass casualties of injured persons. for this purpose, emergency surgeons can be recommended as experts regarding coping with a major emergency and for developing adaptive training concepts. conclusions: new operational concepts coping with mci of infectious patients were developed. in a second step, an emergency plan and a training concept for relevant stakeholders in the port will be developed. these will be evaluated in a full exercise in the port of hamburg and tested for their suitability. the results will be transferred to comparable infrastructures to cope with a major case incident with infected people in the port area. emergency surgeons should be involved in these steps due to their expertise. the work was funded by the german federal ministry of education and research ( n ). no further significant relationships. war surgery training, the use of swine model in military simulation center introduction: due to the international instability, our forces are deployed in many place and our military surgeons have to deal with ballistics trauma and improvised explosive devices related trauma. in order to be well prepared and effective in these isolated situation, the val de grace school (our military health service academy) provide a years course to train the young surgeon. this year surgical courses ended with war trauma surgery simulation on a swine model. material and methods: this use of the swine alive model is incorporated in the cesimco (military surgical simulation center) and also use for the training of our fully registered surgical team. this laboratory responds to all civilian authorizations and ethical considerations as enacted by european rules (felasa). results: the aim of this presentation is to show the different procedures and the teaching provided in this structure to improve surgical skills in war condition. all procedures are approved by the ministry in charge of the animal experimentation and respond to the animal welfare regulation. the number of swine used in these teaching is reduced to the minimum. we think that this animal model and its use in military forward surgical facilities, is the end point of the years military surgical course provided by the val de grace school. conclusions: this model is actually the most reliable and ethically acceptable teaching procedure we've found. during these teaching the students have to deal with open trauma and hemorrhagic lesions in damage control situation. we try to follow the different type of war related lesions observed in french military in order to stick to the reality of the field. this teaching is now mandatory before being deployed as a military surgeon on field. case history: -year-old male, previously healthy, admitted to the er due to shotgun injury to the right hip. during transport, the bleeding open wound was covered, two iv catheters were introduced, and saline and painkillers were administered. on admission, the patient was conscious, eupneic and normotensive, with a gcs score of . clinical findings: after the primary survey and exclusion of cranial, thoracic and abdominal lesions, the limb injury was addressed, showing a cm oval-shaped wound. the right leg was shortened and externally rotated. pulses were present but the patient referred calf and foot hypoesthesia. investigation/results: x-rays showed a comminuted pertrochanteric fracture and the presence of metallic foreign bodies. diagnosis: open right pertrochanteric fracture. therapy and progressions: initially, the wound was covered, and iv antibiotics and supportive therapy were given. in the or, irrigation, surgical debridement, and foreign body removal were performed, followed by orif with one dall-miles cable and a cephalomedullary femoral long nail. after surgery, the patient maintained lower limb hypoesthesia and had plantar flexion and foot dorsiflexion grade motor deficit. during follow-up, soft tissues recovered uneventfully and bone healing successfully occurred. full weight-bearing was tolerated at weeks post-op but the neurological deficits persisted despite physiatric treatment. electromyography confirmed severe partial lesion of the sciatic nerve. comments: generally, clean wound, fracture stability, restoration of circulation and skin closure of neurovascular structures are a priority and should be a reason for delayed nerve repair. introduction: despite mass casualty incidents (mci) are becoming a common concern, particularly regarding the care of paediatric victims, pure paediatric trauma centres (ptc) are still rare in europe. the purpose of this study is to assess the capacity of the hospitals in the metropolitan area of milan in case of mci involving the paediatric population, with focus on the pre-impact planning phase. material and methods: relevant literature and existing guidelines were reviewed by the representatives of four referral centres for the management of either trauma or paediatric patients. minimum standard requirements of care of paediatric trauma and consequently the maximal surge capacities for each hospital were defined based on the severity of injuries and personnel/equipment availability. results: overall, the four hospitals are able to treat patients with the highest priority (t ), to patients with intermediate priority (t ), and patients with deferrable priority (t ). severely injured patients \ years old should be preferentially transported to the hospitals with paediatric expertise, whereas patients between to years of age can be managed in multi-speciality structures. conclusions: in case of mci it is not always possible to rely on the availability of a ptc. hospitals with paediatric trauma care expertise can work in synergy with ptcs, or offer an alternative if there is no ptc, and should therefore be included in disaster plans for mci involving paediatric victims. case history: we present a case of a -year-old male with a proximal radius and ulna gunshot fracture associated with a complete lesion of the brachial artery, which was urgently repaired by grafting in his native country. a partial proximal radius excision was also performed. three months later, after soft tissue recovery, the ulna fracture was fixed with a dcp plate plus iliac crest bone graft. at months follow up x-rays showed hardware loosening, so the plate was removed and an external fixator was implanted. in this situation the patient attended to our clinic months after the initial injury. clinical findings: findings included proximal pin purulence, an elbow varus deformity and a limited joint motion: flexion °, extension °, supination/pronation °. investigation/results: x-rays and ct scan showed proximal ulna pseudoarthrosis. diagnosis: proximal ulna pseudoarthrosis after a gunshot fracture. therapy and progressions: a two-stage procedure was performed. initially we performed a wide debridement and external fixator removal. an ulna nail combined with gentamicin and vancomycin pmma spacer was implanted. s. aureus was identified in intraoperative cultures. in a second stage, year after, the nail and spacer were removed and a vascularized fibula graft with saphenous loop was implanted and fixed with a va-lcp plate. the central band of the interosseous membrane was repaired with a prosthetic device. currently, the patient presents full flexion range, hyperextension of °, active pronation of °and supination of °. x-rays show graft consolidation. comments: gunshot fractures are complicated lesions with significant soft tissue damage and high risk of vascular and nervous injury. a thorough study and initial systematic approach is mandatory in order to avoid later complications. introduction: the purpose of our study was to independently analyze pediatric trauma data, especially that of preschool-aged children, including demographics, injury patterns, the associated mechanism of injury, and outcomes, at a single institution in korea to gain a better understanding of current trends in non-regional trauma centers. material and methods: we conducted a retrospective review of preschool-aged children with trauma, who presented to the emergency department a single center between march and december . results: overall, there were pediatric patients who experienced trauma admitted during this study period. the frequency of admissions was similarly high in all seasons except winter. falls were the most common mechanism of injury at all ages, except , , and years of age, according to comparative analysis by age and mechanism. the most common place of trauma at - years of age was at home, and outside the home at the age of years or older. the most common injury region was to an extremity ( . %). mean injury severity score was ± . , and the mean hospital stay was . ± . days. conclusions: although mortality from trauma is low in pediatric patients, we must continue to improve treatment outcomes for children. it is unlikely for a hospital to have a pediatric trauma specialist, such as a pediatric orthopedic surgeon or plastic surgeon, due to manpower constraints. in order to further improve the outcome of treatment with insufficient resources, it is necessary to recognize agespecific characteristics. question: the new safety situation in europe and the lessons learnt civilian events of damage show that hospitals have to be prepared for mass casualties. the shift of the operational mode to ''emergency medicine'' have to be planed and practiced. the reporting tool for this is the hospital action plan (hap) that every hospital should have. the efficiency of the existing plan is already proven in different largescale exercises. in germany the legislator obligates the hospitals to enable there staff to properly perform the different tasks of the hap. in addition, the have to develop and evaluate proper training and exercises. goal of this study was to establish along the hap of a level one trauma center an modular mass casualty training (manv ) that would help to analyze the tasks to face and to deepen the existing structures of communication. method: we set a scenario with casualties and evolved the different shifting phases of the trauma center (alarming-, mobilization-, constitution phase). setting the concept of training outside the regular service period we took in account that there will be a lack of resources and material. we did not exercise in a large-scale but trained in small groups modular. we also did a screen adaption of the hap of the trauma center to have a mind set for the staff and a starting point to the scenario. to teach our operative procedure we simulated our '' columns concept'' (medical, personal and infrastructure) to the staff. specific to the different task groups (medical doctors, technicians, nurses) we exercised and the different sectors (er, triage, or, command etc.) and the necessary shifts of the different hospital sectors when a mass casualty occurs. before and after we did a query of the staff to see how much impact the modular exercise would have on the hap-knowledge of our staff. results: we were able to simulate realistically an identical mass casualty scenario to different staff groups of our hospital. knowledge about the hap increased significant from to % after the trainings. % of the staff see a clear improvement of information about the hap. also, the specific shifting-phases and the enrolment of the plan to move in an ''emergency medicine mode'' understand % better. % of the staff fell now a much better preparedness than before. % think that through modular exercises and small group training the communication in between working groups improved. conclusion: we could manage to improve a significant increase of knowledge about the hap in our staff. all the small group modular training in the different sector can be easily but together in large-scale exercise and other teams like police, military or fire-department can easily be added. introduction: dstc course focusses on surgical skills for trauma care. it is designed to teach surgical techniques for the definitive treatment of severe trauma. currently, it has evolved into an international trauma team course. our objective was to assess faculty members' opinion regarding course content, educational methods, and incorporation of non-technical skills. material and methods: a descriptive study was designed using an anonymous online survey issued from may to august , . senior international faculties' opinion from countries assessed. the survey inquired views of courses content, duration, adequacy of hands-on practice, need for updates, and usefulness of incorporating non-technical skills to the course. results: from the surveys issued, were ( %) answered. the course content was valued as very satisfactory by %; % were very satisfied or satisfied with courses educational method. % considered the time devoted to lectures, case discussions, and skills lab very adequate or adequate. course duration ( days) was valued suitably by % of responders. the inclusion of non-technical skills was considered as very important by %, important %, of some importance %, of little importance by %, and unimportant by %. this result reflects the insufficient sense of significance, among some, of the importance of trauma team dynamics. course content updates were seen as convenient by % of the surveyed population, suggesting them at least every - years. conclusions: dstc international faculty response to the online survey tool was inadequate, receiving % of the targeted study population. of the assessed faculty, most were satisfied with course content, duration, and educational methods. the surveyed population lacked a uniform perception of the importance of incorporating nontechnical skills. introduction: dstc is an iatsic course emphasizing on teaching surgical skills for trauma care. in many countries, it is an essential course focused on the ''second hour'' beyond atls and teamwork. initially centered on the surgeon, it currently seems to be adopting a trauma team training (ttt) model, incorporating the anesthetist to the program (ds-datc). our objective was to review this changing trend in three countries: spain, portugal, and brazil. material and methods: a descriptive study was designed by faculty from the three countries examining course records and analyzing its evolution during the last five years. number and types of courses delivered in each country from to reported, and the proportion of dstc to ds-datc scrutinized. frequencies and percentages calculated for categorical variables and the proportion of course types also determined. results: during the -year studied period, dstc courses were issued: ( %) in spain, ( %) in brazil, and ( %) in portugal. a total of ( %) ds-datc courses in the three countries, and the percentage of total delivered in each country was as follows; spain ( %), portugal ( %) and brazil ( %). overall ds-datc to dstc ratio was : , detailed as follows: portugal : , spain : , and introduction: thailand is a disaster-prone country with a high dependency on tourism. it has been affected by both natural and manmade emergencies. the thai emergency healthcare system consists of emergency physicians working at hospitals and prehospital levels, emphasizing their essential role in emergency management of any incident. we aimed to investigate the thai emergency physicians' level of preparedness by using tabletop simulation exercises and three different scenarios. material and methods: using the lc (three level collaboration) method, two training sessions were arranged for over thai emergency physicians, who were divided into three groups of prehospital, hospital, and incident command staff. three scenarios of a terror attack and explosion, riot and shooting, and high building fire were discussed in the groups. results: our findings indicate that the initial shortcomings in command and control, communication, coordination, and the ability of situation assessment increased in all groups step by step and after each scenario. new perspectives and innovative measures were presented by participants, which improved the whole management on the final day. conclusions: tabletop simulation exercises increase the ability, knowledge, and attitude of thai emergency physicians in managing major incidents in strategic, tactical, and operative managerial levels, and should be included in their professional curriculum. introduction: non-operative management of traumatic injuries has led to decreased surgical exposure for trauma trainees [ ] . while simulation using cadavers may improve exposure to damage control techniques, tissue handling realism is variable depending on embalmment and perfusion techniques [ ] . objective: to evaluate the feasibility of perfused thiel cadaver use for trauma surgery simulation. material and methods: thiel cadavers were cannulated in the ascending aorta and right atrium to create a left-to-right perfusion system. a magnetic pump was used to achieve a pulsatile flow with a gelatin-based solution, aiming for a flow of l/min. peripheral circulation was improved with arteriovenous fistulas (carotid-jugular, femoro-femoral and brachio-brachial). a left common iliac vein injury was performed laparoscopically through the sigmoid mesentery. the surgical trainee was blinded to the initial injury and assisted by a staff surgeon. results: a trauma laparotomy was performed. the small bowel was eviscerated and all four quadrants were packed with gauze. a left, expanding zone iii hematoma was detected. the left sigmoid colon was mobilized to achieve proximal control of the left iliac vessels. the left common iliac vein was actively bleeding and ligated according to damage control principles. the left ureter was uninjured. the sigmoid mesentery was closed, without active bleeding. the remaining of the abdominal cavity was explored without other injuries. time from laparotomy to closure was min. tissue handling and circulation dynamics were highly realistic due to thiel embalmment and pulsatile perfusion. conclusions: pulse-perfused thiel cadavers represent a realistic simulation option for surgical trainees. widespread implementation may provide accurate simulation for lifesaving procedures rarely performed in an era of non-operative management of traumatic injury. a new concept of intra-operative performance monitoring and self-assessment in hepato-pancreato-biliary surgery and other surgical specialties s. kharchenko , , m. yanovsky colmar civil hospital, university of strasbourg, department of general surgery, colmar, france, hepato-biliary institute henri bismuth, paris, france, interceg, kharkiv, ukraine introduction: currently, the majority of learning curve studies for surgical interventions associated with simple chronometric estimation in a whole: from incision to closure. a selective approach for step-bystep time fixation of all hpb interventions (hepatectomy, others) or other surgical specialties can bring a new vision of correlation between intra-operative timing and the clinical outcome. material and methods: every operation can be divided into step items so standardized worldwide, for example, planned or urgent laparoscopic cholecystectomy e.g. incision to port placement, exposure, dissection to cholangiography, cholangiography, extraction, closure. results: the prototype named chronoi of infrastructure for automated monitoring (simulator of time tracking activities, web-service for request processing, database and knowledge base collection subsystems, learning curve representative and analytics software) is designed and to be implemented. individual self-assessment is available in a real-time fashion. the learning curve changes are shown per procedure. up to our knowledge, we can firstly in the world describe the surgeons, incl. in hpb, as speedy, standard or nonstandard depending on the surgeon's ''individual speed'' in operative performance. it's to be documented in their e-logbooks according to the current fellowship standards or practice re-certification. conclusion: the intra-operative monitoring and worldwide standardization give a new vision of the surgical practice in hpb surgery meaning an introduction of monitoring-based clinical outcomes (timing with morbi-mortality or other). only new trials will approve the role of the presented concept in hpb surgery as well as in general, emergency and trauma. introduction: the management of patients victims of war weapons and collective emergencies represents a major public health issue in france, but also abroad. terrorist events in recent years on the national territory have highlighted the need for training the population and caregivers in the management of these injuries. because of his experiment in the domain, the french military medical service (fmms) was requested to cooperate with the french prehospital teams in order to improve knowledge and teaching in this area. today, a continuing medical education, easily available and free access is needed in this area. material and methods: development of video podcasts (infographics) of a few minutes on the theme of management of patients victims of war weapons and collective emergencies. the working group ensures the production and quality of educational messages. production is provided by the communication establishment of defense. the broadcast is displayed on the channel you tube of the fmms. results: the title of the traum'cast podcast is the contraction of trauma and podcast. twelve episodes are scheduled on a -weeks rhythm. the podcast program is as follows: conclusion: fmms knowledge and experiment in managing patients victims of war weapons is unique. teaching can take various forms, theoretical, practical, academic, or through publications. traum'cast is a major innovation in the dissemination of this knowledge and each episode focuses on a specific skill. traum'cast will highlight the applicability of military medicine concepts in a civilian environment. traum'cast will be translated in an english version. project was supported by grants of french ministry of defense (innovation department). splenectomy in current surgical practice: a tricky and elusive procedure for the surgical resident? introduction: splenic rupture and oncologic resections are the most common indications for splenectomy, but technical expertise is progressively being taken over by non-operative and more conservative approaches. material and methods: retrospective review of all total splenectomies performed between february and january at an italian academic hospital, assessing demographics, diagnosis, operating surgeon, surgical approach, complication rate, postoperative critical care admission, and -day mortality. results: over years, consecutive splenectomies were performed by different surgeons, of whom surgical trainees, with unplanned (i.e. emergency/iatrogenic injury) and planned (i.e. benign/malignant disorders) procedures and an average of . and . procedures per year respectively. over the study period, only surgeons performed at least procedures and only performed at least procedures. laparoscopy was performed in . % of cases, predominantly during planned procedures, with an overall . % conversion rate mostly related to technical difficulties (i.e. spleen dimension, difficult vascular visualization). overall major postoperative complication rate (clavien-dindo c ) was . %, slightly higher in emergency procedures although not significantly different ( . % vs. . %, p = . ). reintervention rate was . %, due to hemorrhage in more than half of cases. overall -day mortality rate was . %, with elective -day mortality rate of . % (p = . ). conclusions: splenectomy may be required ever more rarely but potential risks are not irrelevant. competence for surgical trainees should be achieved elsewhere (e.g. simulated/cadaveric training case history: an year old femal patient underwent changing of the components of the tha because of aseptic loosening. due to circumstances the surgeon decided to implant a cemented femoral component. the procedure was without any significant abnormalities. the first postoperative radiograph was planned after recovery-as usual. the x-ray imaging showed a misplaced femoral component. therefore a ct-scan was performed additionally and the malposition of the cemented femoral component was confirmed. the patient had to undergo another surgery-removing of the cemented femoral component and implantation of a new well placed one. therapy and progressions: after prompt resuscitation, an emergency laparotomy was performed and an anastomotic leak was found, requiring re-do ileo-ileal anastomosis. postoperative course was complicated by intra-abdominal collection treated by antibiotics alone (clavien-dindo grade ). the patient was discharged on th pod. at pathological report, segmental absence of intestinal musculature (saim) was diagnosed. the revision of past specimens confirmed the same finding. comments: usually recognized in neonates/premature infants, saim is generally an incidental finding in adults [ ] , often undiagnosed and more frequently described in the colon [ ] . in such scenario, main differential diagnosis is ischemia. etiology is unclear and can be classified as either primary/congenital or secondary. the former is characterized by acute onset of symptoms, whereas in the latter a longer history of intestinal symptoms is usually present [ , ] . most authors agree upon a congenital pathogenesis. generally, saim is associated with hollow viscus perforation and treated with surgical resection. contrary to our experience, no recurrence of intestinal perforation has been reported [ ] virgen del rocío university hospital, general surgery, seville, spain, hospital regional de málaga, general surgery, málaga, spain, hospital de estella, general surgery, navarra, spain, hospital gregorio marañón, general surgery, madrid, spain, complejo hospitalario de jaen, general surgery, jaen, spain introduction: specific training in the management of trauma patients is essential for surgeons. training through courses in this area (atls, dstc, musec) directly impacts the care of these patients. the aim of this study is to know the specific training in trauma care of spanish surgeons. materials and methods: a national survey has been sent to all member surgeons of the spanish surgeons association. it has evaluated their degree of participation in emergency surgery acute care, and therefore the possibility of attending trauma patients, their participation in the initial care at their hospital, as well as their specific training in this area. results: the survey has been completed by surgeons from spanish regions, and most surgeons who responded were from catalonia and andalusia. ( . %) of those surveyed take calls for the ed. only ( . %) report having a hospital registry of trauma patients. . % of surgeons answer that in their hospital the general surgeon is not involved in the initial care of trauma patients. . % have taken the atls course, . % the dstc course, and . % the musec course (or another course on e-fast). despite this, . % consider the atls course should be mandatory during residency, and . % of those surveyed consider trauma care in their hospital as very bad or deficient. conclusions: according to this survey, specific training in trauma care is still deficient in spain and with many aspects that can be improved. only % of those surveyed have received specific training in definitive surgical management of severe trauma. despite this, a large percentage of surgeons take calls for the ed routinely, and face the challenge of managing these patients. exploring team leaders' decision-making challenges in civilian and military complex trauma introduction: in the nordic countries professionals may work in both civilian and military trauma care. timely and effective decisionmaking in complex trauma is essential in improving survival benefits. the mindset and management priorities differ among medical professionals, and correlate with different experience levels. trauma leaders are usually senior surgeons with extensive experience and well-developed decision-making skills. simulation training has been shown to be effective in practicing decision-making. the aim of this study is to explore the team leaders' decision-making challenges in complex trauma care and structure them with the activity theory framework (at). material and methods: video recordings at a trauma center in johannesburg and live observations of complex trauma training in gothenburg focusing on team leaders' decision-making challenges were analyzed and systemized using the at. results: the team leaders' activities were mapped onto the main elements of at ( fig. ) whereby the decision making challenges were classified into six categories (table ) . conclusions: the at framework may benefit and inform the design of educational interventions by structuring key issues of complex activities. introduction: trauma is one of the main causes of mortality worldwide and prevention stands out as one of the main ways to modify its incidence. a prime example of such initiatives is the prevent alcohol and risk-related trauma in youth program (p.a.r.t.y.). it aims to raise awareness of the population most at risk for trauma, young people from to years. the study objective was to evaluate the program impact on students' knowledge and behavior. material and methods: a quantitative, uncontrolled intervention cohort study was conducted through the responses of the p.a.r.t.y. in and . data collection occurred through the application of a questionnaire to participating and non-participating students of public schools in the city of campinas, after a few months of participation in the program. results: among answers, . % were male, . % between and years, and . % program participants. time between participation and answers was . (± . ) months. regarding the first conducts when facing traffic trauma, . % of those who participated chose the correct answer, against . % of those who did not. about the first care while the service does not arrive, . % of the first group answered correctly, compared to . % of the second. concerning about the service that should be called in the event of a trauma, . % of participants would call correctly against . % of non-participants. in questions related to traffic laws, . % of participants opted for the correct answer as to what should be done in the face of a running over, against . % of non-participants. conclusions: students who had participated in the program had a higher rate of correct answers, a few months after the event, compared with students who did not attend. thus, it is concluded that there is a impact over the time caused by it. introduction: currently, intraosseous (io) devices are necessary for the resuscitation of severe trauma patients. however, opportunities to learn io device insertion are limited for residents. the aim of this study was to conduct a simulation of io device insertion for residents and to evaluate its effectiveness. material and methods: in this simulation, residents inserted io needles into the sternum of pigs under general anesthesia with the instructor's guidance. comprehension tests and questionnaires about satisfaction level and self-efficacy were conducted before and after the simulation. the objective evaluation was the io access success rate, and the subjective evaluation was obtained from points on comprehension tests and questionnaires. results: thirty-six residents participated in this study. just one resident had successfully obtained io access clinically. success rate of establishing io access in the simulation was %. the rate of test completion was % and that of questionnaire with survey response was %. the comprehension test results improved from . ± . to . ± . (mean ± standard deviation, p = . ) out of points. the questionnaires concerning satisfaction level changed from . ± . to ± . (p \ . ) out of points. the questions specifically concerning self-efficacy dramatically increased from . ± . to . ± . (p \ . ) out of points after the simulation. conclusions: the simulation in this study improved the knowledge, satisfaction level, and self-efficacy of the residents for io access. the success rate of confirmation of io access in this study was %. this experience may positively affect their clinical performance in trauma care. case history: case . a -year-old white man presented to the ed complaining of intense abdominal pain and vomiting. he referred at least two previous episodes with associated fever which resolved spontaneously. case . a years old white man consulted at the ed for intense abdominal pain, nausea, anorexia and constipation for the last h. none history of abdominal surgery were registered. clinical findings: in both cases, the abdomen was distended without bowel sounds. investigation/results: case . abdomen xr: distended small bowel loops localized at the right side. ct scan: an encapsulated cluster of dilated small bowel loops into the ascending mesocolon. case . ct scan: an encapsulated nonrotated small bowel in the right side of transverse mesocolon and mesenteric vascular pedicle displaced. diagnosis: intestinal obstruction secondary right paraduodenal hernia therapy and progressions: emergency midline laparotomy that evidenced a rpdh which was reduced before closing the mesentery defect. the postoperative was uneventful. comments: paraduodenal hernias are a type of internal hernia and a rare cause of intestinal obstruction accounting for about . % of all hernias. right paraduodenal hernias are far less common than left ones. symptoms of paraduodenal hernias are nonspecific. preoperative diagnosis of pdh by imaging techniques is difficult. contrastenhanced ct scan is highly recommended as the most specific method of diagnosis for pdh. with the increased use and improved enhancement of ct scans, paraduodenal hernias currently can be diagnosed preoperatively. this advancement in diagnostics coupled with increasing experience and facility of general surgeons in using laparoscopic techniques has led to the initiation of laparoscopic repair of internal hernias. case history: a -year-old female patient who goes to the emergency department due to vomiting and abdominal pain. since the accident, the patient reported post-prandial discomfort and gastroesophageal reflux, as well as self-limited abdominal cramps. clinical findings: soft, depressible abdomen. bowel sounds on left hemithorax. investigation/results: cxr: right hemidiaphragm elevation. lab test: leukocytosis. thorax and abdomen ct: right anterior diaphragmatic hernia and passive atelectasis secondary to ascent of dilated small intestine and colon. diagnosis: intestinal obstruction secondary post trauma diaphragmatic hernia. therapy and progressions: emergency laparotomy due to symptoms compatible with intestinal obstruction secondary to incarcerated diaphragmatic hernia. it is right diaphragmatic chronic rupture chronic with omental incarceration, antrum, small bowel and ascending colon with reversible signs of suffering. chelotomy and content reduction, herniorrhaphy with loose spots with non-absorbable material are performed. endothoracic drainage is left removed at h. the postoperative course is uncomplicated. comments: trauma events should be considered in the diagnostic process to avoid delayed treatment. case history/clinical findings: we present a -year-old male patient with a history of large pelvic mass in the rectum-prostate space under study, since months. he were admitted into the emergency unit, days after the mass biopsy, with fever up to °c and rectorrhagia. the patient rapidly developed septic shock with hemodynamic instability and elevation of acute phase reactants. abdominal ct was performed: pelvic mass of . . cm, of heterogeneous content, with areas of blood density. we decided doing an emergency surgical exploration of this mass as the only suspected origin of infection. investigation/results: in the surgical exploration the mass was protruding on the anterior rectum wall. the mass was drainaged with an output of ml of purulent material mixed with clots and necrotic tissue. foley no. probe was placed inside the cavity. in the postoperative period, the patient showed significant hematochezia, so he was reoperated performing hemostasis and rectal tamponade. it was effective and a new foley catheter was replaced at h. when the purulent drain gave way, the catheter was removed and the patient evolved favorably. diagnosis: cytology analysis: mesenchymal type lesion, morphologically and immunophenotypically compatible with gist (gastrointestinal stromal tumor). ihq profile: cd , dog , c-kit positive. therapy and progressions/comments: the complications of gist are usually acute abdomen due to peritonitis secondary to perforation or hemorrhage. however, the formation of intratumoral abscesses is very inusual, although is described in the literature. emergency surgery is often necessary due to the significant affectation of the general condition of the patient and the difficulty of the diagnosis. fournier's gangrene (fg) is a surgical emergency defined by an obliterating endarteritis of the subcutaneous tissue arteries of infectious etiology, with progressive necrotizing fasciitis of the perineal, abdominal, thoracic or lower limbs, which can lead to multiorgan failure. a years old woman was admitted in our er presenting with a week worsening vulvar pain. clinical exam showed vulvar and mons venus erythema, without lesions, bp was / mmhg and she had a fever of . °c. blood work showed leukocytosis ( . /ll), neutrophilia ( . /ll) and crp of mg/ l. past medical history of obesity, right thp and total thyroidectomy. vulvar cellulitis was the initial diagnosis and empirical atb was implemented. on d , due to an evolution into septic shock and spread of an emphysematous inflammatory process to the right thigh and buttock, the diagnosis of fg was made. during emergent surgery we observed extensive fascial and tissue necrosis from the asis and suprapubic region to the proximal third of the right thigh and perineum. extensive necrosectomy, drainage of purulent exudate and transversostomy were performed. empirical second-line broad-spectrum atb was started. she underwent new necrosectomies and surgical debridements on po days and and needed icu stay for days. daily dressing changes were performed with povidone iodine and later with octenidine. microbiology sample showed polymicrobial infection with gram positive and negative organisms as well as anaerobes, thus confirming the diagnosis of fg type i of vulvar origin. after surgical and hd stabilization, the patient underwent plastic reconstructive surgery, with local flaps and partial skin graft. the postoperative period was uneventful and the outcome was great. introduction: appendicitis is not uncommon in the elderly but may often be mis-diagnosed [ ] . the aim of this study was to explore the specific traits and treatments of this group in a swedish context to better understand where to optimize the management. material and methods: all acute appendectomies registered in the southern general hospital registry between january and june constituted the cohort (n = ). patients were stratified into two groups; c and \ years of age. significances were computed with pearsons chi and anova. results: the older group made up % of the study population (n = ). the elderly population was female to a larger extent (or . , p \ . ), triaged higher in the emergency department (p \ . ) and had higher asa classifications (p \ . ). the elderly were also perceived as sicker at the time of decision for surgery, expressed as having higher priorities for surgery (p \ . ). no significant difference between the groups in time from arrival to decision for surgery was found, nor for the time from arrival to surgery. there was a higher rate of perforations in the elderly group ( . % vs . %, p \ . ), twice the length of hospital stay (p \ . ) but no significant differences in complication rates ( . vs . %, p = . ). twenty-eight day mortality rate was % in the younger group and . % in the older group (p \ . ). conclusions: this study shows that an elderly group of appendicitis patients are more frail and more acutely sick when presenting to the hospital. in spite of higher priority for surgery, the elderly experience longer hospitalization and higher mortality rate, but not more complications. the findings are consistent with antecedent research. introduction: existing evidence points towards the notion that patients undergoing emergency surgery receive a poorer consenting quality when compared to their elective counterparts. with , cholecystectomies in england a year, cholecystectomy is one of the most frequently performed procedures both in the emergency and elective settings. however, to date, no studies have explored the relationship between consenting quality and the setting of cholecystectomy. we aimed to measure the quality of informed consent (ic) for patients who underwent emergency vs elective cholecystectomy. material and methods: the final review included the analysis of ic forms completed between - . percentage proportions were calculated to demonstrate the degree of completeness of consenting against a total of components of information. binary regression was utilised for subgroup analysis. results: patients undergoing emergency surgery were more likely than elective patients to be warned of severe perioperative complications such as cardiac disorders ( . % vs . %, p = . ), fluid collection ( . % vs . %, p = . ), and infected bile spillage ( . % vs . %, p = . ). elective patients were more likely to be counselled about the risk of less serious side effects of cholecystectomy such as diarrhoea ( . % vs . %, p = . ). patients in asa - group were more likely to be counselled about the occurrence of pulmonary embolism. interestingly, patients were more likely to receive a patient information leaflet if they were females and under . conclusions: the results of this study demonstrate multiple inconsistencies in the level of disclosed information to patients undergoing cholecystectomy. the results suggest that the consenting physicians make assumptions regarding the information that the patient would like to receive based on patient demographics and clinical factors, highlighting the need for more consistent consenting procedures. acute calculous cholecystitis and the timing of cholecystectomy: advocating early surgery i. moutsos , r. lunevicius liverpool university hospitals nhs foundation trust, general surgery, liverpool, united kingdom introduction: cholecystectomy cures acute calculous cholecystitis (acc) in nearly all patients and, according to nice, augis, tokyo and wses guidelines, should be conducted at the earliest opportunity, within days of the diagnosis. the present audit aimed to measure whether the care of patients with acc meets the standards of best practice and to assess whether early cholecystectomy was a more beneficial and safer intervention as compared to delayed cholecystectomy. material and methods: a ''snapshot'' sample of patients operated on between / and / with an index admission diagnosis of acc was reviewed. the selected patients were divided into three subgroups according to the timing of their surgery: - (early), - , and[ days. the other measures used in this audit were the rates of conversion to open surgery, subtotal cholecystectomy (stc), perioperative complication-specific morbidity, secondary interventions, and admission to intensive therapy unit (itu). results: nine patients ( %) underwent early cholecystectomy-laparoscopic (n = ) or primary open (n = ); of the other patients-delayed laparoscopic cholecystectomy. the rates of stc were similar in both subgroups- . % ( / ) vs . % ( / ). delayed cholecystectomy was related to five side effects: higher rates of postoperative collections (three patients, . %), external bile leak (one patient, . %), ercp ( . %), emergency re-operations (two patients, . %), and admission to itu ( . %). they all occurred in the delayed [ weeks surgery subgroup of patients. conclusions: although no significant associations were found when comparing early to delayed cholecystectomy, this analysis shows that postoperative morbidity, the rates of secondary interventions and admissions to itu were higher when surgery was delayed. this audit advocates that early cholecystectomy should become a standard of practice as per national and international guidelines. esophagopericardial fistula following primary repair for chronic esophageal ulceration presenting with pericardial tamponade: a case report and outline of management and treatment case history: a -year-old man with chronic esophageal ulcerations presented with substernal pain, fever, and shortness of breath. a radiograph revealed a right pleural effusion and pneumomediastinum consistent with an esophageal perforation (fig. ). he underwent a right thoracotomy, primary esophageal repair with intercostal muscle flap buttress, and gastrojejunostomy feeding access. a post-procedural gastrograffin study demonstrated an anastomotic leak (fig. ) . a right thoracostomy drain was placed for diversion. the patient was discharged home and returned days later. clinical findings: he presented with substernal pain, hypotension, and fatigue. thoracic computed-tomography (ct) revealed a pneumopericardium and an esophagopericardial fistula (epf) manifesting as pericardial tamponade (fig. ) . diagnosis: epf. therapy and progressions: the patient underwent a subxiphoid pericardial window and mediastinal drain placement for decompression. an esophagogastroduodenoscopy revealed an exposed right atrium, thus precluding esophageal stenting. sepsis and antibioticassociated clostridium difficile colitis complicated his post-operative course. once resolved, the patient underwent a partial esophageal resection, epf ligation, and esophagogastrostomy. the postoperative gastrograffin study did not demonstrate an anastomotic stricture or leak. the patient tolerated a regular diet and was discharged home. comments: esophagopericardial fistula is a rare clinical entity most often caused by benign disease. prompt diagnosis and treatmentpericardial decompression and fistula ligation-is critical. due to wide use of proton pump inhibitors and development of interventional radiology (ir), causative reasons are changing. introduction: secondary peritonitis yields high morbidity and mortality rates. besides rapid source control, adequate antimicrobial therapy is essential to improve outcomes. thus initial empiric therapy has to take suspected germ spectrum as well as possible resistance rates into account. microbial selection and resistances may pose problems during prolonged administration of antibiotics. however, a possible negative effect of multi-resistant germs on mortality has not yet been clarified. the choice of a suitable antibiotic and the relevance of its efficacy on isolated germs as well as the relationship between germ spectrum and clinical condition of the patients need to be clarified. material and methods: intraabdominal swabs from consecutive patients from to requiring intensive care due to secondary peritonitis were evaluated retrospectively. patient characteristics and outcomes, germ spectrum and resistance rates were collected. changes over the course of therapy and development of resistance as well as influences on the clinical course were analyzed. introduction: complicated intra-abdominal infections (c-iai) represent challenging diseases with high mortality rates. depending on different selection criteria and therapy strategies the reported mortality rates vary between . and %. usually a distinction between community (cap) and hospital acquired peritonitis (hap) is made. hap can further be classified as postoperative peritonitis (pop) or non-postoperative peritonitis (hap-non-pop). we conducted a retrospective analysis of patients with c-iai requiring intensive care therapy. material and methods: all patients with c-iai requiring surgery and intensive care treated at the danube hospital in vienna from to were retrospectively analyzed. a total of patients where included into the study and grouped as cap, hap-non-pop or pop. for each group comorbidity and patient characteristics, source and cause of infection, hospital and icu stay, apache ii, saps ii and sofa-scores, mortality and outcome were calculated and compared to each other, using fisher exact test or mann-whitney-u-test. results: a total of c-iai were treated, consisting of . % cap, . % hap-non-pop and % pop. concerning the patient characteristics and comorbidities no significant differences were seen between the groups, except for malignant diseases which were significantly higher in pop. the postoperative (source control) apache ii and saps ii values did not differ between cap and pop (apache ii mean: cap . , pop . ) whereas both were significantly higher in hap-non-pop (apache ii mean: . ). mortality rates were not significantly different in cap and pop ( . % vs. . %): however, hap-non-pop was complicated by a nearly doubled death rate ( . %). conclusions: although patients with pop are described to have a higher mortality in the literature, this could not be shown in our study. postoperative survival was comparable between cap and pop patients. hap-non-pop demonstrated a significantly higher mortality. acute appendicitis and acute diverticulitis presenting concurrently treated surgically and conservatively clinical findings: on examination the abdomen was soft but there was tenderness and guarding in the right iliac fossa and suprapubic region. her observations were stable on admission and she was afebrile. investigation/results: laboratory tests demonstrated a wcc . ( /l) and crp of . (mg/l). urinalysis was normal. a ct of the abdomen and pelvis with intravenous contrast demonstrated acute appendicitis with non-perforated sigmoid diverticulitis (fig. , fig. ). diagnosis: concurrent acute appendicitis and non-perforated sigmoid diverticulitis. therapy and progressions: the patient underwent a laparoscopic appendicectomy. intraoperative findings included a retrocaecal inflamed appendix and diverticulitis in the pelvis which was not disturbed. there was no pus in the pelvis. she recovered well postoperatively and was discharged home to complete one week of oral antibiotics the following day. the histology demonstrated acute appendicitis. comments: there are very few reports in the literature of concurrent appendicitis and sigmoid diverticulitis despite these two pathologies being amongst the most common presentations of abdominal pain. this case demonstrates the value of cross sectional imaging, ct imaging is a helpful diagnostic tool and is highly sensitive and specific for both diverticulitis and appendicitis.the challenge in this case is balancing the two differing managements of these two conditions. most cases of diverticulitis are managed conservatively with dietary modification and antibiotics. operative management is only usually considered if there are associated complications such as intraabdominal perforation. this is in contrast to appendicitis where the standard treatment is to undergo surgery. references millions of people die from major trauma annually. - % of these deaths are due to exsanguination, with nearly half dying prior to hospital arrival. when properly managed, these deaths are preventable. this paper summarizes data relating to the extent of hemorrhage as a cause of mortality in the traumatic arena. an overview of the pathophysiological steps occurring during massive bleeding and their clinical implication is presented. a variety of treatment options, both historical and current, is then discussed, including vascular occlusion methods and hemostatic dressings, along with their limitations and complications. finally, woundclot, a new hemostatic gauze, is introduced, which not only requires no compression when it is applied, but allows the first responder to rapidly and effectively treat more than one casualty within seconds. additionally, it is adaptable to a wide array of clinical applications, both traumatic and surgical, including situations where vascular occlusion methods are not practical or are contraindicated. i am the clinical research administrator for core scientific creations treating acute colonic diverticulitis with extraluminal pericolic air; a multi-centre retrospective cohort study background: since the emergence of acute care surgery as an entity encompassing trauma and emergency general surgery there have been several studies evaluating patient outcomes noting a higher unexpected survivorship and expedited operative times, shorter hospital stays, and fewer complications for patients undergoing procedures such as appendectomy; however, these superior outcomes have not been demonstrated across the array of emergency surgical cases. the aim of this investigation is to determine whether patients operated on by acute care surgeons in a trauma center benefit from the trauma model of in-house availability, earlier availability of surgical care, and care dictated by evidence-based protocol. we examined our health care system's data to determine if trauma centers were to able to provide more timely care with improved outcomes, by focusing on truly emergent general surgery cases. this was examined by identifying and quantitatively comparing time to operative intervention, need for re-operation, hospital length of stay, duration of stay spent in intensive care unit, and patient disposition at time of discharge. methods: this is a retrospective cohort study. patients presenting with emergency general surgery conditions (incarcerated hernia, perforated viscus, sbo, necrotizing soft tissue infection) who underwent surgery within h of presentation were selected. outcomes were compared between patients presenting to our two trauma centers versus our two non-trauma centers. n = results: at this time we are nearing the finalization of our data interpretation. we are examining mean time to operation, los, icu los, need for re-operation, and disposition at discharge. discussion: although our data analysis is not complete we feel that the results of our data will shed valuable and needed light onto the care delivered to emergency general surgery patients by surgeons in this increasingly complex population. anastomosis leakage after hartmann removal, with conservative treatment at the beginning but after, bad evolution, a surgery was performed with colostomy and vac system. patient. after h, he develop a compartmental syndrome and a vac system was applied. investigation/results: patient. after the first change the distance between the two layers was cm and botulinum toxin was applied. pat. the distance between the two layers of abdomen was cm and botulinum toxin was applied. patient. the distance between the two layers was cms and toxin was applied. unfortunately, he suffered from a hepatorenal syndrome and died. diagnosis: open abdomen with distance between the two layers: cm, and cm. therapy and progressions: we have added botulism toxin with doses of units in each side of abdominal wall. patient. three changes after, the abdomen wall was closed. months later, the abdominal wall is ok. patient. a reduction of % was got. comments: the use of open abdomen in patients suffer from septic shock or after an abdominal compartment syndrome often poses a challenge in the abdomen closure. we have developed a protocol, dividing our patients according to the distance between the two layers in two group: more than cm or cm or less. in the first group ([ ), we present our first cases in our protocol. conclusions: botulinum toxin can make easier abdomen closure when the distance between the two layers is more than cms incidentally discovered splenic peliosis in a patient with no comorbidity clinical findings: a -year-old man with no comorbidities visited our emergency medical center based on a complaint of chest pain. the chest and abdomen radiographs, electrocardiogram, and cardiac markers showed no abnormalities; therefore, he was discharged from the hospital. two months later, he returned to our hospital with abdominal pain and distension. he was hemodynamically stable, and there were little tenderness and rebound tenderness on his abdomen, although he complained a slight abdomen discomfort investigation/results: no abnormalities were found on the laboratory examinations, including complete blood cell count, cardiac markers, and coagulation profile. an abdomen computed tomography revealed multiple hemorrhagic cysts on spleen with moderate amount of hemoperitoneum. diagnosis: ruptured splenic peliosis with hemoperitoneum. therapy and progressions: laparoscopic splenectomy was done because recurrent rupture of hemorrhagic cysts was strongly anticipated. on histologic examination, the blood-filled cysts were welldemarcated, distributed in red pulp congestion. no vascular-endothelial cells were observed, and normal lining cells were disappeared in the wall. comments: a peliosis is a rare disorder characterized by widespread, blood-filled cystic cavities within the parenchymatous organs. the liver is the most commonly involved organ, and an isolated splenic peliosis is extremely uncommon. patients are often asymptomatic; therefore, early recognition and withdrawal of offending agents is crucial. in cases with the rupture of surface lesions, which can occur spontaneously or by the minor trauma, prompt surgical management is necessarily required. splenectomy offers the advantage of a definite histological diagnosis with the complete elimination of the risk of recurrent hemorrhage. introduction: despite an evident success and advantages of endoscopic surgery, the discussion on reasonability of endoscopic surgeries in children with acute appendicitis is still going on. purpose: to assess the effectiveness of laparoscopic techniques for treating appendicular peritonitis in children. material and methods: children with appendicular peritonitis were operated in our hospital ( ) ( ) ( ) . they aged - years ( ± . ); . % of boys, . % of girls. appendicular peritonitis was registered in . % cases of acute appendicitis. three ports were used for the approach: appendectomy was performed by the ligature technique with roder loop. results: laparoscopic surgery is indicated in all forms of appendicular peritonitis, except appendicular abscess stage , and total abscessing peritonitis. in appendicular abscess stage , we perform a puncture and drainage under ultrasound control. - months later appendectomy is made. total abscessing peritonitis is an indication for laparotomy. laparoscopic surgery in patients with peritonitis has the following stages: diagnostic laparoscopy; sanation of the abdominal cavity by the aspiration of purulent exudate; ligature appendectomy; in diffuse and combined peritonitis a pelvic aspiration drainage is made. in appendicular abscess stage , we additionally put the aspiration drainage in the cavity of destructed abscess. conclusions: laparoscopic technique applied for surgeries in children with acute appendicitis has considerably improved outcomes introduction: nighttime emergency surgery is associated with increased postoperative morbidity and mortality [ ] , and delayed appendectomy due to acute appendicitis is not linked to a higher rate of postoperative complications (pc) [ ] . the aim of this study was to determine whether appendectomy on-call (oc) was associated with higher risk of pc. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . two patients underwent major thigh amputation. negative pressure wound therapy and hyperbaric oxygen therapy were used in and patients, respectively. three patients died (mortality rate = %). conclusions: the mortality and major amputation rates ( % and %, respectively) were lower than those reported previously. in this study, even when patients had multiple organ failure or septic shock, major amputation was not always needed because of effective communication between the infection control team and intensive care specialists, resulting in radical debridement without amputation. material and methods: a systematic search in pubmed/medline, embase, cinahl and central was performed. the primary outcomes were mortality and amputation. these outcomes were related to the following time related variables ( ) time from onset symptoms to presentation; ( ) time from onset symptoms to surgery; ( ) time from presentation to surgery; ( ) duration of the initial surgical procedure. for the meta-analysis, effects were estimated using random-effects meta-analysis models. results: a total of studies ( patients) were included for qualitative analysis, of which patients died ( . %). a total of studies ( nsti patients) were included for the different quantitative analyses performed. mortality was significantly lower for patients with surgery within h after presentation compared to when treatment was delayed more than h (or . ; % ci . - . ). surgical treatment within h resulted in a % mortality rate compared to % when surgical treatment was delayed more than h. also, surgery within h reduced the mortality compared to surgery after h from presentation (or . ; % ci . - . ). patient delay (time from onset of symptoms to presentation or surgery) did not significantly affect the mortality in this study. none of the time related variables assessed reduced the amputation rate. conclusions: average mortality rates reported remained constant (around %) over the past years (fig. ) . surgical debridement as soon as possible lowers the mortality rate for nsti with almost %. thus, a sense of urgency is essential in the treatment of nsti. altemeiers procedure in an emergency setting case history: three patients with irreducible incarcerated rectal prolapsed were referred to our department for treatment. all patients were female and their age was , and years old. all patients suffered from severe co-morbidities. clinical findings: all patients presented with incarcerated rectal prolapse. in one patient there was macroscopic evidence of mucosal necrosis, whereas the other two patients had evidence of ischemia. the former patient was febrile whereas the latter did not exhibit signs or symptoms indicative of sepsis. investigation/results: blood panels demonstrated leukocytosis and elevated levels of c-reactive protein (crp) in all patients. apart from routine imaging upon admission (e.g. chest radiography), no other imaging modalities were performed. diagnosis: irreducible incarcerated rectal prolapse. therapy and progressions: initially manual reduction of the prolapsed was attempted without success. all patients were evaluated as high risk surgical candidates. altemeier's procedure was selected as a safer alternative to an abdominal approach. all patients were successfully discharged after resumption of bowel function. comments: incarcerated rectal prolapse is a rare clinical condition. initial management involves manual reduction of the prolapse. when this is not feasible, urgent surgical management is mandatory. in patients with severe co-morbidities, altemeir's procedure is a safe and effective treatment when performed by an experienced practitioner. introduction: treatment options for sigmoid volvulus are decided by its severity. uncomplicated cases are usually treated by endoscopic detorsion followed by elective surgery and complicated cases or cases can't be detorsioned are treated with emergency surgery. in this study we aim to review a single center experience in long term management of sigmoid volvulus cases. material and methods: data of the sigmoid volvulus cases between - were collected using hospital database. files of patients were reviewed for treatment modalities, demographic info and complications. patients were dropped from the study due to inadequate long term follow-up. results: were men and were women. mean age was , . endoscopic detorsion was attempted in cases. success rate was % (n = ). of these patients were followed up with elective surgery. patients with complicated cases and unsuccessful detorsion patients were managed by emergency surgery. hartman procedures, anterior resections, left hemicolectomies, subtotal colectomy and transverse loop colostomies were done. a stoma was created in cases. patients had their stoma created in the primary surgery and an additional of stomas were created due to anastomosis leakage. mortality rate in the first days was % (n = ) in patients with a stoma (n = ). asa and charlson co-morbidity scores were exceptionally high in the mortality group. in the remaining patient group, stoma closure rate was . %. conclusions: endoscopic detorsion is a powerful and highly successful management option in uncomplicated cases when done by an experienced staff. emergency surgery shouldn't be delayed in complicated cases or after unsuccessful detorsion attempts. introduction: esophageal perforation has high mortality rates when not treated aggressively. treatment options are conservative approach, endoscopic intervention and surgery. purpose of this study is to review cases of esophageal perforation in a single center and to evaluate types of diagnosis and treatment options. material and methods: using hospital database we collected data of patients diagnosed with esophageal perforation between - . we reviewed treatment modalities, demographic data and complications. patient was removed from the study due to insufficient long term data. results: were female and were male. average age was . . average time between the onset of symptoms and admission was . days. the most common etiology was iatrogenic (n = ) followed by consumption of corrosive substances in patients, spontaneous perforation in patients, esophageal tumour in patients and foreign body ingestion in patients. patients were treated surgically, patients were treated with endoscopic stenting and patient was treated with surgery following stenting. patients were managed conservatively with antibiotherapy. average time in intensive care was . days and average hospital stay was . days. mortality was seen in patients treated with surgery and patients treated with stents. conclusions: esophageal perforations are mainly iatrogenic but also can be caused by multiple reasons. especially in cases developed after endoscopy, rapid intervention can be a significant factor that can decrease both mortality and morbidity rates. introduction: spontaneous rupture of liver tumors (rlt) is a rare but potentially life-threatening condition. damage control techniques, namely perihepatic packing (php), is a resource for the most physiologically compromised patients, with more stable patients undergoing transarterial embolization (tae) or immediate resection. decision algorithm depends on patient status, available resources and liver function. the authors present their center experience in managing rlt and propose a management algorithm. material and methods: eighteen consecutive patients who underwent surgery for rlt in our department (january -october ). inclusion criteria: spontaneous rupture and evidence of intraperitoneal bleeding. fourteen patients were male. mean age of . years ( - ). thirteen patients ( %) presented in hemorrhagic shock. mean tumor size was . cm ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . most frequent pathological diagnosis were: hepatocellular carcinoma in cases ( %); adenoma in three cases ( %); metastases in two cases ( %); liver sarcoma in one case ( . %). median of seven units transfused by patient ( - ). statistical analyses with spss tm version . results: six patients ( %) needed immediate surgery (php in three and resection in three). five ( %) underwent urgent ([ h and \ h) and seven ( %) delayed ([ h) resection. hepatectomy was performed on all (fifteen minor and two major) but one patient php only. eight patients ( %) underwent tae prior to resection, two of them ( %) between php and hepatectomy. median length of stay days . major morbidity in three patients ( %); mortality in three patients ( %). number of transfused units associated with increased risk of complications (p = . ). conclusions: rupture of liver tumors is a severe complication. although hepatic resection, with or without preoperative tae, should be considered gold standard, damage control techniques such as php are the only option for physiologically compromised patients (fig. ) . seasonal variability of cellulitis: a five year retrospective cohort study introduction: it is commonly purported that the incidence of cellulitis is highly seasonal but there is little empirical evidence supporting this assertion. this year retrospective cohort study set out to identify whether there is a statistically significant relationship between an increase in temperature and incidences of cellulitis. as a corollary to this proposition, length of hospital stay for cellulitis was examined in relation to the level of inflammatory markers upon admission and micro-organism identified on culture. material and methods: this is a year retrospective single centre cohort study of all patients admitted with cellulitis to tallaght university hospital from to inclusive. the patient cohort was identified via the use of a prospectively managed database of all surgical admissions and corroborated via examination of clinical chart records. dates of admission were correlated with the average temperature of dublin as provided by the meteorological office of ireland. site of infection, inflammatory markers and the prevalent micro-organism were also identified whilst the length of admission was extrapolated from hipe (hospital inpatient enquiry) records. results: there were admissions for cellulitis with cases of necrotising fasciitis. there was a statistically significant (p \ . ) relationship between temperature and cellulitis with admission peaking in late summer/autumn. age correlated significantly with readmission. furthermore, the level of crp had a statistically significant prognostic value as an independent predictor for the length of hospital stay with a high level resulting in a prolonged admission. conclusions: there is a statistically significant relationship between a rise in temperature and the incidence of cellulitis. furthermore age is an independent risk factor for re-admission with same whilst inflammatory markers at time of admission can be used as a prognostic marker for length of stay. case history | clinical findings: a -year-old female patient, with history of type ii diabetes, high blood pressure and major depressive syndrome, was admitted in the emergency room department complaining of abdominal pain. based on the patient's history and physical examination, a presumptive diagnosis of renal colic was initially made. however, after days, the patient showed signs of fever, aggravated abdominal pain and vomiting. investigation/results | diagnosis | therapy and progressions: a ct scan showed the presence of a radiopaque foreign body near the duodenum, the presence of air bubbles outside the intestinal lumen and an hepatic abscess. we agreed to perform a laparoscopy, drainage of hepatic abscess and fish bone removal after successfully identification. after days, the laboratory findings showed persistent leukocytosis and raised cpr, which led to a second ct scan with maintenance of the hepatic abscess. the decision was to perform a percutaneous drainage. after the second drainage, the patient had an uneventful recovery. comments: foreign body ingestion into the gastrointestinal (gi) tract is rare and typically accidental in adults. most ingested foreign bodies pass through the gastrointestinal tract without the need for any intervention. gi perforation is rare and can occur at any site. surgical intervention is required in less than % of the cases. fish bones are the most commonly ingested objects. preoperative diagnosis, when possible, is made with ct scan, identifying a linear high-density structure. high level of suspicion is of paramount importance. in cases of delayed diagnosis, perforation may lead to intraperitoneal abscess formation. reports of hepatic abscess secondary to fish bone perforation has been limited to isolated case reports in the literature. case history: description of two cases of appendicular goblet cell carcinoid tumors, which debuted as acute appendicitis. patient a was a -year-old woman with a -h evolution of classic symptoms of acute appendicitis. patient b was a -year-old female that consulted for chronic abdominal pain in rlq that recently increased pain intensity and fever. clinical findings: patient a had pain and defense in rlq without a fever. patient b had a chronic painful fluctuating mass in rlq, with fever over °c. investigation/results: patient's a lab test showed leukocytosis and us findings of acute appendicitis. the patient's b ctscan showed an intra-abdominal abscess fistulized to the abdominal wall, along with formation of a phlegmonous mass related to appendicular plastron. diagnosis: the anatomopathological reports for both patients were informed as appendicular goblet cell carcinoid tumor. therapy and progressions: both underwent laparoscopic exploration. after appendicectomy in patient a, when the diagnosis of gcct was made, the case was discussed at our mdt meeting and a right hemicolectomy was indicated and performed shortly after. in the patient b a right hemicolectomy was performed in the initial surgery due to the magnitude of tissue involvement. currently, both are receiving chemotherapy with xelox without signs of recurrence or tumor spread on follow up. comments: the gcc is a rare entity of appendicular tumors with a less favorable prognosis than the appendicular pure neuroendocrine tumors. it behaves like a low-grade adenocarcinoma and often presents as disseminated disease. therefore, sometimes surgical treatment with appendicectomy is not enough, needing the right hemicolectomy to avoid recurrence. this is recommended for tumors [ cm, pt or t and higher grade histology. introduction: among the post-pancreatoduodenectomy complications post pancreatoduodenectomy hemorrhage (pph) is the least common complication, but severe form may be life-threatening without an urgent treatment. late pph are more likely due to a complex physio-pathological pathway secondary to different etiologies. the understanding of the etiology and such a pathway could therefore be of great interest to guide the treatment of potential lifethreatening late severe pph. results: during the aforementioned period patients underwent pd, of whom ( . %) developed pph. early pph was reported in one patient ( . %) with severe bleeding from the gastric stapler line. late pph were reported in of these patients ( . %). the most common causes were bleeding from a vascular pseudoaneurysm reported in patients of which, one had mild and had severe hemorrhage and bleeding from gastro-enteric anastomosis marginal ulcer in patients, all with mild hemorrhage. no etiology was fond in patients with mild hemorrhage. a significant association was found between the severity of late hemorrhage and the vascular pseudoaneurysm as a cause of bleeding (p \ . ). all pseudoaneurysm bleeding occurred in cases complicated by a postoperative pancreatic fistula (popf) with a significant statistical association (p \ . ). conclusions: the most common cause of pph was bleeding from a vascular pseudoaneurysm, most of them were severe bleeding with late presentation and all were associated with a popf. in these cases, early detection by cta is mandatory, allowing an urgent treatment by angiography of such a bleeding vascular complication following pd. ventral hernia in hostile situation introduction: there is no consensus about the benefit or harm derived from adding a mesh hernioplasty at the same time as an urgent intraperitoneal surgery for another cause. the use of a prosthesis in contaminated fields is controversial, but suture repair has a high risk of recurrence. the main objective has been to analyze the impact of the simultaneous repair of uncomplicated midline hernias at the same time as emergency surgery for another cause, in relation to the presentation of complications, the surgical site infection rate (isq) and recurrences. material and methods: retrospective, observational study of all urgently operated patients (surgery open and laparoscopic) in the period between - who underwent a simultaneous midline primary ventral hernioplasty. the background, circumstances of the surgery and postoperative complications during the first month and long term through the basis of prospective data of emergency surgery and complications of our surgery department. results: a total of patients ( female) met the inclusion criteria with a mean age of . years (sd = . ), average bmi of . kg/ m (sd = . ). the most frequently performed interventions were: appendectomy ( . %); cholecystectomy ( . %); and lysis of adhesions ( . %). the . % of all interventions were performed by laparoscopic approach. they presented associated peritonitis in . % of the cases. the . % of patients presented some complication, in . % surgical site infection ( . % organ space). during the followup three recurrences were detected ( . %), no patient has presented chronic infection related to the use of prostheses. conclusions: in our series the simultaneous performance of hernia repair of the midline in the context of emergency surgery for another cause has been safe and not associated with long-term complications and low recurrence rate. the open abdomen: our experience introduction: ''open abdomen'' refers to a solution in which the abdominal content is left deliberately exposed under a temporary cover for a variable amount of time. since this method has been used more and more for the treatment of severe intra-abdominal infections. starting from the s the concept has been also applied in trauma surgery. material and methods: between / we have treated patients with this technique. in cases the etiology was traumatic, in the remaining cases the abdominal pathology was inflammatory. in the last years we also started to use it in some cases of treatment of surgical complications. the techniques we used were different and changed during the time. at the beginning of the experience we've completed drainages of the abdominal cavity according to mickulizt, laparostomies with mesh, bogota bags. these techniques have been abandoned since the negative pressure therapy came out. we started with the barker vacuum pack ( cases), followed by the vac (vacuum assisted closure) and ab thera kci Ò ( patients) systems and in the last three years we used the cnp suprasorb Ò of lohmann and raucher ( patients case history: year old lady presented at the a&e with few days history of constipation, faeculent vomiting, abdominal distension and pain in the lower abdomen. she had hysterectomy many years ago through a lower midline incision. her urgent ct scan of the abdomen and pelvis confirmed an incarcerated right obturator hernia containing a small bowel loop causing bowel obstruction. clinical findings: elderly, frail patient with mild tachycardia, distended abdomen and lower abdominal tenderness with guarding in the left iliac fossa. per rectal examination was unremarkable. investigation/results: inflammatory markers were raised, lactate, liver and kidney function was in normal limits with only mild hypokalaemia and hyponatraemia. ct abdomen and pelvis confirmed small bowel obstruction at the mid ileal level due to right obturator hernia. diagnosis: incarcerated right obturator hernia causing small bowel obstruction. therapy and progressions: patient was taken to the operating theatre for urgent laparotomy. dilated small bowel loops and incarcerated right obturator hernia was found with proximal ileal loop in it. after blunt stretching and dilatation of the obturator foramen, the involved ileal loop was reduced. it was deemed viable, therefore no bowel resection was required. the defect at the right obturator foramen was closed with suture. post-operatively the patient was transferred to the intensive care unit for further management. comments: obturator hernias are a rare type of pelvic hernias. their real incidence is unknown but it is thought to be less than % of all hernias worldwide and due to its non-specific symptoms and late diagnosis, they require bowel resectional surgery in nearly % of the cases. howship-romberg sign is helpful in diagnosing such a hernia, but the ultimate diagnostic choice is ct scanning which is the only way to find this condition early and avoid bowel ischaemia. case history: a -year-old woman without previous medical history presented to the emergency department with abdominal pain and dysphagia associated with nausea, vomiting and absolute constipation. during previous months, she reported having ingested hair. clinical findings: abdominal examination revealed a distended abdomen with rebound tenderness and tinkly bowel sounds. investigation/results: ct-scan showed a distended stomach with a mussel-shaped, heterogeneous and non-enhancing mass. an esophagogastroduodenoscopy revealed hair inside the lower esophagus and the stomach. diagnosis: high intestinal obstruction due to a gastric trichobezoar. therapy and progressions: the patient underwent laparotomy, gastrotomy and trichobezoar removal (fig. ) . the postoperative period was uneventful and she was discharged home on the th pod with a psychiatric evaluation scheduled. comments: bezoars are rare conditions consisting of compacted material that is unable to pass through the gastrointestinal tract. this condition usually involves the stomach; rarely, it can extend into the small bowel and even the colon, giving the so-called rapunzel syndrome. bezoars could be composed by vegetable material (phytobezoars), hair (trichobezoars), drugs (pharmacobezoars), or other materials. , a trichobezoar is the result of trichotillomania, trichophagia or other psychiatric disorders. always consider bezoars in differential diagnosis. introduction: the effectiveness of different step-up approaches is increasingly evaluated but results are controversial. we assessed the results of a standardized step-up approach protocol in the treatment of acute severe necrotizing pancreatitis, with a special focus on patient stratification to obtain an early identification of those deserving a more aggressive strategy. matherials and methods: this is a retrospective analysis of patients with acute severe pancreatitis over a period of years. the variables taken into account were: etiology and severity of the disease, sepsis, organ failure, hemodynamic stability, treatment, los, morbidity, mortality. since , patients with infected necrosis underwent a standardized step-up approach: percutaneous drainage only; percutaneous and endoscopic procedure; surgery. the results were compared with the standard care delivered from to . results: among patients, ( . %) were identified as affected by severe necrotizing disease. overall mortality was . %. the initial management was non operativein all patients. mortality in the step-up group was % ( / ) vs % ( / ) in the standard care group. conclusion: a standardized step-up approach protocol offers better results than standard care in the management of acute severe necrotizing pancreatitis. however, a better stratification of patients. introduction:the appendix stump closure in complicated appendicitis has been widely practiced in different ways such as metal clip, hem-o-lok clip, endoloop and endostapler. the treatment of complicated appendicitis with necrosis and perforation of the appendix base is controversial. we aimed evaluate the efficacy of laparoscopic partial caecum resection with endostapler in complicated appendicitis with necrosis and base perforation. material and methods:from january to october , we evaluated consecutive patients who underwent a laparoscopic partial caecum resection in complicated appendicitis with necrosis and perforation of the appendix base. partial caecum resection was performed with the endostapler to close the appendix base at ileocaecal junction. results:the laparoscopic partial caecum resection with endostapler was used in % . of the cases. the mean operative time was . ± . min. there were necrosis of appendix base in , perforation of appendix base and diffuse peritonitis in , perforation of the appendix base and localized peritonitis in of the patients. the wound and intra-abdominal infection rates were . % and . %, respectively. there were no operative complications and the conversion rate was . %. the average length of hospital stay was . ± . days. there was no leakage on the stapler line. conclusions:the laparoscopic partial caecum resection with endostapler in complicated appendicitis with necrosis and perforation of the appendix base, is a safe and effective technique. introduction: the term ''''volvulus'''' comes from the latin ''''volvere''''meaning twist. if left unattended, sigmoid volvulus can compromise the blood supply of the involved segment,leading to ischemia,gangrene,perforation and death. the mainstay of sigmoid volvulus management has been through proctoscopic or colonoscopic decompression when feasible, followed by surgery either during the same admission or electively. the aim of our study is to identify patients which can benefit of immediate surgical approach and prognostic factors associated with failure of conservative/endoscopic treatment. materials and methods: charts of patients admitted for sigmoid volvulus to our institute were retrospectively analysed. we revised ct scan images and laboratory tests of all the patients to identify risk factors for conservative treatment failure. results: patients underwent surgical procedures; in cases after a failure of an initial conservative approach; patients were managed with endoscopic approach only. elective surgery was performed in patients. case history: we report a -year-old male case presenting left hand middle finger pain after pressured paint gun shot in volar proximal phalanx clinical findings: on physical examination swelling and tenderness on the volar side of the hand was observed (fig. ) investigation/results: pain was remarkably more intense with passive finger extension. distal nerurovascular status was unscathed. there was no fracture reported on radiography. leukocytosis and acute phase reactants rise was observed on laboratory examination diagnosis: after physical, radiological and laboratory examination the diagnosis of acute flexor tenosynovitis was made. therapy and progressions: open debridement and irrigation following bruner incisions on middle finger was undertaken within h of injury. paint impregned in tissues could be observed in subcutaneous tissue, palmar fascia and flexor tendon sheath. paint affected tissues samples were analysed in microbiology laboratory (fig , ) after checking nerurovascular indemnity, g drainage was left in deep tissues and skin suture was performed with - monofilament non absorbable suture. the patient followed days intravenous antibiotical therapy followed by weeks oral treatment. he attended physiotherapy program postoperatively, reporting no functional disability or wound complications after weeks. comments: chemical flexor tenosynovitis is an important emergency which must be correctly diagnosed and treated due to quick progression and potential morbidity if not treated effectively ( ) in our experience, case was managed by open debridement and irrigation but different treatments can be followed depending of patientsclinical situation, such as iv antibiotics with serial examinations or percutaneous drainage. it should also be noted that australia does not have a specific subspecialty in emergency surgery. the acute surgical unit at the tch was set up in in order to provide a dedicated acute unit to service the ever increasing demand acute surgery. previous model was that the acute surgical service was integrated into the elective work. additional beds were provided to the unit including the positions of a dedicated director and chief nurse. the achievement of the unit has been the decreased time to theatre, less after-hours operating, standardised treatment approaches, and dedicated emergency surgery medical staff. the difficulties have included clinician engagement, competing resources with elective surgery, emergency surgical presentations increasing by - % each year, and the unit''s beds being used for non-acute patients as the hospital approaches regular %. the acute surgical unit has evolved into a specialised acute care that enables rapid assessment and treatment of patients with staff dedicated with skills in this area. treating pyogenic liver abscesses secondary to diverticulitis in a patient using immunosuppressants for crohns disease by performing a sigmoid colectomy introduction: pyogenic liver abscess (pla) formation due to microbial contamination of the liver parenchyma is often seen secondary to intra-abdominal infections. pla formation due to crohn''s disease (cd) is a rare complication and not well-documented in current literature. as symptoms often mimic a cd exacerbation, diagnosis is often delayed and severe disease may develop. optimal treatment for this group of patients remains debatable. case presentation: a -year-old man was admitted to the hospital with a -week history of overall malaise, fever and night sweats. patient''s history solely stated a -year treatment of cd that was stable over the past period with infliximab and azathioprine. investigations and treatment: biochemical analysis revealed a c-reactive protein of mg/l and a white blood cell count of . /l. an abdominal ct scan showed multiple abscesses in the right lobe of the liver and a thickening of the wall in the transition of the descendent colon to sigmoid. the patient''s immunosuppressants were paused, intravenous antibiotics were administered and a percutaneous drainage of the biggest pla was performed. however, the clinical condition of the patient did not improve. colonoscopy and pet-ct scan did not reveal any other sites of infections. as patient remained septic and previous imaging revealed mild diverticulitis rather than active cd, an emergency hartmann''s procedure was performed. hereafter, the patient recovered rapidly and the plas resolved completely. conclusion: diverticulitis of the sigmoid colon should be considered as causative pathology in patients presenting with multiple pyogenic liver abscesses and a history of crohn''s disease that is in full remission with immunosuppression. when the abscesses exceed cm in size and are multilocular, resection of the inflamed colon can be a treatment option of value. clinical findings: epigastric pain and recent episode of hematemesis. pain at deep palpation of the epigastrium, no signs of peritoneal irritation investigation/results: abdominal x-ray and ct showing a large right sided strangulated paraesophageal peh, with pneumatosis of the gastric wall diagnosis: right sided strangulated peh therapy and progressions: emergent laparotomy. peh reduced, ischemic portion of the stomach recovering viability. closure of diaphragmatic defect with non-absorbable suture, reinforcement of lower esophageal sphincter with round ligament (ligamentum teres hepatis) and anterior partial fundoplication (dor). postoperative course uneventful, patient discharged on th pod. comments: peh are mediastinal displacements of abdominal organs, most often the stomach, associated with laxity or a hole in the phrenoesophageal membrane, large enough to allow the gastric fundus to herniate. because the stomach is attached to the gastroesophageal junction, it tends to rotate around its axis leading to organoaxial volvulus. occurrence and size increases with age. peh account for - % of all diaphragmatic hernias. in patients without prohibitive operative risk, they should be surgically corrected, avoiding the risk of acute and potentially life-threatening complications when emergent surgical repair is required. the risk of developing these complications is less than %/yr and associated mortality rate is approximately %. case history: patient was a previously healthy -year-old female with an unremarkable past medical history, non-smoker with a high body mass index (bmi [ ). she first presented to a level medical facility with acute left upper leg pain and swelling. one week prior to this she had a progressive cough, swinging fever, and malaise. clinical findings: patient was transferred to our hospital haemodynamically unstable, acidotic, hypoxemic and delusional. tachypnea and oliguria were present. she continued to deteriorate clinically with pyrexia (t , oc), resistant shock, and toxaemia. on examination her left leg was found to be paresthetic below the femoral-inguinal fold. investigation/results: abg samples showed lactic acidosis with a ph of . and lactate of . mmol/l. hypoxia and hypocapnea were present.her biochemical profile showed acute kidney injury (aki) with raised creatinine kinase (cpk) and serum creatinine (cr) . . chest x-ray illustrated bilateral lung infiltrations (ards image). diagnosis: patient was urgently referred to a ct scan of the left femur with i.v. contrast for suspected necrotising fasciitis. ct findings highlighted a deep muscular femoral abscess with multiple regional fluid collections and necrotizing inflammation from the femur diaphysis to the patella. therapy and progressions: the patient was immediately transferred to or for emergency surgical exploration and debridement. almost the entire anterior compartment of the femur was necrotic and hence an extensive excision of the dead tissues and packing with npwt was performed. comments: severe snm can cause marked systemic toxic effects, namely, the streptococcal toxic shock syndrome (stss). stss secondary to snm is a life-threatening host response to gas superantigens with a mortality rate as high as %. clinical findings: patient had a diffusedlty tender abdomen and had not passed flatus proceeding his admission to the a ? e department and was vomiting. investigation/results: ct abdomen showed small bowel dilatation with abrupt cut-off point proximal to the icv diagnosis: a diagnosis of small bowel obstruction was made based on the clinical and ct findings. therapy and progressions: patient was taken to theatre for laparoscopy ? -proceed and a 'slipped' bowel lopp was noted within the peritoneal flap that had been created a week prior during the original hernia repair. the 'v lock'' suture line was found to be loose which is thought to have led to this complication. the bowel loop was reduced, deemed viable and an internal hernia repair was performed. post-operative period was unremarkable and the patient was discharged day posy-operatively. comments: during lap tapp hernia repair, there are currently at least options avaiable for peritoneal flap closure; (sutures, tackers and glue.) suregons prefernce prevails over the chosen approach. when sutures are chosen, most surgeons prefer the self-locking v-lock stitch. by adopting this technique, meticulous periotneal closure is impoartan, as loose suturing of the peritoneum can lead to post operative complications of internal herniation and small bowel obstruction, as described in this case. a multi-centre prospective study would be welcomed, to compare efficacy and safety of all types of peritoneal closure devices. introduction: peer review assessment of medical treatment has been shown to be a robust way of improving quality of care in trauma in our institution and globally. in we introduced regular morbidity and mortality meetings at the department of gastrointestinal surgery. severe complications (revised accordion classification [ ) after surgery were identified on a weekly basis, evaluated and data included in a local quality registry with the aim of revealing suboptimal surgical quality and continuously improving our results. material and methods: retrospective analysis of collected data from the described quality registry. all adult patients who had undergone gastrointestinal surgery in were assessed. results: of surgical procedures performed, % were emergency procedures. a total of % ( / ) experienced a severe complication after surgery and % ( / ) required reoperation. in the group of upper gastrointestinal surgery [n = ( %)] % were emergency procedures. anastomotic leak (al) was identified in % ( / ) undergoing thoraco-laparoscopic esophagectomy and in % ( / patients) after gastrectomy. of laparoscopic cholecystectomies, % were emergency procedures with % ( / ) reoperation. of hernia repairs, % required reoperation. in the group of lower gastrointestinal surgery [n = ( %)] % were emergency procedures. al was diagnosed in % of colonic resections and % of patients after rectal resection. in emergency colorectal resections(n = ) there were no al. of appendectomies, patients ( %) required reoperation. the most frequent cause of reoperation was revision of stoma ( ), followed by reoperation for al ( ), abscess ( ), and wound dehiscence ( ). patients died after surgery of which were emergency surgical patients. conclusions: systematic assessment of all severe complications helps reveal surgical procedures which can be improved but also to identify surgical procedures with low complications rates. plans are being developed to improve the quality of the identified procedures. all surgical departments should have regular and thorough assessment of their activity. acute surgical patients operated by emergency surgeons has less risk of post-operative complications and mortality d. gumaa east kent hospitals university nhs foundation trust, general surgery, ashford, united kingdom introduction: in england and wales, we perform over , emergency laparotomy every year. days mortality rate is around - %. in our study we are trying to demonstrate if have dedicated emergency surgery service will make a difference in the outcome of emergency laparotomy. material and methods: retrospective study on prospectively collected data from nela database done in a large district general hospital. all patients over years old who underwent emergency laparotomy for acute surgical condition between november and january were included in the study. mortality and post-operative complications were the primary outcomes. results: total of patients were included in the study, operations were performed by emergency surgeons (es). days mortality rate was %, while it was . % for the none emergency surgeons group (nes) post-operative complications were . % compared to % for patients operated by nes. there was shorter itu stay with average of . days, while the itu stay for the other group was . days, but the es group had higher chance of unplanned return to theatre. . % of the patients went back to theatre compared to % of the other group. reasons of unplanned return to theatre was mainly post-operative collection or wound dehiscence. conclusions: emergency surgeons has better outcomes when they perform emergency laparotomy, may be because they perform higher number of laparotomy compared to their peers. emergency surgery has been a growing subspeciality recently, and with no doubts having surgical emergency units has improved the patient's care around uk. the advantage of g over g of prophylactic cefazolin in surgical site infections in trauma surgery below the knee introduction: the rate of surgical site infections(ssi) after foot/ankle surgery remains high, despite the implementation of antibiotic prophylaxis ( ) . recently guidelines suggest a single dose of g instead of g of cefazolin for implant surgery, this decision is largely based on pharmacokinetic studies ( ) . however, the clinical effect of this higher dose has never been investigated in this region. this retrospective cohort study therefore investigated the effect of g compared to g of prophylactic cefazolin on the incidence of ssis in foot/ankle surgery. material and methods: all patients undergoing trauma-related surgery of the foot, ankle or lower leg between september and march were included. primary outcome was the incidence of a ssi. ssis were compared between patients receiving g and g of cefazolin as surgical prophylaxis. results: a total of patients received g and patients received g of cefazolin. the groups did not differ in gender, age, weight, co-morbidities or intoxications. the overall number of ssis was ( . %) in the g group and ( . %) in the g group. corrected for the confounders ''age'', ''smoking'' and ''blood loss'' this was not statistically significant (p = . ). conclusions: even though the decrease in ssi rate from . to . % was found not to be statistically significant, it might be clinically relevant considering the reduction in morbidity, mortality and healthcare costs. research linking pharmacokinetic and clinical results of prophylactic cefazolin is needed to establish whether or not the current recommendations and guidelines are sufficient for preventing ssis in foot/ankle surgery. introduction:right-sided colonic diverticulitis (rd) is much rarer than left-sided (ld) and subsequently, controversies concerning the most appropriate treatment remain unsolved. our experience let us believe that mild rd can benefit from an outpatient management. material and methods: we performed a single center retrospective comparative study in which we included all our diverticulitis patients that were treated as inpatient in our unit. we divided in two groups:rd and ld group. the ld group was created by randomization from a prospective ld patients database. results: we included rd and ld patients treated in our unit from july to july . median age was . in rd and . in ld, with a . % of females in rd vs . % in ld. asa classification was significantly lower in rd (asai: . % vs %, asaii: . % vs , , asaiii: vs . %, asaiv: vs . % p = . ). the presence of neumoperitoneum in ct scan was significantly higher in ld . % vs . % p = . ) surgery was performed in . % of the left-sided diverticulitis compared to of the rd group (p = . ). antibiotics of third line (imipenem and meropenem) were only required for ld ( vs . % p = . ). length of hospital stay was significantly shorter (p = . ) in rd ( . ± . ) than in ld group ( . ± , ) conclusions: in our series, patients with right diverticulitis had fewer perforations in the ct scan, they required lower spectrum antibiotics and did not required any surgical treatment with a shorter length of hospital stay. we consider that mild right diverticulitis could benefit from an outpatient treatment with oral antibiotic following similar recommendations to those followed for mild ld patients. when surgery should not be immediate, a night of hospitalization in a specialized environment is performed and surgery deferred overnight. in some selected patients, a return home is possible with a scheduled emergency surgery the next day. the pa.r.c.o.ur protocol is set up in the surgical emergencies of the university hospital of lille after a suitable medical treatment and enlightened information. this retrospective study assesses whether this deferred surgical management allows a return home on the day of the operation. methods: between / / and / / , records of patients operated for an abscess, appendicitis, cholecystitis or symptomatic inguinal hernia were reviewed. patients who did not have criteria for immediate surgical management (peritonitis, occlusion, sepsis, cellulitis, intravenous treatment need) agreed to return to their home for an os the next day. results: / % interventions were performed in os and allowed a return home at day , within a median time of h [iqr - ]. conclusions: the pa.r.c.o.ur protocol makes it possible to reserve the availability of the entire technical platform (operating rooms and beds) to the most serious pathologies with a failure rate of %. the medico-economic benefits, the efficiency in the management of the beds and the satisfaction of the patient and medical staff of this protocol must be evaluated prospectively. a years old woman was admitted in our er presenting with a h sharp epigastric and ruq pain, fever, nausea and vomiting, hd stable. the patient had a past medical history of tachyarrhythmia, open-angle glaucoma and lower limb venous insufficiency. her past surgical history included an hysterectomy and bilateral salpingooophorectomy, appendectomy and left inguinal hernioplasty. during clinical examination, signs of peritoneal irritation were present. ct scan revealed a small pneumoperitoneum in the luq and multiple small and large bowel diverticula, without free peritoneal fluid. blood work showed mild leukocytosis and neutrophilia. we performed an urgent exploratory laparoscopy in which dozens of small intestine diverticula were found, increasing proximally in number. one of them, cm distally from the treitzs angle, showed signs of perforation, with a small abscess and surrounding fibrin. the affected bowel was externalized through a cm laparotomy for segmental resection and a manual double-layer terminoterminal jejunojejunostomy was performed. in the perforated jejunal diverticulum, a mm cod fishbone was identified as the cause of the perforation. the histopathological examination of the extracted cm tissue sample, found several diverticular structures of the muscular wall, one of which with a mm perforation and a granulocytic infiltrate with serosa involvement. complicated cases of small bowel diverticulosis are best managed by segmental resection surgery. despite being quite rare, every surgeon should be aware of such acute abdomen presentation. asymptomatic cases benefit from a watch-and-wait approach. case history: a -year-old female consulted to the emergency department for a h epigastric pain. it was accompanied by nausea without vomiting. clinical findings: the patient was hemodynamically normal and the abdomen was soft with minimal distention. investigation/results: x-rays showed large gastric dilation. the abdominal ct scan showed mesenteric axial gastric volvulus with minimal free fluid. suddenly, the patient presented diffuse abdominal pain with diaphoresis, mucocutaneous pallor, hypotension and tachycardia. diagnosis: a gastric volvulus with gastric ischemia was suspected. broad-spectrum antibiotic therapy and resuscitation measures were started. emergency surgery was indicated. therapy and progressions: a decompressive gastrostomy, gastric reduction and devolvulation, transverse colon resection due to ischemia and splenectomy were performed. after h, she required total gastrectomy and right hemicolectomy due to ischemia secondary to severe septic shock associated with disseminated intravascular coagulation. comments: the gastric volvulus is an uncommon entity, being the mesenteric-axial type so rare. there are very few cases described whose manifestation is accompanied by hypovolemic shock secondary to splenic laceration, which occurred due to the great gastric distention. early diagnosis is the key to start treatment as quickly as possible, due to high mortality the main mechanism of death is usually vascular involvement, perforation and multiorgan failure. results: we analyzed , pediatric ogis, and . % of pediatric cases occurred in the - age group, . % in - , . % in - , and . % in - . the average age of the cohort was . years and . % of cases occurred in boys. racial distribution revealed . % of cases in caucasians, . % in african americans, and . % in hispanics. most ( . %) cases were documented in the southern united states. of our , cases, . % underwent vitrectomy, . % underwent enucleation, and . % developed endophthalmitis. the rate of endophthalmitis development after ogi was highest ( . %) in the asian/pacific islander group. the average length of stay for the entire cohort was . days, and the average cost per day was $ , . . table contains a breakdown of our statistics. conclusions: as documented in the nis, ogi occurs more commonly in boys than in girls at a ratio of approximately : . the rates of vitrectomy and enucleation are higher in boys. we noted a higher of rate of enucleation in asian/pacific islanders and african americans. the plurality of ogis occur in the - age group; this age group also has the highest relative rate of enucleation. with respect to location, ogis occurring in the western united states had the highest average cost per day of inpatient stay. autologous tissue from intramedullary channel parietes for femur nonunions management introduction: a reamer-irrigator-aspirator (ria) method is deeply reliable for getting high volumes of bone graft/mscs. high rates of successful outcomes have been reported after the use of ria bone fragments to cure non-unions. material and methods: being supported by histomorphological examination of the material acquired while drilling intramedullary channels of patients with femur nonunions ( -hypertrophic, oligotrophic), we have discovered that nevertheless, expressions of the dystrophy and necrosis in bone tissue and marrow in pseudoarthrosis areas depend on time since fracture occurrence, the microscopic study of the material cm above and below a fracture line has demonstrated ordinary structures of bone tissue and marrow in all cases. introduction: this study aimed to evaluate the outcomes of ankle fractures with posterior malleolus fragments (pmfs) involving \ % of the articular surface treated with or without screw fixation. material and methods: among patients with ankle fractures and pmfs who underwent surgery between march and february , with type pmfs involving \ % of the articular surface were included. of these patients, underwent screw fixation for pmfs and lateral and/or medial malleolar fracture fixation (group a) and underwent internal fixation for malleolar fractures without screw fixation for pmfs (group b). ankle joint alignment and fracture healing were measured using plain radiography and computed tomography (ct). clinical outcomes were determined using the american academy of orthopaedic surgeons foot and ankle questionnaire, short form- , and american orthopaedic foot & ankle society scale. results: nonunion was not noted in either group. however, we detected union with a step-off of mm or more in cases from group b. with regard to ankle joint alignment, case in group a and cases in group b showed mild asymmetry of the medial and lateral clear spaces on ct at months. clinical outcomes at and months after surgery were better in group a than in group b. conclusions: screw fixation of pmfs was effective for fracture healing and maintaining ankle alignment. additionally, it improved short-term clinical outcomes, which we believe was due to stabilization of ankle fractures with pmfs involving\ % of the articular surface. references: level ii, prospective comparative study. how accurate can gaps and step-offs be determined in acetabular fracture treatment? introduction: the assessment of gaps and steps in acetabular fractures is challenging. studies evaluating the value of various imaging techniques to enable accurate quantification of acetabular fracture displacement are limited. this study aimed to assess the inter-and intraobserver variability of gap and step-off measurements using pelvic radiographs, intraoperative fluoroscopy and computed tomography (ct). material and methods: sixty patients, surgically treated for acetabular fractures, were included. five observers measured the gap and step-off on all the pre-and postoperative pelvic radiographs and ct scans. intraoperative fluoroscopy images were reassessed to determine the presence of gaps and/or step-offs. the inter-and intraobserver variability were calculated for the measurements using pelvic radiographs or ct scans. kappa was calculated for the intraoperative fluoroscopy assessment. results: for the preoperative displacement, the intraclass correlation coefficient (icc) was . (gap and step-off) using pelvic radiographs, and . (gap) and . (step-off) using ct scans. for the postoperative displacement the icc was . (gap) and . (step-off) using pelvic radiographs and . (gap) and . (step-off) using ct scans. the average kappa for the intraoperative gap and/or step-off assessment using fluoroscopy was . (- . to ) both for the inter-and intraobserver assessment. conclusions: there is little agreement between the observers regarding the measurements of the preoperative displacement, the presence of gaps and step-offs intraoperatively and the measurements of the postoperative displacement. a possible explanation for this is that the acetabulum has a three-dimensional spherical shape with multiple fracture lines and fragments going in different directions. single radiographic or ct-based gap or step-off measurements do not seem to be representative for the fracture characteristics, therefore the use of d measurements should be considered. introduction: long-term intake of glucocorticoids leads to pathologic changes in bone and cartilage tissues. material and methods: to understand how to prevent the occurrence of the pathology, we studied the use of vitamin d, vitamin e and a combination thereof on the background of the intake of prednisolone, . mg/ g of body weight. the experiment involved male rats of wistar linear breed. the animals were months old and weighted . ± . g. the experiment included series of animals, rats in each, namely: the first group-intact animals; the rest of the animals received prednisolone, . mg/ g of body weight. the rats of the third series received additionally iu of vitamin d . the animals from the fourth group also received . iu ( . mg) of vitamin e. results: long-term administration of prednisolone to the experimental animals has caused significant structural and functional disorders in their bone and cartilage tissues. they can be construed as simulated glucocorticoid-induced osteochondropathy. the combination of the vitamins d and e has demonstrated its ability to promote restoration of histomorphologic features of bone and articular cartilage in proximal femur epiphysis and epiphyseal cartilage of proximal femur epimetaphysis in animals with simulated glucocorticoid-induced osteochondropathy. the combination of the vitamins d and e has demonstrated a better effect on the background of the glucocorticoid-induced osteochondropathy, compared to the vitamin d alone. conclusions: preventive administration of the vitamins d and e while treatment with prednisolone leads to avoidance of the majority of pathologic changes, resulting otherwise from glucocorticoid-induced osteochondropathy. konyang university hospital, orthopaedic, deajeon, south korea introduction: the purpose of this study was to evaluate clinical, radiological and functional outcomes of patients had osteochondral autograft harvested from the ipsilateral femoral head for a femoral head defect after posterior hip fracture dislocation material and methods: this study was approved by irb at our institution. a retrospective chart review of a prospectively performed operation was performed at two university hospital between march , , and june , . all fracture was classified by the ao/ota classification. we included the patients had minimum months of follow up periods. ten displaced head fractures were addressed through posterior surgical dislocation and two patients had no posterior dislocation was operated using smith-peterson approach. an osteochondral graft was harvested from inferior non-weight bearing articular surface and grafted to osteochondral defect. all patients were full weight bearing by months results: we had femoral head fracture dislocation. patients were excluded due to lost to follow up. twelve of with type i/ii pipkin fracture dislocation with the articular defect and reduced within h of injury was identified for review. the patients were followed up for a mean of . months. there was no osteonecrosis. decreased joint space was identified in two patients. all fractures achieved union. the mean harris hip score of last follow up was . ( - ) one patient who operated using the smith-peterson approach had femoral nerve palsy. conclusions: the clinical and radiological results after treatment of femoral head fracture dislocation with articular defect by osteochondral autograft harvested from its own non-weight bearing articular surface show good outcomes. hospital universitario fundacion jimenez diaz, madrid, spain, hospital universitario de octubre, madrid, spain, hospital universitario la paz, madrid, spain introduction: preoperative computerized tomography scan provides important information about ankle fractures associating posterior malleolus, helping us distinguishing fractures affecting distal tibiofibular joint. the aim of our paper is to describe our series of patients suffering an ankle fracture with posterior malleolus involvement. methods: fifty-two consecutive patients, with ankle fracture involving posterior malleolus were evaluated prospectively. all of them were assed with a preoperative ct scan, demographic data, fracture mechanism, surgical approaches, posterior malleolus size measured classification and treatments were analyzed. results: most frequent posterior malleolus pattern according to bartonicek classification was type ii, twenty-two patients ( . %). an alternative surgical approach was performed in thirty-three patients ( %) as a consequence of information provided by ct scan. no statistical differences were observed when measuring posterior malleolus in conventional x-rays or ct scan. analysis of variance showed a p value less than . when comparing pm size and haraguchi and bartonicek classifications. discussion and conclusion: ct scan is required to perform an adequate preoperative study of ankle fractures involving posterior malleolus, using this information to provide a better outcome to our patients. effect of atorvastatin and losartan on gene expression and cell count in a rat model of posttraumatic joint contracture of the knee-a blinded and randomized animal study introduction: myofibroblasts have been associated with increased posttraumatic joint contracture, which has a massive impact on articular function. atorvastatin and losartan have shown to reduce the proliferation of cardiac, hepatic and pulmonary myofibroblasts. the aim of this study was to evaluate the effect of atorvastatin and losartan on gene expression, cell count and collagen deposition in the posterior joint capsule , and weeks after trauma in a rat model of posttraumatic joint contracture of the knee. material and methods: posterior capsular injury and kirschner-wire immobilization of the knee were performed in sprague-dawley rats. atorvastatin, losartan, or placebo was administered daily orally. the rats were sacrificed at either (n = ), (n = ) or (n = ) weeks after initial surgery. rats euthanized at week had their k-wire removed at week , followed by a remobilization period of another weeks. the results were evaluated via qpcr and immunohistochemistry. results: losartan reduced the number of myofibroblasts in comparison to the control at week and , whereas atorvastatin lowered myofibroblasts only at week (p \ . ). atorvastatin reduced the collagen deposition at week , whereas losartan had no effect on collagen deposition. losartan decreased gene expression of connective tissue growth factor (ctgf) at week and of tgf-b at week . clinical findings: positive anterior drawer test, grade iii valgus instability, and a palpable gap below the patella were assessed. no neurovascular alterations were found and ankle-brachial index scored [ . . investigation/results: initial immobilization with a splint was performed. radiographs showed a high patella with no other lesions. mri revealed a complete rupture of the patellar tendon and a complex multiligamentous injury with complete anterior cruciate ligament (acl) tear, avulsion of distal medial colateral ligament (mcl), and a complex rupture of both meniscus. diagnosis: knee dislocation with patellar tendon rupture. therapy and progressions: definitive treatment was performed days after the initial lesion, with arthroscopic resection of the posterior horn of the external meniscus and reconstruction of the acl with posterior tibial tendon allograft, as well as open repair of the patellar tendon and the internal meniscus, with subsequent mcl distal reinsertion. immediate partial weight-bearing with an extension orthosis was allowed. the patient is currently progressing with rehabilitation. comments: knee dislocation is a rare injury, and most cases are due to highenergy trauma. concomitant rupture of the patellar tendon is very unusual, and most cases are described in the context of open injuries. surgery is mandatory in order to restore full stability of the knee, with either one intervention or a staged surgery, including repair of the collateral ligaments and the patellar tendon followed by arthroscopic reconstruction of the cruciate ligaments. postoperative management consists on early rom restoration and weight-bearing as tolerated. introduction: apophyseal anterior inferior iliac spine (aiis) fractures are rare injuries. they most commonly occur in athletes in adolescence period. because the ossification of pelvis is not completed, apophyses are the weakest part of musculo-tendinous unit during this period, thus avulsion fractures are more frequent than muscle ruptures. aiis avulsions are the result of sudden and forceful contraction of rectus femoris muscle concentrically or eccentrically. material and methods: we report a clinical case of a aiis avulsion fracture in a young male football player, after being misdiagnosed as muscle strain. results: our patient was treated with conservative treatment including bed rest, analgesia, using crutches and toe-touch weight bearing, progressing to full weight bearing as tolerated and nonsteroidal anti-inflammatory drugs. at follow-up, he showed relief from his pain and mechanical symptoms and regained full range of motion and returned to his previous levels of activity. conclusions: diagnosis requires careful attention to the physical examination and imaging. in this case, the fracture was managed successfully with a conservative approach. good results and return to previous levels of activity can be achieved with conservative treatment. when misdiagnosed as a simple strain, the late diagnosis may cause chronic pain with decreased sportive performance in the future. therefore, a carefully taken anamnesis and physical examination with comparative anterior-posterior pelvic x-rays are needed not to miss avulsions in adolescents; also in some instances, more advanced scanning methods must be considered. introduction: the problem of meniscus damage in children is due to unsatisfactory treatment results, which is associated with the frequent execution of meniscectomies. amount of unjustified meniscectomies and the incidence of osteoarthritis can be reduced if menisci are repaired. material and methods: during the period january -august children with injuries of the meniscus were treated in morozov children's clinical hospital. children underwent meniscus repair by suturing using three techniques: ''all inside'', ''inside out'' and ''outside to inside''. meniscus suture decision was made taking into account the assessment of the severity of the damage. the period from the moment of injury wasn't taken into account. the technique of meniscus suture was determined depending on the location and type of damage. we met children with damage to the discoid meniscus who underwent partial resection and meniscus suture. children underwent a meniscectomy due to severe traumatic and degenerative changes. children had mri of the knee after months and x-ray after months. results: children achieved a satisfactory functional result; operated children are at the rehabilitation stage. we faced a complication-limitation of flexion in the knee joint in child. in all children on the control mri, the absence of synovitis, the safety of the reconstructed meniscus contour and the decrease in the intensity of the hyperechoic signal in the gap zone in dynamics are determined. conclusions: the introduction of a technique for repair meniscus integrity in the daily practice of an arthroscopist makes it possible to reduce the number of meniscectomies, which will reduce the number of unsatisfactory treatment results for this pathology and prevent the development of early osteoarthritis of these, children revealed a fracture-dislocation of the patella. in children, a tangential fracture of the lateral condyle of the femur was noted. in children, the dislocation was repeated. we met children with bilateral damage. all children with complete damage to the medial patellofemoral ligament, fracture-dislocation of the patella and dysplastic dislocation were performed tendon plastic using the quadriceps femoris tendon. the technique includes: transplanting a graft quadriceps tendon graft without cutting off the patella. next, the transplant is subfascial carried out in the medial direction and is fixed with a bio-integrated screw in the femur. results: the rehabilitation period was months. % of children have a satisfactory result (there is a limitation of flexion in the knee joint to °). % have an excellent clinical result: the full range of motion in the knee joint, the absence of pain and a return to sports. none of the operated children had relapses of dislocation. conclusions: it is recommended to consider the technique of tendon plasty of the medial patellofemoral ligament using the quadriceps femoris tendon as a method of choosing the treatment for patellar dislocation in children. case history: a -year-old boy who was injured while playing baseball. he was playing as a catcher and was bumped into the runner, therefore his ankle got twisted. he was immediately taken to the hospital. clinical findings: x-ray the distal tibial epiphyseal growth plate was irregular. although the ankle joint was not dislocated. in the ct, the proximal fibular fragment was caught behind the posterior edge of epiphysis of the distal tibia and was trapped there. investigation/results: the patient must be operated in order to repair the ankle. but the reduction of the entrapped distal tibia epiphysis was not easy without open. diagnosis: we diagnosed with bosworth like fracture. therapy and progressions: reduction was not easy, however we performed it by the pulling the fibula towards to outside, pulling out the curled anterior tibiofibular ligament, and then pushing into the tibia. we performed screw fixation after reduction of distal tibial epiphysis. furthermore, we fixed the fibula with plate. we made him to do range of motion exercise and toe touch gait from next day, and full weight bearing from weeks. we removed the implant months after the surgery. he did well subsequently, and at years after injury, he had normal function of the ankle, and normal x-ray. and he has returned to sports without pain. introduction: judo is the most popular martial art in the world and the first martial art recognized since as an olympic sport. worldwide, the international judo federation has registered countries with about million judo practitioners. like martial arts, judo mainly involves grip and throwing techniques. the competition rules in judo have been subject to constant adjustment and optimization in recent years. injuries prevalence is an important factor in the contact martial arts. material and methods: a prospective cohort study of all registered international athletes ( ) at three different european judo contests in germany were accomplished with the aim to investigate the injury rate as well as the pattern of injury. the age of the athletes ranged between and years. injury incidence rates were calculated per athlete-exposures (iirae) and per min of exposure (iirme). independent variables were sex and weight division. subgroups were compared by calculating the injury incidence rate ratio. results: severe injuries by judo tournaments are rare. the most frequently injured regions were the hand and head. the fights of the main block are riskier than the finals. the incidence of injury in heavyweight division differed with lightweight competitors. the risk of injury for female and male competitors differed slightly. conclusions: further studies are needed to determine a judo specific injury patterns and factors especially in the pre-competitional phase. investigation of prevention-strategies like the adaptation of competition rules etc. makes sense. does garden''s classification of femoral neck fracture match between orthopedic specialist and clinical resident? t. inoue , s. inoue , t. muraoka prefectural miyazaki hospital, orthopedics, miyazaki, japan introduction: garden''s classification is the most popular classification of femoral neck fractures. femoral neck fracture should be operated^ h; however poor agreement make waiting time longer because it takes more time to prepare implants and biological clean room. we investigate the agreement of the garden''s classification (non-displacement type or displacement type) between clinical resident and orthopedic specialist. material and methods: the examiner are a clinical resident ( nd year) and an orthopedic specialist ( th year). the subjects were cases of femoral neck fractures treated at our hospital between january and december . first, the examiners classified them into a non-displacement type and a displacement type (test ). second, the examiners studied the literature about unclassifiable type. third, the examiners classified cases month later once more (test ). finally, we compared the first test with the second test using the agreement (the number of matched patients/total) and kappa coefficient. results: the test showed that the agreement and kappa coefficient were . % and . . the test showed agreement was . %, . . the intra-observer agreement of clinical resident was . % and kappa coefficient was . . the orthopedic specialist was . %, and kappa coefficient was . . at test , cases did not match. cases of those were unclassifiable type, which were valgus type with medial fracture line. with slight displacement, agreement will get lower; some doctors consider it displacement type. conclusions: unclassifiable type makes us confused. it makes agreement better to discuss about unclassifiable type. introduction: the aim of this retrospective study was to describe the profile of missed hand and foot fractures in multitrauma patients and to elucidate risk factors for the delayed diagnosis. material and methods: from to , there were included patients. missed fractures were defined as fractures, which were not diagnosed during primary and secondary survey. patients were assessed for age, sex, glasgow coma scale, injury severity score, and length of stay in hospital (los). timing of hand or foot diagnosis related to admission date (measured in days) was noted. results: overall, . % of patients had a delayed diagnosis of hand fracture, . % ha a delayed diagnosis of foot fracture. the mean gcs for patients with delayed diagnosis was , whereas patients with diagnosis the day of admission had and mean gcs of (p \ . ). patients with delayed diagnosis had a mean iss of . versus . for those diagnosed the day of admission (p \ . ). furthermore, patients with delayed diagnosis had a mean los of . days, whereas those diagnosed at the time of admission had a mean los of days (p \ . ). concerning delayed diagnosis hand fractures, metacarpal and phalangeal fractures were the most common injuries overall ( . % and . %, respectively). concerning delayed diagnosis foot fractures, metatarsal fractures ( cases) and calcaneus fractures were the most common injuries overall, followed by talus fractures and toe fractures. conclusions: this study revealed that with a decreased gcs and increase in iss, polytrauma patients are increasingly at risk for delayed diagnosis of hand and foot fractures with a concomitantly increased los. as a delayed diagnosis has significant impact on the final functional outcome, correct and careful primary, secondary and tertiary survey is essential. introduction: the aim of this study was a) to determine the methods of hemorrhage control currently being used in clinical practice and b) to analyze pelvic fracture mortality rates before and after initiation of a multidisciplinary pelvic fracture protocol. material and method: between and , we included trauma patients with pelvic fractures (group ). a similar retrospective examination was performed on a number of trauma patients without pelvic fractures (control group). there were collected injury severity score (iss), the highest abbreviated injury scale (ais) score in each anatomic region and methods of pelvic hemorrhage control. there were also recorded hospital lengths of stay (los) and in-hospital mortality. results: the average follow-up was -months. the average iss in group and group was respectively . and . . in both groups the commonest mechanism of injury was motor vehicle crash ( . %). in group , angioembolization and external fixator placement were the commonest used method of hemorrhage control. patients underwent diagnostic angiography with contrast extravasation noted in patients. patients with pelvic fracture had a mean hospital los of . days. the overall in-hospital mortality rate of patients with pelvic fractures was . %, while in group the overall in-hospital mortality was . %. age, shock, severe head injury and increasing iss, are all significantly associated with mortality in the pelvic fracture group. conclusions: the findings from this study demonstrate no clear relationship between the choice of hemorrhage control intervention used and the patient's clinical status. in healthier patients with unstable pelvic fractures, the mortality rate was similar to that of patients with stable fracture patterns. introduction: various percutaneous screw placement for pelvic and acetabulum fractures is often difficult because of complex anatomical morphology, however, it becomes very beneficial to set enough fixation stability if we can insert the long screws. d-ct navigation system for the screw placement is beneficial for precise screw insertion. we investigated the accuracy of screws with d-ct navigation. material and methods: our retrospective case series were assessed by the accuracy of screws with d-ct navigation for pelvic and acetabulum fractures. twenty-six patients who sustained pelvic fractures and thirteen patients who sustained acetabular fractures were included in this study and . mm cortical screws or . mm cannulated screws were inserted with d-ct navigation. we investigated the number of screws and screw positions which is measured by postoperative ct scan and classified by smith criteria. results: we inserted tits (transiliac-transsacral) screws and is (iliosacral) screws for pelvic fractures. of screws ( . %) were placed in correct position (grade or ). screw for s lesion was placed in incorrect position. meanwhile we inserted antegrade pubic screw, anterior column screws, posterior column screws and infra-acetabular screws. of screws ( . %) were placed in correct position (grade or ). screws were in incorrect position and they were all cortical screws. and there was no complication related to screw insertion. conclusions: our study highlights that d-ct navigation system reduced the malposition rate of screw insertion for pelvic and acetabular fractures. however, we sometimes had difficulty in inserting tits screw for s lesion and cortical screw for acetabular fractures. we assumed that this was caused by narrowness of s corridor and flexibility of drill or inserting cortical screws in wrong position manually. we should pay much more attention even using d-ct navigation. is operative therapy still warranted for dislocated acetabular fractures in elderly patients? introduction: the incidence of acetabular fractures in elderly patients is increasing. there is no consensus about the right treatment for the impaired elderly patient with an acetabular fracture. the aim of study was to investigate acetabular fractures in the elderly patient and the risk of a secondary tha. material and methods: a retrospective study was performed from till in the radboudumc nijmegen. all patients with an acetabular fracture were reviewed. they were divided into two groups, younger than and or older. ct scans were used for classification according to letournel and for the quality of the reduction according to matta. there was a follow-up of minimal years. results: in total, patients attended at the radboudumc with an acetabular fracture, of which were years or older. in the younger group, patients received surgery and elderly patients. according to matta, an anatomical reduction was achieved in % of the young patients and % of the elderly patients. imperfect reduction was achieved in % of the younger patients and % of the elderly patients. thirteen percent of younger group and % of the older group needed a tha based due to the posttraumatic arthritis, the younger group after months and the older group after months on average. one younger patient with anatomical reduction needed a tha, none of the elderly patients. twenty-three percent of the younger patients and % of the elderly patients, all with a poor reduction, needed a tha. age, the complexity of the fracture and the quality of the reduction were important factors leading to a secondary total hip arthroplasty. conclusions: elderly patients are two times more likely to need a secondary total hip arthroplasty. after an anatomical reduction, the risk is very low, even in the elderly. surgery for dislocated acetabular fractures is a good option when there is a possibility for a good reduction. references: letournel e. matta jm. introduction: in japan, as a definition of basicervical fractures of the proximal femur, a fracture line is placed into and out of the joint capsule of the hip joint. however, in fact there are various fracture types.we classified these fracture types based on treatment methods and reported on these results. material and methods: cases of proximal femoral fractures treated in our hospital from january to december . basicervical fractures occurred in cases ( . %). all cases diagnosed with x-ray and d-ct, and observed for months or more after surgery. results: there are two types of basicervical fractures: the fracture line exists around the just inside of the intertrochanteric part: normal type(n type); cases ( . %), and fracture line exists subcapital at ventral side, the coronal plane in the center of the neck and the trochanteric fossa at the dorsal part: coronal shear type(c type); cases ( . %).c type was further classified by treatment method depending on existence of posterolateral fragment and anterior wall fracture. c type without comminution ( part:c- type) was cases ( . %). with posterolateral fragment ( part:c- type) was cases ( . %), with posterolateral fragment and anterior wall fragment ( part:c- type) was cases ( . %).n type and c- type were treated by sliding hip screw (shs) with anti-rotation screw. c- type: shs with trochanteric stabilizing plate, c- type because of the bony contact area is very small: hemi-arthroplasty with calcar replacement was performed. cut out occurred in cases of c- type and case of c- type, but others obtained union.. one case of c- type occurred peri-prosthetic fracture intraoperatively. conclusions: we classified cases of basicervical fractures, and according to its classification, treatment method was decided and good clinical results were obtained. strategies aimed at preventing chronic opioid use after trauma: a scoping review c. cô té , m. berube université laval, faculty of nursing, québec city, canada, chu de quebec research center, université laval, trauma, emergency, critical care medicine, québec city, canada introduction: a high incidence of chronic opioid use (up to %) has been documented after trauma. solutions are urgently needed considering the importance of this public health issue. we aim to identify strategies to prevent chronic opioid use in the trauma population and to assess their level of evidence. material and methods: we initiated a scoping review of literature to identify research articles and guidelines on preventive strategies. several databases and websites of trauma were searched. strategies were classified according to their types and targeted trauma populations. the level of evidence was summarized according to an adaptation of oxford center for evidence-based medicine classifications and strategies effectiveness. results: close to items have been screened until now from which studies - and one guideline were found eligible. two studies - combined education with mandatory limit of opioid prescriptions (level iii) in the orthopaedic trauma population and the other study used tailored physical training after whiplash injury (level i). findings showed reduction of opioid use or complete weaning at and weeks after trauma, however the effect was not maintained beyond weeks. guidelines on orthopaedic trauma made the following recommendations: prescribe the lowest effective dose for the shortest period (strong, high-quality evidence), avoid long-acting opioids in the acute setting (strong, moderate-quality evidence), and prescribe precisely (avoiding ranges of dose and duration) (strong, low-quality evidence). conclusions: chronic opioid use is an important issue in trauma patients. findings highlighted the need for more research to reduce the burden associated with chronic opioid use in this population. references material and methods: we analyzed clinical cases: men- and women- , mean age years. trauma circumstances: habitual trauma- cases, traffic accident- , precipitation- , sport- , aggression- . for cohort analize schatzker classification was used: especially type i was meet in cases, ii- , iii- , iv- , v- , vi- ; close, open. for paraclinic examination were used x-ray and ct. surgical management consisted of: close reduction, internal fixation- cases ( -percutaneus canulated screws arthroscopic assisted, -external fixator), open reduction, internal fixation- cases. bone graft was done in cases. results: postoperative follow up was performed at , , , weeks. patients were evaluated according to the lysholm knee scoring scale, obtaining an average score of points. bone healing was achieved in a period of between to weeks. postoperative complication developed in cases. results were depending on the stability of osteosynthesis, precocity, rightness of functional reeducation and patient compliance. conclusions: favorable functional results and less complication were met in cases of individual approach of surgical management, a good choice of implants and minimally invasive surgical techniques. fractures of the shoulder processes-a case report case history, clinical findings and diagnosis: -year-old male, low-speed motorcycle crash with subsequent polytrauma. he presented with right shoulder pain, swelling and pain to the touch. articular ct revealed a type i fracture of the coracoid base, type iii acromion fracture and scapular body fracture without displacement. results, therapy and progressions: he was submitted to surgical treatment days later. a superior ''sabercut'' approach with open reduction and osteosynthesis of the coracoid process was performed with a cancellous screw and washer and fixation of the acromion with k-wires and tension band wire. fracture of the scapular body followed a conservative treatment. immediate postoperative period was uneventful and he presented with favourable evolution in the subsequent -week, -week and -month follow-up. at present time, at -month follow-up, maintained anatomical reduction in radiological control, complete arm abduction and no limitation with efforts. comments: conservative treatment is generally indicated for all shoulder body fractures without displacement. fractures of the coracoid or acromion with [ cm displacement are described as an indication for surgical treatment. fractures of the acromium without displacement may follow conservative treatment with sling immobilization. surgical fixation can be achieved with screws, plate and screws or tension band wire. although controversial, surgical treatment for coracoid fractures is preferred, especially in active young patients with open reduction and fixation with screws or, if necessary, with plate and screws. the treatment applied in the present case, all approaches described in the literature as being effective and with good results, is in agreement with the options described in the literature and constitutes a corroborative example of its efficient results. case history: a -year-old male, hand worker, attended to our emergency department after a traffic accident complaining about pain and swelling in his left wrist. initial radiographs revealed an isolated dorsal dislocation of the lunate that went unnoticed. two and a half months later he was referred to our clinic. clinical findings: findings included dorsal wrist deformity and pain. he presented a decreased passive wrist flexion and extension range of motion, with normal finger tendinous function. investigation/results: plain x-rays showed persistence of the lunate dorsal dislocation without any associated injuries. diagnosis: chronic isolated dorsal dislocation of the lunate therapy and progressions: open reduction was performed using a dorsal approach. the scapholunate, lunotriquetal and scaphocapitate spaces were stabilized with a compression screw and kirschner wires respectively. the patient persisted with pain and functional limitation after the surgery, showing an insufficient reduction of the scapholunate space on the x-ray. nine months after the initial surgery, he developed a purulent fistula on the ulnar edge of the carpus. after it was resolved, a total wrist arthrodesis was performed using the mannerfelt technique. at the months follow up, he was clinically stable, consolidation of the arthrodesis was documented and he had returned to his previous normal activities. comments: isolated dorsal dislocation of the lunate is a rare lesion. the delay in the diagnosis of carpal dislocations is frequent. this compromises the final outcome of reconstructive techniques and the risk of residual instability, hence increasing the risk of chronic pain associated with posttraumatic osteoarthritis. in the case of chronic lesions, treatment with palliative techniques such as proximal carpectomy or joint arthrodesis should be taken into consideration. references: siddiqui n., sarkar s. isolated dorsal dislocation of the lunate. open orthop j. ; : - is ultrasound-guided regional anesthesia safer than landmark technique? one-hospital experience introduction: according to the literature the application of ultrasound (us) in performing regional anesthesia had a significant impact on patient safety by increasing the success rate [ ] . in a donated ultrasound device became available in the institute of emergency medicine, chisinau, republic of moldova. due to lack of equipment both us guided and landmark techniques have been performed. the aim of this study was to analyze the two methods of performing regional anesthesia, in order to estimate the potentials benefits of of us guided techniques (succes rate and doses). results: the bivariate analysis showed that, out of anesthetics in lmg, a number of were reported as unsuccessful, compared with a number of in usg. the v test with corrections for continuity did not determine significance (test value . , df = , p = . , effect size = . ), rr being . ( % ci . - . ). linear regression for dose (lidocaine) modeling, in patients included in the research, showed a decrease of the dose by mg in lmg, the confidence interval being quite wide ( % ci -. , -. ). that is, the actual decrease is within the limits of and mg. conclusions: the tendency towards higher failure rate in successfully performing an us guided regional anesthesia and relative ''uncertain'' decreasing of dosage are in contradiction with the international statistical data. this in turn evidenced probable deficiencies in the training of the practitioners in field of ultrasound guided techniques in our country. the prospective research to confirme/infirme these results and estimate the complication rate follows. references: . barrington mj, uda y. did ultrasound fulfill the promise of safety in regional anesthesia? current opinion in anaesthesiology ; ( ) results: average age years old ( - ).all were active labour patient. the most frequent mechanism was high energy trauma (traffic accident), of who presented gustilo grade iiib open fractures operated in the country of origin. most frequent pattern of fracture was -c. ( cases) and -c. ( cases). initial conservative treatment was performed in of the cases. one persistent pseudoartrhosis with osteosynthesis material failure. in every case, preoperative ct and early surgical intervention were carried. in cases, an additional procedure was associated at the radioulnar distal joint. in all cases consolidation occurred. one patient required reintervention for persistent pseudoarthrosis. average consolidation time months ( ) ( ) ( ) ( ) ( ) ( ) ( ) .average follow-up of months ( - ). average active joint balance: flexion °( °- °), extension °( °- °), pronation °( °- °), supination °( °- °). average dash . ( - . ).force reduction greater than % compared to contralateral in of the cases. radiological parameters:radial height . mm ( - ),radial inclination °( - °),volar angulation . °( . °- °), ulnar variance . mm ( ) ( ) ( ) ( ) ( ) . conclusions: malunion of the distal radius is an uncommon and severe complication with increasing incidence that requires early and personalized surgical treatment to achieve the correction of the deformity, preserving mobility acquiring consolidation with acceptable functional results case history: isolated ulnar translocation of the carpus is unusual. when the translation occurs without injury of the radius, ulna or carpal bones are often misdiagnosed. early diagnosis is key, to avoid further complications such as redislocation of the carpus ( ). clinical findings: in our case a young male patient suffered a high energy motorcycle accident. he had no a b c d problem investigation/results: the ulnar translation of the left carpus was evident but comparison x-rays were taken on both wrist for further evaluation. the distance between the line, drawn through the axis of the radius and the center of the capitate bone was measured bilaterally. the results were . mm vs . mm. diagnosis: isolated, open ulnar translocation of the radiocarpal joint, dumontier type i, was diagnosed. treatment: the primary treatment was debridement, reposition and fixation with ex fix. after the wound healing on th days we made reconstruction. volar approach was used, we re-reponate the carpus and fixated the position with two mm smooth kirschner wires. the radioscaphocapitate and long radiolunate and radioscaphoid ligaments were reattached to the volar margins of radius using mitek mini anchors. we put the ex fix and left the bended wires percutaneously. after weeks the ex fix and the k wires were remove. wrist motion exercises were initiated under supervision of physiotherapist. comments: after weeks the wrist was in good alignment, the flexion-extension were - , the deviations were - °. the radiographic signs of this injury are unusual and often misdiagnosed. it can be useful to compare with contralateral x-rays. the radiolunate and radioscaphocapitate ligaments is considered crucial in prevention of ulnar translation. in our opinion the radiolunate arthrodesis can be reserved for failed ligament repairs. introduction: within the orthopaedic paediatric population, there is a distinct paucity of literature in regard to post-operative paediatric analgesic regimes. supracondylar humeral fractures account for % of all paediatric limb fractures and there has been a marked divergence in recent literature concerning the most appropriate choice of analgesia for this cohort with recent studies recommending the routine inclusion of an opioid agent post-operatively on prescription. opioids have deleterious side effects pertinent to paediatrics. in our institution, patients'' only receive a prescription for acetaminophen and nsaids upon discharge. our study assessed postoperative analgesic satisfaction rates in all paediatric patients who underwent crpp for supracondylar humeral fractures in our institution from january to december . material and methods: this is a retrospective multi-surgeon case series of all paediatric patients who underwent crpp from january to december . patient data was extrapolated from theatre records and clinical charts. for each patient, all analgesic agents given were identified, the dosage, route and frequency of administration in addition to the length of their hospital stay and time from injury to operation. following discharge, patients'' guardians were contacted retrospectively and a questionnaire was administered which ascertained the efficacy and duration of analgesia used by the patient postoperatively. results: fifty patients were identified for inclusion within the study who met the inclusion and exclusion criteria. there was a % satisfaction rating amongst the responders with the analgesic regime recommended-acetaminophen & nsaids. conclusions: in stark contrast to papers which we discuss throughout our paper, our study conclusively demonstrates that opioid prescriptions are not required upon discharge for supracondylar fractures within a paediatric population case history: a -year old man suffered an isolated injury of his right hand in a motorcycle accident. clinical findings: the patient presented with a swollen hand, a subtotal amputation of the middle finger at the level of the middle phalanx and lacerations to the other fingers (fig. ) . investigation/results: after excluding injuries to other body regions, radiographs and a ct of the hand were performed (fig. ) . diagnosis: closed fracture dislocation of cmc joints from ii. to v. finger, comminuted fracture of the middle phalanx of the middle finger, closed fracture of the proximal phalanx of the middle finger, other lacerations to the iv. and the v. finger. therapy and progressions: urgent open reduction and internal fixation (orif) with k wires of the cmc joints. exploration of the middle finger reviled heavy contamination and comminution of the phalanx, with injury to one neurovascular bundle. a phalangectomy with acute finger shortening was performed with creation of a new ip articulation (distal to proximal phalanx) (fig. , ) . progression after the surgery was uneventful. there was no sign of infection. the shortened finger was sufficiently perfused and the patient reported a sense of touch. k wires were removed after weeks and physical therapy was started. the patient has limited rom in his neo ip joint with minimal pain (vas - ) (fig. ) . comments: middle phalangectomy of the hand was described in the literature only in two papers which report treatment of chronical or congenital diseases. the authors propose this method as an alternative to amputation in selected trauma cases. results: patients ( m, f, mean age y) with fractures were included. kidney-tpl, lung-tpl, liver-tpl, heart-tpl, kidney/pancreas-tpl. all patients got treated with at least two immunosuppressive drugs. cause of accident: . % sports/leisure, % work/household, . % traffic accidents, % without trauma. the operation was performed under perioperative long-term antibiosis, often with a combination of two or three drugs. patients were hospitalized for an average duration of . days and were also examined by the particular organ specialists. osteosynthesis: in % primary operative fracture treatment, in % two-step procedure. plates distal radius and ulna [healing period (h) conclusions: the fracture healing was possible but significantly delayed. the wound healing took longer. the immunosuppressive therapy may be responsible for these problems. the rehabilitation of movement and weight bearing has to be adapted to the slowed fracture healing. introduction: the prevalence of fragility fractures of the pelvis (ffp) increases, including in up to % a lesion of the posterior pelvic ring. an operative therapy is indicated in cases of prolonged or immobilizing pain or in a displaced dorsal fracture. methods: patients suffering an ffp treated with a minimal-invasive trans-sacral bar through s from to were included. the patients or their relatives were contacted to ask about mortality, the present mobility and place of residence. % of all patients still alive could be included in follow-up. results: females and males with a mean age of . ± . years ( - ) were included. concomitant stabilization of the anterior pelvic ring was performed in %. . % underwent an operative revision ( % evacuation of hematoma, % peri-implant infection, % hardware removal-combinations possible). the trans-sacral bar was removed in one case due to malpositioning. the length of stay was ± days. at discharge, % were mobile on the ward, % in their room, % for transfer to sitting position and % were bedridden. % were discharged to their home, % in geriatric rehabilitation unit, the remaining to other rehabilitation or to a nursing home. during follow-up, mortality was %, one patient died during hospital stay. the patients died in average ± weeks after discharge. after a follow-up of ± weeks, % lived at their home, thereof one-third with assistance. % needed a walking aid, % were mobile without walking aid, % were bedridden or only mobile to sitting position. conclusion: the trans-sacral bar in s is a valuable minimal-invasive stabilization method to recover mobility in elderly with an ffp. a relatively long in-hospital stay could be explained by the initial trial of conservative treatment and due to intra-and inter-departmental cogeriatric services. the high mortality and need for assistance reflects this geriatric, multi-morbid patient collective. case history: a -years-old woman was admitted in the emergency room after being run over by a bus. clinical findings: at the emergency room, she was conscient and hemodynamic stable. head, thoracic or abdominal trauma were excluded. the patient presented with an open wound in left popliteal area with massive bleeding with exposure of gastrocnemius and soleus muscles and achilles tendon investigation: radiologic images didn't show any fracture. a limb angiography showed complete perfusion of the leg, without any lesion on major arteries. diagnosis: open aquilles tendon avulsion through the popliteal fossa therapy and progressions: the patient was taken to the operating room. we approach the popliteal area and found a small laceration of popliteal vein, which was sutured with prolene / . then, we reference the achilles tendon, and tunneled the posterior face of the leg, and passed the tendon through the tunnel. a distal approach, above the insertion of achilles tendon was done, and two suture anchors preloaded with sutures were inserted in the medial and lateral sides of the calcaneal tuberosity, then we did an krackow suture. we also did a fasciectomy on the lateral side of the leg, to prevent compartmental syndrome. the patient was put in a posterior cast with of flexion for weeks. the immediate post-operative time was in an intermedia unit care, to control possible multiorgan failure. in days, she was discharged to orthopedics nursery. due to the degloving of subcutaneous tissue, she evolved with some blisters which made her stay inpatient about weeks. after some time, she developed some areas of skin necrosis, which needed some intervention by plastic surgery with skin graft. now, she has skin completely healed, some loss of strength in the leg, with loss of plantarflexion, and is under prolonged rehabilitation program. therapy and progressions: she was rushed into the or and submitted to external fixation of the humerus and bones of the forearm, debridement, and primary closure of the forearm and hand. successive dressings and debridement was maintained and, at th postoperatory day(po) the external fixator of the left humerus was removed and a nailing was performed as well as an osteosynthesis of the clavicle fracture with anatomical plate. at thpo the external fixator of the forearm bones was removed and an open reduction and internal fixation of the radius with lcp plate and closed reduction and internal fixation of the ulna with an anterograde ten nail was performed. at thpo, she underwent an autologous skin graft of the forearm and hand wounds. good clinical evolution of the wounds and fractures, all of which evolved to consolidation, although m fracture malunion was verified as well as deficit of thumb abduction and extension of rd- th fingers. uefi of / . comments: the approach of polytrauma patients should be sequential, according to the atls protocol, preserving life, limb and function. treatment of these lesions is complex and, if poorly managed, can be associated with high morbidity, as most patients combine severe and contaminated lesions, extensive skin loss, open fractures, postoperative infection. a sequential approach is required, which involves injury assessment, infection prevention, soft tissue treatment and fracture stabilization. introduction: pelvic fractures, though rare ( - %), are often associated with high mortality ( - %). the factual outcomes in polytrauma patients with the additional burden of pelvic fractures are unknown. the purpose of this study is to provide an in-depth analysis of pelvic fractures in seriously injured patients. material and methods: this is a retrospective analysis of prospectively maintained trauma registry from to . we included all trauma patients with iss c . group i, which had an additional burden of pelvic fractures, was compared with group ii, consisted of patients without pelvic fractures. a double-adjustment propensity score match (psm) analysis was utilized to minimize confounding and unbiased estimation of the impact of pelvic fractures. . ± . , asmd = . ).patients in group i had higher number of genitourinary surgery (p = . ), exploratory laparotomy (p = . ). therequirement of angio-embolization was similar in between two groups (p = . ). while there were no difference in mortality (or . , % ci . - . , p = . ), group i had higher odds of severe sepsis (or . % ci . - . , p = . ) and ventilator-associated pneumonia (or . , % ci . - . , p = . ) conclusions: pelvic fractures in polytrauma patients did not translate into higher mortality. however, there was an increased risk of sepsis and vap. evidence-based management at tertiary care specialized centers can further enhance the outcomes. investigation/results: ap pelvis x-ray reveals a complex left proximal femur fracture with neck and trochanteric extension. a ct-scan was obtained and showed a complex fracture pattern with subcapital and trochanteric extension. blood analysis showed a hemoglobin of . g/dl. diagnosis: therapy and progressions: at admission, patient refused erythrocytes'' concentrate transfusion and was hospitalized for pain control and hemodynamic stabilization. despite alternative measures such as intravenous iron supplementation and erythropoietin, hemoglobin values remained lower than . g/dl, thus preventing any surgical procedure. at day , patient finally decided to accept packed red blood cells and was then transfused. at day and with a hemoglobin of . g/dl, the patient was finally submitted to a total hip arthroplasty with an uncemented revision femoral stem. at day , the patient initiated the rehabilitation protocol with hospital discharge at day with a hemoglobin of . g/dl. comments: proximal femur fractures arise as one of the major problems of present traumatology. comorbidities frequently prevent surgical treatment within the golden hour (first h) and thus limiting the postoperative results. in this particular case, a timely surgical approach would have made it possible to try a more conservative procedure with femoral osteosynthesis. the surgical delayed due to low hemoglobin values limited the surgical options and forced a more aggressive procedure. routine versus on demand removal of the syndesmotic screw; a multicenter randomized controlled trial on functional outcome introduction: syndesmotic injuries are common, being present in approximately - % of surgically treated ankle fractures . one of the most commonly used ways of fixation is the syndesmotic screw (ss). traditionally, this screw is removed after - weeks as it is thought to hamper ankle function and cause pain. however, a recent study showed that implant removal does not always result in improvement of functional outcome . with the relatively high complication rate of implant removal in mind, retaining sss could be beneficial. we therefore aimed to investigate the effect of retaining the ss on functional outcome. material and methods: in this multicenter rct, patients were randomized between routine and on demand removal (upon patients request). the primary outcome was functional outcome at months after ss placement, measured by the olerud-molander score (omas) with a non-inferiority limit of points ( % power, a = . ). secondary outcomes include quality of life, range of motion, complications and costs of ss removal. results: a total of patients were randomized, of which for routine removal and for on demand removal. the mean age was years old and % was male. follow up of all participants will be completed in march . results of the primary outcome analysis are therefore not yet available, but will be at the conference. conclusions: if on demand removal of the ss is non-inferior to routine removal in terms of functional outcome, this will offer a strong argument to adopt this as standard practice of care. this means that patients will not have to undergo a secondary procedure, resulting in fewer complications and subsequent lower costs. introduction: treatment options for pertrochanteric fractures of the hip are extra-or intramedullary fixation. the aim of this study is to identify risk factors for the development of complications: varus deformity, neck shortening, revision and cut-out. material and methods: retrospective cohort study in which radiographs of patients with pertrochanteric fractures, treated at the uz brussel between and , were reviewed. fracture type, type of the device, cut-out and revision where noted. measurements for the centrum-collum-diaphyseal angle (ccd) of the two hips, impaction, tip apex distance (tad), parker''s ratio were realized. statistical analyzes were made with logistic and multiple linear regression analyzes. results: patients were included. bmi (p = , ), type of osteosynthesis (p = , ), dhs ? plate (p = , ), short nail (p = , ) and the tad (p = , ) are independent risk factors for the development of varus deformity after consolidation. for impaction are bmi (p = , ), short nail (p = , ), long nail (p = , ) and fracture type a (p = , ) independent risk factors. we identified a marginal statistical significant risk factor for cut-out: tad (p = , ). conclusions: , % of the patients had varus deformity after consolidation. the risk of varus deformity rises with a higher bmi and a higher tad. the risk for this complication was higher when using a nail. neck impaction was shown more together with a high bmi and less in fracture type a and with the use of a short or long nail. in the prevention of cut-out, it is important to keep the tad low. case history: -year old female with previous distal femoral plating ( years ago) and ipsilateral proximal femoral nailing ( months ago) presented with a diaphyseal femur fracture. clinical findings: extremity was swollen, painful, neurocirculatory intact, no shortening or external rotation was seen. she was unable to lift her leg. scars showed no sign of infection. investigation: x-ray revealed a spiral fracture including distal pfna locking screw, unhealed proximal femur fracture without loss of reduction, protruding pfna blade and a healed distal femoral fracture. diagnosis: peri-implant fracture classification proposed by the singapore group presented a discrepancy between nail type subtype b and plate type subtype. by simplification, we disregarded the distal (healed) fracture to choose the first option. therapy: firstly, the distal femoral plate was removed as the preoperative simplification dictated. secondly, pfna distal locking screw was removed and the pfna blade shortened. after open reduction cerclage wires were applied. a long lcp plate was initially fixed through the plate and pfna locking hole, adjusted in line, fixed proximally with screws through a locking attachment plate and cerclage, distally locking screws were used. comments: distal femoral callus prevented the use of a long nail. as the proximal fracture was not yet healed, we avoided full implant removal. as the pfna was unstable, fixation through the plate and pfna distal locking hole enabled implant coupling to strengthen the construct. the plate covered the entire bone to bridge the possible loci minori left by the plate removal and minimize stress risers. background: we have been reported the usefulness of intra-medullary antibiotics perfusion (imap) and intra-soft tissue antibiotics perfusion (isap) for suppressing open fracture and bone infection. imap and isap was a method of antibiotics delivery with the continuous administration of high-dose aminoglycosides. however, the best dose was not obviously. the purpose of this study was to evaluate translation of aminoglycosides from imap or isap. as follows: males and females, average age was . years old, intramedullary nails and plates. one dialysis patient was including. we measured concentration of gentamicin from imap, isap and in blood, outflow. results: average administration concentration of all cases was . lg/ml. average blood concentration of all cases was lg/ml and outflow concentration were . lg/ml. average blood and outflow concentration of each dosage were shown as follows: lg/ ml: . lg/ml, lg/ml, lg/ml: . lg/ml, . lg/ml, lg/ml: . lg/ml, lg/ml, lg/ml: . lg/ml, . lg/ml. in dialysis patient case, lg/ml administration lead concentration of blood as . lg/ml, outflow as lg/ml. side effect were not observed. discussion: local antibiotic administration using imap and isap showed increasing blood concentration depend on administration dose. under lg/ml administration dose showed safe blood concentration(\ lg/ml). on the other hand, lg/ml administration dose achieve trough concentrations over - times of minimum inhibitory concentration. furthermore, we need to pay attention for administration dose in dialysis patient case. conclusion: lg/ml administration dose achieved safe and effective local concentration. introduction: distal radius fractures and supracondylar humerus fractures are two of the most common fractures seen in children. most can be treated with non-operative treatment but a small number require operative reduction and surgical stabilisation, often with percutaneous kirschner wires. this study aims to identify whether an early review is required before planned removal of the wires. materials and methods: retrospective review of paediatric patients undergoing surgical reduction and stabilisation with percutaneous kirschner wires for upper limb injuries. data collected over threemonth period (june-august ). number and type of outpatient reviews, imaging episodes and clinical interventions recorded. results: consecutive patients with mean age years (range - ). distal radius fractures and supracondylar humerus fractures. patients transferred to another unit. / patients received a week check and then a second review where the wires were removed. mean time to first outpatient review . days (sd . ). at initial appointment all patients had a change of cast and a satisfactory radiograph. mean time to second outpatient review was . days (sd . ). at the second appointment / patients had the wires and cast removed and subsequent satisfactory radiograph. / required a further period of casting. / had a third appointment. / required formal physiotherapy after cast removal. there was one transient anterior interosseous nerve palsy after supracondylar fracture stabilisation. clinical union of the fracture and good functional outcome was seen in all cases. conclusion: the initial outpatient review at - weeks allows a lighter weight cast to be applied but in this series the radiograph taken after the cast was changed did not alter management. our findings support a cast change alone at weeks and then clinician review with radiographs at the time of wire removal. introduction: the aim of this study was to describe surgical technique, report on patient-based functional outcomes and complications following open reduction and internal fixation in patients with scapular fractures. methods: the study comprised patients who were treated with open reduction and internal fixation (orif) of a scapular fractures between september and july . surgical indications were as follows: medial/lateral displacement greater than mm; shortening greater than mm; angular deformity greater than °; intraarticular step-off greater than mm and double shoulder suspensory injuries (including fracture of clavicle, coracoid or acromion with displacement greater than mm). all patients underwent x-ray examination (true ap, y scapular view) and computed tomography (ct) scans. fractures were classified according to the revised (ao/ota) classification system. functional outcome were measured using the constant-murley score. results: seven patients had glenoid fossa fracture, six patients had scapular body fracture and one patient had acromion process fracture. all glenoid fossa and scapular body fractures were exposed via the judet approach. eleven of patients were reviewed with constant-murley score at the final follow-up examination, three patients were lost for follow-up. the mean follow-up after injury was months ( - months). we found in four patients infraspinatus muscle hypotrophy. mean constant-murley score was . (± . ) for injured arm and . (± . ) for uninjured arm. mean score between injured and uninjured arm was . (± . ) which is excellent functional outcome according to grading the constant-murley score. conclusions: open reduction and internal fixation of displaced scapular fractures is a safe and effective treatment option that results in reliable union rate and good to excellent functional outcome. introduction: the aim of this study was to evaluate clinical and radiological results of intramedullary radius and ulna nails in treatment of adult forearm fractures. methods: the retrospective study included patients who were treated with intramedullary nailing of forearm fractures between january and september . the medical records and radiographic images of all patients, taken preoperatively and postoperatively, were reviewed. fractures were classified according to the ao/ota classification system by reviewing the radiographs. we analayzed time to union, union rate, clinical outcome and complications. results: primary intramedullary osteosynthesis were performed in patients with forearm diaphyseal fractures. the average time to union was months (range, - months) in primary osteosynthesis cohort. secondary intramedullary osteosynthesis were performed in four patients following removal of plates and screws due to pseudoarthrosis. the average time to union was months (range, - months) in secondary osteosynthesis cohort. overall union rate was , % in forearms with fractures or pseudoarthrosis of the radius, ulna, or both bones, which were treated with intramedullary nail with compression screw. overall complications were one nonunion, one postoperative rupture of the extensor pollicis longus tendon and one postoperative transitory radial nerve palsy. conclusions: intramedullary nailing of adult forearm fractures is a safe and effective treatment option that results in reliable union rate and good to excellent clinical outcome. key words: forearm fractures, intramedullary nailing, biological fixation, union rate results: transverse or short oblique fractures of the middle third of the humeral shaft were treated using a retrograde approach. spiral fractures of the middle third of the humeral shaft were treated through the antegrade approach. comminuted fractures of the proximal third of the humeral shaft were treated mostly through the antegrade approach. comminuted fractures of the distal third of the humeral shaft were usually treated using the retrograde approach. whenever possible, we prefer retrograde insertion because the approach through the shoulder joint is avoided. reduction with retrograde nailingnis easier because upper arm was placed on the radiolucent operating table extension. interlocking screw insertion by freehand techique is also easier to perform because there is no danger of radial nerve injury. nonunion was found in eight patients ( , %). there were five patients ( , %) with postoperative transitory radial nerve palsy that fully recovered within months. conclusions: the choice of approach to the medullary canal depends on the fracture type and the fracture site. therefore, antegrade nailing should be performed for proximal third humeral shaft fractures and complex middle third humeral shaft fractures, while retrograde nailing should be perforemd for distal third humeral shaft fractures and simple transvese or short oblique middle third humeral shaft fractures. keywords: humeral shaft fractures, intramedullary nailing, radial nerve palsy, nonunion the diaphyseal aseptic tibial nonunions after failed previous treatment options managed with the reamed intramedullary locking nail i. kostic , m. m. mitkovic clinical center nis, university hospital, orthopaedics and traumatology, nis, serbia, university of nis, serbia, orthopaedics and traumatology, nis, serbia introduction: in this article, we present our approach to the surgical treatment of noninfected tibial shaft nonunions. material and methods: between and , patients with aseptic diaphyseal tibial nonunion was treated by reamed intramedullary nailing and were retrospectively reviewed. all patients, preoperatively, were evaluated for the signs of the infection, by the same protocol. results: the time that elapsed from injury to intramedullary nailing ranged from to months (mean months).open intramedullary nailing was unavoidable in cases ( , %), while closed nailing was performed in patients ( , %). all patients were followed up in average period of years postoperative (range - years), and ( , %) patients achieved a solid union within the first months. conclusions: in conclusion, a reamed intramedullary nail provides optimal conditions for stable fixation, good rotational control, adequate alignment, early weight-bearing and a high union rate of tibial non-unions. percutaneous figure of suture as a novel technique for treating closed tendinous mallet injuries following failed splinting therapy. t. eltantawy , a. yousif , k. maheshwari , a. hartpinto bedford hospital, plastic surgery, bedford, united kingdom introduction: mallet injuries are common injuries affecting the hand. majority of them are managed using conservative method, however a small percentage of patients that do not do well on conservative treatment need an operative intervention. we wish to evaluate the efficacy of percutaneous figure of suture as a new technique for treating closed tendinous mallet injuries resistant to splinting therapy, as a minimally invasive treatment option. material and methods: we present a case series of patients who had persistence of more than degree extensor lag, despite splinting minimally for weeks. all of these were treated with a percutaneous figure of suture placed across the dorsum of dipj, which provided splinting for further weeks. this technique provides fixation for the dipj in hyperextension position by going through the periosteum on both sides and was done under local anaesthesia. results: the mean age of our patients was years, with a single digit involved in all patients. all the five cases had nearly fully straight dipj with less than °extensor lag following weeks of percutaneous stitch placement. there was no further recurrence with mobilisation or overlying skin necrosis. conclusions: percutaneous figure of suturing technique can be an effective, minimally invasive and safe technique to treat closed tendinous mallet injuries not responding well for conservative splinting. introduction: osteosynthesis of pertrochanteric fractures (pf) is a frequently performed procedure in orthopaedic trauma care. dynamization of the osteosynthesis during fracture healing can lead to dynamization of the lag screw. which can cause debilitating complaints. a spontaneous femoral neck fracture (sfnf) after implant removal was seen in patients over a month period. based on these cases we evaluate the different aspects of the pathophysiological and mechanical mechanisms of lag screw dynamization, complaints and complications in pf healing. material and methods: pubmed search on incidence of chronic pain, gait impairment associated with dynamization of osteosynthesis, risk factors for dynamization and complications after implant removal. based on research data preventive recommendations are suggested. results: literature describes complaints as reduced mobility, gait impairment and chronic pain in association with lag screw dynamization. an important risk factor is the ao-classification of pf, a type fractures are significantly associated with more dynamization and the onset of trochanteric pain and gait disturbances. partial implant removal can reduce complaints in the majority of symptomatic patients, and induce symptoms in % of asymptomatic patients. literature study shows a sfnf after lag screw removal with an incidence of %, affecting mostly vulnerable elderly patient resulting in a high mortality rate. risk factors associated with an increased risk of this complication are pre-existing systemic osteoporosis, stress-shielding, pre-loading of the implant. most importantly the removal itself, a sfnf with the implant in situ is very uncommon. conclusions: the clinical indications for implant removal in healed pf are not well established, and should be restricted to specific cases. after removal, partial weight bearing and good patient counselling is extremely important. replacement with shorter lag screw should be considered. metal osteosynthesis of pathological bone fractures with metastatic lesion of plates with a spray on their surface of hydroxyapatite and % silver v. protsenko , a. abudayeh , v. chornyi , y. solonitsyn institute of traumatology and orthopedics of nams of ukraine, onco-orthopedics, kiev, ukraine, bogomolets national medical university, kiev, ukraine introduction: surgical intervention in the case of pathological bone fracture against the background of metastatic lesion involves performing osteosynthesis. for more effective integration of the metal plate with the bone, a material based on bioactive glass was sprayed on their surface. bioactive glass-based material is an osteoinductive and osteoconductive biomaterial that integrates quickly with bone, forms a bone-ceramic complex, and is transformed into bone over time. material and methods: metal osteosynthesis of pathological bone fractures with metastatic lesion of plates with spraying on their surface of hydroxyapatite and % silver was performed in patients. the functional result of the operated limb was calculated on the msts scale. evaluation of pain was performed on the scale of r.g. watkins. the quality of life of patients was evaluated using the eortc qlq-c system. the evaluation of the integration of the plate with the bone was performed by radiological examination and by osteoscintigraphy. results: postoperative complications were found in ( , %) patient, recurrence of metastatic tumor was noted in ( , %) patients. the functional result of the operated limb after metal osteosynthesis was , %. the degree of pain decreased from , % to , %. the quality of life of patients after metal osteosynthesis improved from to points. x-ray examination revealed the formation of callus within a shorter timeframe, as evidenced by the more intense accumulation of radioisotope during osteoscintigraphy. introduction:the aim of this study was to evaluate the results in patients who had heal intertrochanteric fracture but did not receive adequate mobilization and rehabilitation support. material and methods:sixty patients over years old age were included in our study. the rehabilitation emphasized pain relief, muscle strength, range of motion, endurance, balance challenges, and proprioceptive enhancement for all patients. it started postoperative first day and was delivered twice a day by the physical therapist until discharge. patients were discharged on average . days ( - days) after surgery. the mobilization of patients was evaluated with the parker and palmer mobility scoring system, the clinical evaluation was performed with the haris hip scoring and daily living activities were evaluated with the barthel life index before and at the end of the fracture. results: female male patients were included in our study. the mean age was , ( - ) years and the mean follow-up period was , ( - ) months. patients had a type, patients had a type intertrochanteric femur fracture. in the last follow-up, all patients had fracture union. patients' mobility, daily life activity and clinical evaluations were found to be statistically significantly worse in the last control than before surgery. conclusions:the success of the surgical treatment and the union of the fracture after fixation are not sufficient for the successful mobility,daily life activity,and clinical results.the success in the functional results are significantly related with the ambulatory ability.although early mobilization and rehabilitation support are important in intertrochanteric femur fractures after surgery,the continuity of mabilization and rehabilitation support after hospital discharge is more important.the rehabilitation which administered by the patient''s ralations after hospital discharge is not sufficient.therefore,the importance of home-based rehabilitation is increased. the prognostic value of the hip screw position in trochanteric fractures i. gárgyán , î csonka , t. ecseri university of szeged, department of traumatology, szeged, hungary introduction: in our study, we analyzed one of the hungarian population's most frequent injuries, the hip fracture, focusing mainly on the lateral femoral neck and the pertrochanteric fractures. according to the classification of the swiss association for ostheosynthesis (ao), we focused on -a and -a fractures, the incidence of which increases by ageing. material and methods: between and , we analyzed the data of patients. all of the fractures were stabilized with intramedullary nails. patients received stryker gamma Ò , whereas patients' fractures were solved with synthesis pfna Ò nail. in all cases, closed reduction method was used with fluoroscopy on an extension table. the surgeries were done in general or epidural anesthesia and performed by traumatology residents or specialists using standard lateral exploration. data were collected using gepacs software and statistical analysis was done with ms excel. results: cut-out occurred in cases ( , %): out of that ( . %) were left sided and were ( , %) right sided. ( . %) patients were treated with gamma nail, and in ( , %) cases pfna nail was used. the average tad-index was mm. conclusions: according to recommendations of the tad-index value, when using dynamic hip screw, it should be mm or lower. the average index value was mm which was equal in the complicated and non-complicated groups. our study shows that the cutout is independent from the tad-index value, thus this recommendation cannot be applied for intramedullary nails. oita university hospital, acute trauma, emergency, and critical care center, yufu, japan, oita university, orthopaedic surgery, yufu-city, oita, japan introduction: dome impaction fragments (difs) in acetabular fractures are typically accompanied with anterior column fragments and recognized as the gull sign on plain radiographs. meanwhile there are some difs which do not fit into typical difs. the aims of this study were to define atypical dif and describe tips for diagnosis and intraoperative visualization. material and methods: this study was a retrospective case review. we defined atypical difs as the fragments which were independent of anterior column fragments and did not show the gull sign on plain radiographs. from jan to july , there were patients of acetabular fractures, and patients ( . %) had difs. among them, patients ( . %) were identified as the cases with atypical difs. all of them were male. the ages were from to . results: the atypical difs were not obvious on x-rays (fig. ) . all three atypical difs were located at posteromedial weight bearing zones of the acetabulum. case and were displaced in accordance with posterior column fragments, and were visualized clearly on the sagittal view of ct images (fig. ) . case was impacted posteriorly into a posterior part of the ilium as a free fragment, and well visualized on ct sagittal and coronal views. anterior intrapelvic approach was chosen in all patients to treat atypical difs. the iliac oblique view was useful to visualize the atypical difs intraoperatively in case and . in both cases, the reverse gull sign appeared after reduction of posterior column fragments (fig. ) . in case , the inlet view was useful to visualized the atypical dif intraoperatively.the fragments were reduced and fixed with supra-acetabular screws (fig. ) . results: we found prospective two to years after acetabular osteosynthesis , % complications. avn of the femoral head was present in , % of the hips reduced within h and , % of the hips reduced more than h after the injury [p = , ; = , ; or = ( % ci = , - , ) ]. post-traumatic oa of the hip we found in , % (fig. ) infections we found in , % ( deep, superficial), iatrogenic nerve palsy in ( , %), traumatic nerve palsy in , % ( ), dvt in , % ( ) , and ho in , % ( ) cases. in one case ( , %) revision surgery was done. conclusions: acetabular fractures are followed with complications. some complications depend on surgery, meanwhile others cannot be affected on (type of fracture, impaction of acetabulum, injury of the femoral head, dislocation of femoral head). good knowledge of acetabular anatomy, surgical technique, experienced surgical team, early surgery, anatomical reduction and stable orif, early mobilization, can significantly influence excellent/good functional outcomes and reduce possibility for complications. introduction: reduction is one of the important factors in surgical treatment of femoral trochanteric fractures. in this study, postoperative reduction status was examined and the relationship between this reduction status and unsatisfactory cases was investigated. material and methods: cases of femoral trochanteric fractures over years treated with pfna-ii were investigated. postoperative reduction status was evaluated in ap and lateral view of x-ray and ct. anatomical reduction means medial or anterior cortex is reduced anatomically (abbreviation am and aa). intramedullary reduction means medial or anterior cortex of proximal fragment is inside the shaft (im, ia). extramedullary reduction is medial or anterior cortex of proximal fragment is overlapped to cortex of shaft (em, ea). unsatisfactory cases were ununited cases until months and excessive sliding cases over mm. reduction status of these cases was evaluated. results: postoperative status was classified with combination of medial and anterior reduction status. so there are nine groups and number of each group are as follows; im-ia: case, im-aa: cases, im-ea: case, am-ia: cases, am-aa: case, am-ea: cases, em-ia: cases, em-aa: cases, em-ea: cases. non-united cases until months were cases. reduction status of non-united cases were; im-ia: cases, im-ea: cases, am-ia: cases, am-aa: cases, em-ia: cases, em-aa: cases. there was no case in extramedullary reduction of anterior cortex. excessive sliding of blade over mm was cases. there was also no case of extramedullary reduction of anterior cortex in these cases ( cases were cut out). conclusions: our results show there are no ununited cases and excessive sliding cases in extramedullary reduction of anterior cortex. this means extramedullary reduction of anterior cortex is important to reduce unsatisfactory results in surgical treatment of femoral trochanteric fractures. male injured open lateral condyle fracture of femur by to be bitten by a pig. after months from initial debridement, i confirmed the size of bone defect was cm( ) cm in depth. the same size of bone was harvested from iliac crest and transplanted in the bone defect area of lateral condyle of the femur. after months from bone transplantation, i confirmed bone union and two . mm diameter osteochondral grafts and . mm diameter osteochondral graft were transplanted for the chondral defect lesion. case ; seventy year old male injured open lateral condyle fracture of femur by traffic accident. after months from first debridement, i confirmed the bone defect (size cm( ) cm in depth) and the same size of bone was harvested from iliac crest and transplanted in the bone defect area. and simultaneously two mm diameter osteochondral grafts were transplanted for the chondral defect lesion. case ; year old male injured open lateral condyle fracture of femur by traffic accident. i confirmed the size of bone defect was cm( ) cm in depth. the same size of bone was harvested from iliac crest and transplanted in the bone defect area of lateral condyle of the femur. after month from bone transplantation, he had undergone autologous chondrocyte implantation. investigation/results: at last follow-up, average flexion angle of knee was degrees. in all cases, lysholm knee scoring scale was good. diagnosis: large traumatic osteochondral defect of the weightbearing articular surface of the knee comments: treatment of large traumatic osteochondral defect of the weight-bearing articular surface of the knee is a difficult condition to treat. combination of bone transplantation and osteochondral autograft transfer or autologous chondrocyte implantation is useful strategy for the injury. references: tegner y., lysholm j., clin orthop relat res., , - , pr treatment of double tension band wiring method with ai wiring system for transcondylar distal humeral fractures m. uchino hakujikai memorial general hospital, orthopaedic surgery, tokyo, japan introduction: as ai wiring system is united the pin with the cable due to compressed sleeve, the pin is never deviated. we review the treatment of transcondylar distal humeral fractures with ai wiring system in geriatric patients. patients and methods: were identified as receiving this surgery. all patients were female and their mean age was years. they were assessed union rate, range of motion for elbow joint, postoperative complication and functional outcome for japanese orthopedic score. results: union rate was %. the mean arch of motion was °at latest follow-up. the complications were detected cases which were temporary ulnar palsy for cases and hardware failure for case. the average of functional outcome was points ( / ). conclusion: tension band wiring of transcondylar distal humeral fractures with ai wiring system provides stable fixation for osteoporotic bone and tiny fragment. introduction: the purpose of this study was a comparative evaluation of the complications related to the treatment of trochanteric fractures using -screw proximal femoral nail (pfn) versus proximal femoral anti-rotational blade nail (pfna). material and methods: a retrospective review was conducted between march and march . the study included patients treated surgically for trochanteric fractures. the mean age was , ± , ( - ) years. patients were treated by pfn ( patients, , %) or by pfna ( patients, , %). implant related complications were the primary objectives. infection and revision surgery were also recorded. results: complications were observed in ( . %) patients in pfn group and ( , %) patients in pfna group (p = . ). screw backout (n = ) and cut-out (n = ) occurred in , % patients treated with pfn. in the pfna group, cut-out occurred in , % (n = ) of cases. infection (n = ) represented , % in pfna patients and , % (n = ) in pfn group. there were no statistically significant differences in both groups considering implant-related complications (p = , ) and infections (p = . ). revision surgery was performed in ( , %) patients. soft tissue problems are more likely in fractures due to high energy impact than low energy type fractures. high energy type present with horizontal fractures of tibia and fibula (i.e. on the same level), whereas in low energy type tibia fractures they present with spiral or oblique fracture patterns often associated with concomitant fractures of the posterior rim of the distal tibia (i.e. volkmann's triangle). posterior malleolus fractures occur regularly but are often missed and seen only on ct scans obtained either for preoperative planning or to verify postoperative rotation. in literature these mostly undisplaced fractures are treated with screw fixation mostly from anterior. but is this really necessary? material and methods: we retrospectively analysed consecutive tibia shaft fractures operatively treated over the past years at our regional hospital analysing the fracture pattern. results: out of patients with tibia shaft fractures patients presented with a posterior rim fracture of the tibia. no routine stabilisation of the volkmann fragment was performed, in all cases the posterior rim fragments healed uneventful. angles of °and above seem to present themselves with a concomitant fracture of the posterior malleolus. they are mostly undisplaced and the trauma mechanisms is low energy and torsion. none out of the patients had known osteoporosis. conclusions: low energy and torsion-type tibia fractures with an angle of [ °seem to have an accompanying undisplaced fracture of the posterior malleolus. these fractures are usually undisplaced and do not need to be addressed. as a consequence there seems to be no need to actively rule them out with ct scans prior to surgery. concomitant ankle fractures including posterior rim fractures should be addressed like isolated ankle fractures. the dangers of bouncing: a prospecive cohort study of injuries associated with trampolines and bouncy castles over a month period in a paediatric population. introduction: within the orthopaedic paediatric population, there is an increasing incidence of presentation of fractures associated with both trampolines & bouncy castles. whilst this phenomenon has been depicted frequently within the media in recent years given the dramatic upsurge in trampoline and bouncy castle usage, there have been few studies documenting either the incidence of fractures associated with either. materials and methods: this was a prospective cohort study conducted within our institution over a month period june to august inclusive . all paediatric patients who sustain a fracture and present to the national childrens'' hospital are referred to the orthopaedic department either whilst as an inpatient or as an outpatient depending on the assessment of the severity of injury. a standardised mixed questionnaire was given to all parents''/guardians which recorded the type of injury, type of trampoline/bouncy castle, inherent awareness of safety precautions governing the usage of either and application of same was recorded. the type of fracture was corroborated via examination of x-ray in addition to the recording of any complications via examination of clinical chart records. results: there were patients who sustained a fracture directly related to the usage of either a trampoline or bouncy castle for which the majority required operative intervention. there was wide variability in the nature of injuries recorded; supracondylar/radial fractures were the most common whilst more complex injuries such as an open fracture of the femur was rarer. conclusions: awareness and application of necessary safety precautions was low ( %) amongst parents'' supervising parents''/guardians highlighting the need for greater public awareness of same. furthermore, the incidence of severe injury relating to usage of trampolines/bouncy castles is not uncommon highlighting the high risk activity that trampolining is. introduction: conventional plate fixation (pf) of distal fibular fractures in elderly patients is associated with a high risk of wound and implant related complications. intramedullary fixation (imf) using a fibular nail is a minimally invasive alternative to pf that provides superior biomechanical strength and allows immediate full weight-bearing postoperatively. aim: to compare the postoperative complications of minimally invasive intramedullary nail fixation to conventional pf for lauge-hansen supination external rotation type fractures in patients aged years or older treated in a single geriatric trauma unit in the netherlands. methods: a retrospective cohort study was performed including unstable ankle fractures in patients aged years or older treated with either imf or pf between january to january . the primary outcome measure was the total number of wound related complications. results: a total number of patients were included with a mean age of . years (range to ). the imf-cohort (n = ) had a significantly higher mean age ( . versus . years, p = . ) and charlson co-morbidity index ( . versus . , p = . ) compared to the pf-cohort (n = ). the total number of postoperative complications was lower after imf ( %) compared to pf ( %), although this relative difference was not statistically significant (p = . ). all complications observed in the imf-cohort were wound related but demanded no debridement or implant removal. wound related complications did not differ significantly from pf ( % versus %, p = . ). no implant related complications, hospital-acquired complications or mortality were observed after imf. conclusion: despite the higher mean age and co-morbidity status of patients treated with a minimally invasive intramedullary nail, the total number of postoperative complications was lower after imf compared to pf. this technique might be a promising alternative in a selected group of patients. the authors declare that they have no commercial associations that might pose a conflict of interest. no funding or other compensation was received for the research, authorship or publication of this article. gustilo type ii and gustilo type iii fractures. the treatment protocol was external fixation at admission and definitive osteosynthesis with plate at ± days. a single approach to the tibia was performed in patients, and a combined anterior and posterior approach was used in . the incidence of complications was %: cases of poor soft tissue evolution, of which were infections. patients evolved to nonunion. osteoarthritis appeared in % of patients ( . % grade ), and only one patient needed arthrodesis. . % had a valgus ldta (\ °) and . % a varus deformity ([ °). we found a significant relationship between the history of open fracture and the development of complications (p \ . ). we found no relationship between the incidence of complications and the approach. conclusions: tibia ao c fractures have a high percentage of complications and evolve to well-tolerated osteoarthritis. open fracture seems to significantly influence the poor postoperative outcomes of these patients. clinical findings: a -year-old male, who suffers a closed chest trauma with pneumothorax, right pulmonary contusion and poor pneumoperitoneum. also a grade iiia open fracture of the right femur, with a cm bone defect. investigation/results: upon arrival at the hospital, he needs orotracheal intubation, as well as blood transfusion with red blood cell concentrates. external fixator is placed on the right femur. diagnosis: a iiia grade diaphyseal open fracture of the right femur with cm bone defect, bearing external fixator with one broken proximal pin and positive culture for s maltophila in the distal pin. therapy and progressions: antibiotic treatment and medical optimization are performed, cemented intramedullary nailing (t -stryker) with antibiotic (vancomycin-tobramycin), as well as cement spacer with antibiotic (masquelet's first stage) in the defect area. in second time, withdrawal of spacer and contribution of ria autograft of contralateral femur and allograft respecting membrane. the patient begins the protected weight bearing with two crutches immediately, without using them months after the surgery. bone consolidation without pain or limitation after year. comments: the induced membrane technique is a simple and effective technique for the reconstruction of segmental bone defects and can be used as a first time technique together with the initial stabilization, leaving the defect ready for graft delivery in the second time. introduction: carpal metacarpal dislocation is a rare entity that accounts for less than % of all carpal injuries. dorsal dislocations are the most common and occur most frequently after violent trauma in young individuals and are easily overlooked and may lead to longterm sequelae. material and methods: we present the case of a carpal metacarpal dislocation from d to d . male, years old, no relevant personal history. brought to the emergency service after a motorcycle accident with projection. he had a symphysis pubis diastasis, a distal radius fracture on the right wrist and a fracture of the left forearm bones. no other apparent injuries associated. at week , he presented edema and dorsal deformity of the left hand associated with limited finger movements. neurovascular assessment was normal. the radiological evaluation showed a carpal metacarpal dislocation from m to m . it was an unstable reduction so open reduction was performed, with debridement of fibrous material, until exposure of the articular surfaces, and reduction and fixation with k wires of the three metacarpals (from d to d ). similarly, m was stabilized with a k-wire due to clinical instability observed intraoperatively. results: it is necessary to reduce and stabilize these lesions to avoid vasculonervous compression and skin distress. open reduction is indicated in irreducible cases allowing debridement and excision or os of small osteochondral fragments and fixation of associated fractures. conclusions: combined dislocation of multiple metacarpals is a rare lesion that compromises the functional prognosis of the hand in the absence of adequate treatment. instability and post traumatic arthrosis are among the sequelae of this lesion. identify the lesion to allow the appropriate treatment usually leads to good results. case history: -year-old suffered direct trauma to his right hand after falling off his bicycle. clinical findings: on physical examination showed edema and bruising from the base of the thumb and thenar eminence, tenderness over the cmc joint and functional disability speacialy in pincer grasp. no neurovascular injuries investigation/results: the x-ray revealed a comminuted fracture of the base of the thumb metacarpal. diagnosis: we identifed a rolando fracture. therapy and progressions: on the day after the trauma, he was submited to open reduction and osteosinthesis with lateral-palmar plate and screws, through radiopalmar aproach of the thumb base. intra operatively no dorsal fragments werefound to be left undisplaced. two months after surgery, the patient went back to the hospital for sudden pain and inability to extend the thumb. clinically with rupture of the long extensor of the thumb. on the x-ray, the fracture was aligned. the latero-lateral tenorrhaphy with kessler suture was preformed and intraoperatively a bony spicule was identified in the proximal stump of the tendon, which was removed. months after the initial trauma, the patient has a consolidated neck and no limitation of the mobility of the thumb. comments: rollando fracture is relatively rare in adolescents. the aim of treatment should be exact reduction usually with open technics. the main complications are stifness and early arthrosis. there are also records of conflicts with the plates and even rupture of the extensor tendon, so the radiopalmar placement of the plate was chosen. nevertheless, the rupture occurred due to conflict with an unidentified bone fragment during surgery causing an unexpected complication in this case. the immobilization necessary after tenorrhaphy could have caused joint stiffness, but in this case the teenager fully recovered after physical therapy case history: periprosthetic and periimplant femoral fractures are an increasingly frequent pathology. in many cases they are a challenge with limited or too aggressive therapeutic options. it is important to investigate new approaches that increase the arsenal of the orthopedic surgeon. the recently described mipo (minimally invasive plate osteosynthesis) approach for the medial aspect of the femur may seem like a dangerous procedure because of the anatomical structures that run along the medial aspect of the thigh, but it is a viable and useful option in selected cases. clinical findings: we present the case of a -year-old patient with a total hip replacement who presented a first periprosthetic vancouver b fracture of the femur that was treated with a lateral blocked plate. subsequently the patient presented a second supracondylar femur fracture below the first plate (vancouver c). investigation/results: after thinking over the possible therapeutic options, we decided to treat our patient by means of the medial femoral mipo approach with a long medially placed blocked plate, managing to stabilize the fracture and superimpose the plate on the previous implants without the necessity of removing the previous lateral plate. diagnosis: periprosthetic and periimplant supracondylar left femoral fracture. therapy and progressions: we used the surgical technique of the medial femoral mipo approach as described by apivatthakakul . comments: we consider that the medial femoral mipo approach is a useful therapeutic tool to consider. it seems a safe and low-invasive option for the resolution of cases in which the lateral mipo approach is not a feasible option. references: c. jiamton y t. apivatthakakul, « the safety and feasibility of minimally invasive plate osteosynthesis (mipo) on the medial side of the femur: a cadaveric injection study » , injury, vol. , n.o , pp. » , injury, vol. , n.o , pp. - » , injury, vol. , n.o , pp. , nov. . posterior knee dislocation with neurovascular injury associated-a case report case history, investigation and diagnosis: a -year-old male was brought in after h following a heavy straw bale fall. he presented with a posterior knee dislocation that had already been reduced and an open wound in the popliteal fossa. the limb was flushed and pale on the extremity, with absence of the pedis and posterior tibial pulses. stability tests revealed unstable knee in all axes. an anterior shoulder dislocation was diagnosed and reduced. therapy and progressions: an emergent surgery was performed, involving a transarticular external knee fixation and a femoro-popliteal bypass above the knee (angiogram revealed a stop sign at the level of the interarticular popliteal artery). he developed circulatory shock and was admitted to the intensive care unit. on the stpostoperative day(po) was diagnosed a compartment syndrome that was treated with fasciotomies. these incisions showed a slow but progressive evolution, that required vacuum dressings and underwent autologous skin graft on the thpo day. the external fixator was removed on the stpo day and rehabilitation was started. on a -month follow-up, the patient had a good evolution of the wounds, but a knee with valgus and anteroposterior laxity and severe complete peroneal, tibial and sural neurological injury, confirmed with electromyography, and neuropathic pain. introduction: isolated iliac wing fractures represent only a small part of all pelvic fractures. these fractures are associated with severe injuries, but are considered benign. the literature lack information about the function and quality of life of these patients. our objective was to evaluate the long-term effects of isolated iliac wing fractures. material and methods: patients with pelvic fractures treated at oslo university hospital, ullevaal, in the time period - , were extracted from the local fracture registry. patients were registered in this period. a search was also made in the hospital''s administrative electronic database for patients registered with diagnose code s . in icd- in the same period. patients were identified. in total, patients had an isolated iliac wing fracture, and these were invited to a follow-up examination, including proms (eq- d- l and majeed score), clinical examination, and pelvic x-ray. results: nine patients agreed to participate in the study, median years after the fracture (range - ). all of them were injured from high energy trauma, with mean niss , (range - ) . four of the fractures were open, and seven of the patients had associated injuries. five were treated with internal fixation. the mean eq- d vas was (range - ). five patients reported pain, one of them related to the pelvic fracture. the mean majeed score was (range - ). seven patients had sensory deficit in the lateral thigh. one patient had difference in range of motion between the two hips. the x-rays showed healed fractures in all the patients. eight of them showed ectopic ossification. conclusions: our study confirms previous studies that isolated iliac wing fractures are results of high energy trauma with severe associated injuries. however, the majority of this group of patients seem to have a good general state of health, which is in accordance with the general assumption of the injury as a benign one. fenton's syndrome-a case report of a common underdiagnosed entity case history: a right handed -year-old male, construction worker, was admitted in our emergency department, after a meters fall. the authors report a case of fenton's syndrome in a politrauma scenarium. clinical findings: both right elbow and left wrist were painful, swollen and with a remarkable restriction of the range of motion (rom). patient also reported lower back pain. no neurovascular injuries were detected. investigation/results: x-ray and ct scan confirmed a fracture of a lumbar vertebra, fracture of the right olecranon and, on is left wrist, a carpal fracture-luxation mayfield of both scaphoid and capitate associated with rotation of the last one proximal pole-fenton''s syndrome. diagnosis: this syndrome is an atypical presentation of perilunate fracture dislocation and, therefore, difficult to diagnose. few reports were found in literature. after an open reduction of the fractures, a definitive fixation with headless herbert screws was achieved. percutaneous kw and immobilization of the wrist were performed to further stabilization of the lunotriquetral joint. weeks later consolidation was noted. a decrease of °in extension and flexion were detected when compared with the contralateral wrist. grip strength test was similar on both hands. osteosynthesis of the right olecranon was also realized. comments: a careful neurovascular assessment is important. although it is rare, injuries of median nerve were already reported associated to this complex fractures. open reduction and osteosynthesis are necessary due to the great instability and the risk of nonunion and osteonecrosis of the rotated proximal segment. introduction: intramedullary nailing has been popularly applied for the femoral shaft fractures. the current study aimed to analyze the femur geometry for development of implant design with dimensional skeletonization. material and methods: we acquired computed tomography (ct) images of both femur reviewed in a single center from to . the total participants were enrolled and they were divided into subgroups according to age (decades) and gender. each subgroup included persons, respectively. these images are used to produce d samplings. with the skeletonization, we obtained the geometry parameter; ( ) femur shaft length from the tip of the greater trochanter to the bicondylar line, ( ) the minimum diameter of the medullary canal and its location, ( ) anteroposterior (ap) diameter and lateral diameter of the entire femur, ( ) radius of curvature (roc) of the femur (bowing). we compared all parameters according to sex and age. results: the average age of the participants were . years (range - years) and the number of each gender was exactly same. the femur length was . ± . mm (range, . - . mm) and the femur shaft length was . ± . mm (range . - . mm), both of them were longer in male (p = . , \ . ). the minimum diameter of the medullary canal was . ± . mm (range . - . mm). the roc was . ± . mm (range . - . mm) . the rate of the minimum diameter less than mm and mm was . % and . %, respectively. the rate of roc with less than mm and mm was . % and . %, respectively. conclusions: this geometry analysis showed that there are mismatch problem between the current nail and the medullary canal in . % and the roc of the femur was smaller than that of the current nail systems ( - mm). the result indicates potential mismatch problem in clinical cases and the problem can be resolved with newly designed nail system. the study was funded by national reserach foundation of korea (nrf- r d a b ). safe zone of the infracacetabular screw: virtual mapping of three-dimensional hemipelvises for quantitative anatomic analysis introduction: an infra-acetabular screw can provide increased stability in fixating acetabular fracture. we conducted this study to define the incidence of the safe corridor for infra-acetabular screw and to determine the correlation between the safe corridor and other demographic factors such as age, sex and height. material & methods: pelvis computed tomography (ct) of participants was extracted with evenly age-and sex-allotted. virtual three-dimensional ( d) model was generated. a search was performed to find the maximum-with corridor connecting two points. the entry and exit point was displaced in the template. the maximum diameter of each corridor was measured in automatic procedure. a minimum mm corridor diameter, sate corridor, was defined as a cutoff for placing a . mm cortical screw in clinical setting. all data were presented as mean and range or mean and standard deviation. two-sample t test and regression analysis were used to compare difference between groups based on sex, age, and height. results: among hemipelvis, hemipelves ( . %) satisfied a minimum safe corridor diameter of mm. when divided into a subgroup by the patient's gender, the incidence of the safe corridor of a male group was statistically higher than a female group ( . % vs . %), with the mean corridor diameter of . mm ( % ci, . ) and . mm ( % ci, . ), respectively (p \ . ). in correlation analysis, only the height showed a positive correlation with the diameter of the safe corridor of a total population (r = . ; p \ . ). conclusions: the study provided the safe corridor was found in % of male and % of female, and the taller had the higher incidence of the safe corridor. the patient''s height was correlated with the corridor diameter of the infra-acetabular screw, whereas the patient''s age did not correlate with the corridor diameter. introduction: femoral neck fractures in middle-aged and older patients represent one of the most common orthopedic conditions. osteosynthesis, as a primary treatment option for femoral neck fractures has shown to have successful outcomes. however, this is not the case for old fractures. the purpose of this study was to evaluate the outcomes of treatment of femoral neck fractures in which cementless total hip arthroplasty was indicated. the aim of our study was to analyze the prosthetic failure, i.e., the reasons for unsuccessful outcome, in order to suggest the indications for primary osteosynthesis which could guide the femoral neck fracture management. material and methods: a total of patients were analyzed in this study, with femoral neck fracture treated with osteosynthesis. reviewing the radiological findings, as well as the course of the treatment, we set up the criteria, on the basis of which we could advice the immediate implantation of total hip prosthesis for the femoral neck fracture. results: old fractures, varus deformity of the femoral head and neck, dislocation, as well as the comminuted fractures, are all factors affecting the surgical outcomes of osteosynthesis. additionally, medical and technical equipment of medical institution, personnel competence, and minutious surgical technique affect the treatment outcomes. introduction:proximal ulnar fractures are usually osteosynthesized by means of angle stable plate osteosynthesis. despite good functional results of this procedure, complications such as high access morbidity and disruptive osteosynthesis material with a high rate of material removal are described. the aim of our study was the development of a new locking nail and test setup for comparison with a plate osteosynthesis on artificial bones. material and methods: in our biomechanical laboratory, a jupiter b fracture of the proximal ulna was standardized on sawbones and stabilized by means of the newly developed nail or anglestable posterior plate osteosynthesis. a servopneumatic testing machine, the specimens were flexed under a cyclic load ( - n) in the physiological range of movement of the elbow from °to °.the maximum elastic deformation of the specimens and the loosening of the implants were evaluated after test cycles. results: the primary stability of the constructs at the anterior cortical bone after nail osteosynthesis was significantly greater ( . ± . mm) than in the angle-stable plate osteosynthesis ( . ± . mm, p \ . ).after passing through the test cycles, both implants showed a low loosening rate. in the area of the anterior cortex, the locking nail showed a significantly lower rate of loosening (nail . ± . mm, plate . ± . mm, p \ . ). at the dorsal cortex, there were no differences between plate and nail in both series of measurements. conclusions: intramedullary implants provide biomechanical benefits in fracture stabilization. good biomechanical results have already been shown in the literature after nailing olecranon fractures . nevertheless, due to the complex anatomy and the resulting difficult implantation technique, ulnar nails could not prevail in practice. the presented nail allows a safe stability with simple surgical technique. introduction: adequate treatment of tibial plateau fractures is crucial to minimize patient disability, development of posttraumatic arthritis and subsequent need for a total knee arthroplasty (tka). however, due to the complexity of the fracture, adequate reduction cannot always be achieved which could result in the early conversion to a tka. in this study we introduce a quantitative d fracture assessment method and investigate whether it could help to identify patients that are at risk of conversion to a tka. material and methods: we retrospectively included patients, who were treated for a tibial plateau fracture between and . patients developed severe posttraumatic arthritis and underwent conversion to a tka. from all patients, d models were created using the pre-operative ct-scans. for each patient, the d gap area between the fracture lines, representing an innovative combined gap and step-off measurement in d, was determined in order to quantify the displacement (figure ). roc curve analysis was performed to determine a critical cut-off value for the d gap area. kaplan-meier survival curves were created to assess the association between d fracture anatomy and risk on a tka at follow up. results: a critical cut-off value of mm was found to give highest combined sensitivity and specificity for d gap area and the risk of tka at follow-up. kaplan-meier survival curves showed . % knee survival (no tka) at year follow up in the group with a gap area of \ mm , whereas in the group with a gap area of c mm a knee survival of . % was found. at year follow up knee survival was . % and . %, respectively, for the two groups (\ mm and c mm ). conclusions: we developed an innovative method to quantify the amount of displacement in d. pre-operative d fracture assessment could be used as an addition to the current fracture classification methods to help identify patients who have a high risk on conversion to tka at follow-up. introduction: soft tissue sarcomas (sts) in the anterior compartment of the thigh are frequent. the extent of quadriceps resection is controversial. the aim of the present study is to communicate our results in complete quadricectomies due to high-grade sts. material and methods: we present sts, in stage iiib of the ajcc, with a mean craniocaudal diameter of cm ( - ). there were women and men, with a mean age of years ( - ). six were undifferentiated pleomorphic sarcomas, myxofibrosarcoma and clear cell sarcoma. in every case, total quadricectomy was performed with wide margins. posterior reconstruction with local muscle transfers was performed, expect for the younger patient, who received a vascularized contralateral vastus lateralis transplant. in all cases, complementary radiotherapy was indicated, and in patients adjuvant chemotherapy. results: three patients required friedrich due to necrosis of the edges of the surgical wound. one patient died months after the intervention as a result of multiple metastasis, and two due to medical complications after week and months, respectively. the average follow-up time for the rest was months , with no local recurrence. as for functional outcomes, mean msts score was ( - ), with deficit of active knee extension in most of them. the functional result of the patient with the vascularized muscle transplantation was excellent. all of them were satisfied with the results of the treatment. conclusions: quadricectomy provides good functional and acceptable cancer results, although it is not exempt from complications in frail patients. vascularized muscle transplantation, though complex, can improve functional results, especially in younger patients. introduction: operative treatment is a valuable option in displaced proximal and/or middle one-third diaphyseal humeral fractures. although plate osteosynthesis is preferred to intramedullary nailing, surgery can be complicated by radial nerve palsy. a helical plate could avoid this high-impact complication. to date there is however a lack of published evidence in literature, although recent asian case reports show promising results. material and methods: we retrospectively reviewed patients who were treated with open reduction and internal fixation with a helical plate consecutively from october until august at az groeninge, kortrijk. a deltopectoral approach was used in combination with a distal anterolateral incision, whether or not in continuity. a self-molded long philos plate was used in the first patients, while in our last patients the a.l.p.s plate (zimmer Ò ) was used. standard radiographs were obtained pre-and postoperatively. we retrospectively searched for complications, e.g. radial nerve palsy, infection and/or loosening. in autumn , patients were reassessed. patient''s general health status was evaluated using the eq- d- l score. constant-murley scores and dash scores were used for evaluating shoulder function and disability measures consecutively. results: all humeral fractures consolidated at months. there were no radial nerve palsies due to surgery. one plate was removed after year due to a late infection. with a minimum follow up of year, the mean dash score was ( - ) and the mean constant-murley score was ( - ). the dash score was inversely proportional with the constant-murley score and patient''s general health status. conclusion: a helical plate avoids neurological complications with similar healing rates and good to excellent shoulder function at year follow up in the treatment for proximal and/or middle one-third diaphyseal humeral fractures. the use of antibiotic-impregnated cancellous bone grafts in onestage surgery for chronic orthopaedic infection: preliminary clinical results k. dendoncker , g. putzeys , az groeninge, tissue bank, kortrijk, belgium, az groeninge, orthopaedic center, kortrijk, belgium introduction: the use of cancellous bone allografts is an established technique in reconstructive orthopaedic surgery. unfortunately, its use is generally avoided in the presence of a local infection. antibiotic impregnated cancellous bone grafts has shown its effectiveness as an local antibiotic delivery system [ ] [ ] [ ] . in this clinical study, we report our first personal experience with the use of vancomycin-impregnated cancellous bone grafts in one-stage surgery for periprosthetic joint infections (pji) and fracture-related infections (fri). material and methods: between december and march nine patients were treated during a one-stage surgery with vancomycinimpregnated cancellous bone grafts, containing g vancomycin per cc bone. regular clinical, laboratory and radiographic follow-ups were performed for at least months after surgery. results: the procedures included revision of pjis (hip and humerus) and fris (tibia, femur and clavicula). one tibia required further revision because of recurrent infection and one hip has an uncertain infection state, however the remaining patients stayed free from infection during a follow-up of at least months. interestingly, in one patient the vancomycin concentration could be determined in the drainage fluid from the wound. radiographic examination revealed no signs of osteolysis or loosening, good incorporation of the bone graft and progressive consolidation. conclusions: within the limits of the study, the use of vancomycinimpregnated cancellous bone grafts in one-stage surgery to treat pji and fri yielded positive outcomes in terms of clinical, laboratory and radiographic follow-up. this technique might offer new treatment strategies in often devastating injuries. references: . putzeys g., et al. orthopaedic proceedings. ; -b:supp_ , - . with the modified arthroscopic approach (group b). the prospective follow-up included the lysholm score, the subjective questionnaire of the ikdc score and the specifically extended oak score for clinical evaluation. the rolimeter Ò was used to test the translational mobility of the knee joint. the statistical significance level was set at %. results: the follow-up was . ± . months and . ± . months postoperatively in group a and b, respectively. the subjective scores were tested. group a and b achieved a mean lysholm score of . ± . and . ± . points respectively. in the subjective ikdc assessment, group a achieved . ± . points and group b . ± . points. the clinical oak score was . ± . points in group a and . ± . points in group b. the following values could be recorded for the stability of the posterior cruciate ligament: the side difference in the rear drawer test was . ± , mm in group a and . ± . mm in group b. in the reversed lachman test, a difference of . ± . mm and . ± . mm was measured in group a and b, respectively. all values mentioned were comparable between the two evaluated groups. conclusions: the results of the two surgical techniques were comparable. therefore the arthroscopic approach is the preferred method in our institute. simple correction technique of femoral malrotation after pfn-a osteosynthesis of trochanteric fracture k. pavotbawan , p. stillhard , c. sommer kantonsspital graubünden, department of trauma surgery, chur, switzerland introduction: malrotation after intramedullary nailing in femoral shaft fractures are well known. but malrotation after nailing of trochanteric fractures is an underestimated problem. during surgery the axial alignment can easily be evaluated by fluoroscopy in both planes. but the torsional alignment is difficult to assess especially with the patient placed on the traction table. in literature a malrotation after pfna is described in up to % of the cases. a revision with replacement of the blade, especially in patients with poor bone quality, may result in a reduced stability. to our knowledge there is no publication till to date to give a treatment pathway for this problem. we developed a rather easy technique to derotate a malrotated femur after pfna fixation. material and methods: the basic idea is to leave the usually well placed blade insitu in the femoral head, just rotating the distal main fragment around the nail. therefore, a small u-shaped osteotomy with a chisel is performed in the femoral cortex just anterior of the entry site of the blade. the length (l) of this osteotomy can be calculated, following the formula: l = d x p x a/ (d = diameter of femur, a = angle of malrotation). then the distal locking bolt is removed, the leg derotated and finally locked again. the procedure is controlled by two schanz''screws separately inserted in both main fragments angulated to each other in the angle ''a''. results: since patients were detected with a clinically relevant femoral malrotation. all patients had an internal malrotation from to degrees confirmed and measured by ct scan. all of them were successfully revised in the above described technique - days after initial fixation. conclusions: first, we believe that malrotation after trochanteric fracture fixation is an underestimated problem. and second our method is a simple salvage procedure for malrotated trochanteric fractures after pfna, leaving the blade in situ in the femoral head. optimal intramedullary nailing for trochanteric fractures: the importance of distal locking screw and reduction position t. waki , t. yano , k. ito , s. matsushima akashi medical center, orthopaedic surgery, akashi, japan introduction: distal locking issue for trochanteric fractures is still controversial. therefore, the purpose of this study was to investigate the complications between distal unlocked group and distal locked group. further, the relationships were evaluated between these complications rates and their reduction positions after operation. material and methods: operations were performed for trochanteric fracture (ao a ?a ) from to . of these, patients with f/u periods [ month were . gamma im nailing system (stryker) was used for all patients. patients (unlocked group) from to operated without distal locking screw. patients (locked group) from to operated with distal locking screw. we retrospectively analyzed those patients who suffered complications such as delayed healing and postoperative periimplant fractures and cut-out of the lag screw. further, in lateral view of their radiographs, we evaluated the position of the proximal fragment compared with distal fragment. the reduction positions were divided into groups: anterior (subtype-a), neutral (subtype-n), and posterior (subtype-p). results: in unlocked group, complication was shown in patients (complication group). delayed healing was shown in / ( . %) in unlocked group and / ( . %) in locked group. peri-implant fracture was shown in / ( . %) in unlocked group and / ( %) in locked group. cut-out of the lag screw was shown in / ( . %) in unlocked group and / ( . %) in locked group. in complication group, subtype-p was more than non-complication group. conclusion: in the current study, higher number of complications was seen in the distal unlocked group. and, our study showed the reduction position might be associated with post-operative complications. we concluded that nailing without distal locking screw might be dangerous and subtype-p should be avoided. introduction: heterotopic ossification (ho) after acetabular fracture surgery has been one of the common complications and often limits function with the range of motion severely. surgical resection is challenging and only effective treatment for established ho. we herein report four cases who underwent surgical resection and mobilization for ho after acetabular fractures surgery. material and methods: four cases with severe ho after acetabular fracture surgery were included in this study. the mean age at operation was years old, and all patients were males. in judet-letournel classification, there were three cases classified as posterior wall fracture, and one case as transverse and posterior wall fracture. two of four cases were combined with posterior dislocation of the hip. in all cases, the first operation was performed using with the kocher-langenbeck (kl) approach. results: surgical resection of ho was performed using with the kl approach at . months (range - months) after the first operation. the median operating time and intraoperative bleeding were respectively . h and ml. intraoperative d navigation was used in one case. as postoperative complications, one case developed sciatic nerve palsy and another case sustained the iatrogenic femoral neck fracture. all cases have no recurrence with a follow-up of . years after the surgical resection. conclusions: surgical resection is the only treatment for symptomatic ho. but that requires preoperative planning and must be performed carefully because the extent of resection is still controversial and that may develop severe complications such as nerve palsy and iatrogenic fractures. by using navigation, we can determine the extent of resection easily and operated safely. case history: -year-old male, previously healthy, turned to the hospital after a motorbike crash, resulting in high energy direct trauma of the right wrist. clinical findings: upon admission, cranial, thoracic, abdominal and other traumatic injuries were excluded. the patient presented with pain, swelling and visible deformity of the right wrist and hand, hypoesthesia of the th finger, and no perfusion deficits. investigation/results: x-rays showed volar perilunate carpal dislocation with associated comminuted scaphoid fracture, radial styloid avulsion, and metacarpal phalangeal dislocation of the th digit. under sedation, closed reduction of the metacarpal phalangeal joint was accomplished, and reduction of the carpal dislocation was attempted unsuccessfully. the wrist was temporarily immobilized in a cast and taken to the or. diagnosis: transcaphoid-transradial-styloid-perilunate volar dislocation therapy and progressions: surgical treatment comprised loose bodies removal, reduction of the perilunate dislocation, orif of the scaphoid using a herbert screw, and stabilization of the carpal rows using two percutaneous kirschner wires. after surgery, a thumb spica cast was applied. post-operatively, neurovascular status was normal. at weeks, x-rays showed signs of bone healing, the cast and k wires were removed, and physical therapy was initiated. at months, scaphoid fracture consolidation was achieved. the patient remained with a mild deficit in wrist extension but reported no pain nor important limitation in daily living activities. comments: perilunate injuries with displacement or dislocation usually require surgery. persistent instability is a described complication, often progressing to secondary post-traumatic arthritis of the wrist and carpus, termed scapholunate advanced collapse. introduction: this study was conducted to study the patient characteristics, classification, treatment, complications and functional outcome of operatively treated displaced intra-articular calcaneal fractures (diacf) in a level trauma center in the netherlands material and methods: patients with an diacf, classified as sanders c and operatively treated with percutaneous screw fixation (psf) or open reduction and internal fixation (orif) between january and december were identified. pre-and postoperative radiological assessment was performed. functional outcome, range of motion and change in footwear were evaluated with the use of the american orthopaedic foot & ankle society (aofas) score and the maryland footscore. general health and patient satisfaction was assessed using the short form- (sf- ) and the visual analogue scale results: in total, patients with an operatively treated diacf were identified. patient with diacf completed the questionnaires. there were males and females, mean age at trauma was years. average follow up was years. were classified as sanders type , and as respectively type and . were joint depression and were tongue-type fractures. there were no differences in sanders classification between the group treated with orif and psf. for orif and psf there were ( - %), ( - %) and ( - %) for respectively sanders type , and fractures. mean aofas, mfs, sf- and vas was ( - ), ( - ), ( - ) and ( - ) for respectively orif and psf. mean pre-and post-bohler angle was ( - ) and ( - ) for respectively psf and orif. underwent an ankle arthrodesis. surgical site infection and deep infection occurred in ( , - %) and ( , - %) in respectively psf and orif conclusions: long-term comparison shows no significant differences between orif and psf in treatment of sanders fracture type, bohler angle reduction, on functional outcome or complication rates introduction: the prevalence of hand injury in the pediatric population is attributed to their curiosity, limited fear of pain and diminuted motor coordination. the seymour fracture, which was first reported by seymour in , represents a transverse extra-articular open fracture of the distal phalanges associated with nail bed injuries. the fracture includes salter-harris type i and ii fractures as well as juxta-epiphyseal injuries. material and methods: the aim of this report is to present a case of a seymour fracture in a young boy and describe the injury mechanism associated with misuse of the newly emerging vehicle, the hoverboard. results: our patient was treated promptly and provided with appropriate management following the standard of care in our hospital for such injuries: disimpaction and repair of the nail bed, reduction of the fracture, and k-wire fixation across the distal interphalangeal joint. the patient was discharged with a volar slab and was prescribed an oral antibiotic. the patient recovered well with no major deficits. conclusions: the timely recognition and management of seymour fractures is crucial. the surgical treatment has good results however, conservative management can be an option in some specific cases. antibiotics are always required. we report a case of a fracture pattern resulting from the improper use of an hoverboard. although improper use was a factor, design fault also plays a role in causing the injury. hoverboards are a new transport technology that has been introduced in recent years. because of the number of injuries that have resulted from hoverboards, they should be used in the most controlled way possible to prevent any unnecessary injuries. case history: we report the case of a years old male from bangladesh, with months of progressively increasing pain, limited range of motion and swelling on his left knee, with kg of weight loss and inguinal lymph nodes. clinical findings: knee radiography and mri of the knee demonstrated a voluminous soft tissue mass surrounding the distal femur with intraarticular and posterior extension. a toracic-abdominal-pelvic ct showed supra and infradiaphragmatic lymph nodes. c-reactive protein level was , mg/dl. investigation/results: the clinical picture suggested a lymphoproliferative syndrome. a biopsy was performed, revealing cm of purulent material. synovial fluid had leucocytes/ul, % of polymorphonuclear cells, % of mononuclear cells and undetectable glucose. acid-alcohol resistant bacilli test and pcr test for mycobacterium tuberculosis were positive. diagnosis: mycobacterium tuberculosis knee arthritis therapy and progressions: the patient was treated with polytherapy consisting on rifampin, isoniazid, pyrazinamide and ethambutol. months later, the patient reports no pain, and tumor size has decreased. comments: mycobacterium tuberculosis infection is not a common disease in developed countries. however, the incidence in europe is increasing due to immigration. even though the lung is the most affected organ, osteoarticular tuberculosis represents around % of extra-pulmonary cases. tuberculosis simulates several diseases. because of non-specific symptoms and radiological signs, it can be difficult to diagnose. in a patient with chronic knee pain and limited range of motion, tuberculosis infection should be kept in mind, among other differential diagnoses, such as fibromatosis, pigmented villonodular synovitis or soft tissue sarcomas. clinical findings: the patient presented with a valgus deformity of the knee, the medial femoral condyle protuding on the medial side of the knee. neurovascular status was intact. investigation/results: xray revealed lateral dislocation of the knee. mri revealed mcl, pcl and acl rupture. diagnosis: knee dislocation (kd) grade iii (schenck). therapy and progressions: the patient underwent emergent closed reduction. neurovascular status was intact after resuction. due to important oedema and blisters, the lower limb was immobilized with a brace to allow for skin surveillance. after weeks, the brace was replaced by a long leg cast for more weeks. after months, the patient maintained residual pain, rom - / and minor instability. comments: kd are unusual injuries, associated with high energy trauma, therefore they often result in disruption of at least major ligaments and associated injuries, from soft tissue to vascular structures. emergent reduction is mandatory, and definitive treatment can be conservative, or early/late surgical repair/reconstruction of the ruptured ligaments. there is a lack of large prospective clinical studies comparing the different types of treatment. even so, data tend to associate early surgical treatment with better functional outcomes, though there is no statistic evidence supporting its improvement of the range of motion or stability. long term complications most frequently include residual pain, instability or rigidity. rarely the knee returns to its pre-injured state, independently of the treatment used. references: dwyer, t., et al. ( ) . outcomes of treatment of multiple ligament knee injuries. the journal of knee surgery, ( ), - . advising a reduction after a fracture of the distal radius, reliability with and without use of expert based criteria introduction: distal radius fractures (drf) are common, however many aspects of its management remain subject of debate . this study assessed the interobserver reliability of surgeons concerning the recommendation for a reduction and the improvement of expert based criteria for reduction. material and methods: we sent out surveys to members of the science of variation group. the first survey divided participants in groups, each rated - radiographs of drf. resulting in rated fractures by participants. each observer indicated whether they would advise a reduction or not. the second survey randomized participants ( surgeons) to either receive or not receive criteria for reduction and participants indicated if they would recommend reduction. results: the reliability for advising a reduction was poor, kappa . ( % ci . - . ). multivariable linear regression analyses indicated that each additional degree of dorsal angulation increased the change of recommending a reduction by % (beta . , % ci . - . p \ . ). criteria for reduction did not increase interobserver reliability for recommending reduction (no criteria kappa . % ci . - . vs. criteria . % ci . - . ). the likelihood of recommending a reduction was higher in the group using the criteria ( . vs . , p = . ). conclusions: poor interobserver reliability is associated with greater practice variation. dorsal angulation is the main drive for recommending a reduction. the liberal use of the criteria in combination with a specific focus on dorsal angulation leads in our opinion to less variation in treatment recommendation for distal radius fractures. this is something future study could assess for distal radius fractures in actual practice introduction: the number of pertrochanteric hip fractures increases proportionally to the increase in life expectancy. currently, the most used treatment in these fractures is the antegrade nailing. suffering a second fracture in the same femur around an antegrade nail is an uncommon complication, but it has a great impact on the patient. the aim of this study is to describe the type of perinail femoral fractures observed in our center, the treatment performed and the medium-term results. material and methods: between and , patients presented a perinail femoral fracture. were women and one was male, with an average age of . initial fractures were classified according to the ao classification: were a , were a and were a . of them were synthesized by short pfn-a (synthes), with short pfn (synthes) and with gamma (stryker). the average time since osteosynthesis of the proximal femur fracture and the perinail fracture was . years ( month- years). results: of the peri-implant fractures occurred at the level of the nail tip or the distal locking screw. the remaining fractures occurred in the distal femur. these supracondylar fractures and of the fractures at the level of the nail tip were synthesized with a va condylar plate (synthes), overlapped with the nail. in the rest of the fractures around the tip of the nail, the short nail was removed and replaced by a long pfn-a nail. one of the patients died in the immediate postoperative period. two patients died during the first year. in the rest of the patients, a complete consolidation of the fracture was observed, and their previous baseline situation was recovered. conclusions: peri-implant femur fracture is a rare but very severe condition, which requires good surgical planning, and is not without complications. gamagori city hospital, department of orthopedics, gamagori, japan, nagoya daini redcross hospital, department of orthopedics, nagoya, japan introduction: hip fracture is a leading worldwide health problem for the elderly. a missed diagnosis of hip fracture on radiography leads to a dismal prognosis. the application of a computer-aided diagnosis (cad) system using artificial intelligence (ai) to detect hip fracture can potentially improve the accuracy and efficiency of hip fracture diagnosis. material and methods: cad system using ai was trained using cases, plain frontal pelvic radiographs (pxrs) between and from each institution. the accuracy, sensitivity, falsenegative rate, and area under the receiver operating characteristic curve (auc) were evaluated on independent pxrs. the authors mixed resnext as classification algorithm and ssd as object detection algorithm to train cad system. results: the algorithm achieved an accuracy of . %, a sensitivity of . %, a false-negative rate of %, and an auc of . for identifying hip fractures. the visualization algorithm showed an accuracy of . % for lesion identification. conclusions: our cad system using ai not only detected hip fractures on pxrs with a low false-negative rate but also had high accuracy for localizing fracture lesions. the cad system using ai might be an efficient and economical model to help clinicians make a diagnosis without interrupting the current clinical pathway. medical faculty university of nis, orthopaedic surgery, nis, serbia, clinical center nis, orthopaedic and traumatology clinic, nis, serbia, orthopaedic word of medical center, cuprija, serbia introduction: bone reconstruction and limb lengthening usually refers to application of ilizarov or other ring external fixation devi-ces . we present here series of posttraumatic reconstruction and limb lengthening, by the use of new concept of d unilateral external fixation device. material and methods: as a clinical material, we present series of patients with different posttraumatic deformities ( ) and limbs discrepancy ( ) as a result of severe traffic accidents and wars. all patients have been treated by specially designed unilateral d external fixation system. that system is not bulky and it is more comfortable in comparison to ring fixators. procedure is relatively simple, so patients handle the device by themselves. during biomechanical testing, it was found that stability of this device is similar to ring systems. the last version of the device includes computer program and two sensors. results: all deformity corrections have been achieved successfully. sliding graft procedure has successfully been performed in all patients with bone defect reconstruction from to cm. in one patient with complex deformity and shortening, correction couldn''t be achieved during one procedure, so additional operations, by the use of the same system have been performed and correction completed. superficial pin tract infection rate was . % and we didn''t have deep infection. there were no other complications including dvt, joint stiffness, neurovascular injuries. conclusion: unilateral external fixation device with balanced d stability provides the same success of bone reconstruction and limb lengthening as ring fixators, but it is more comfortable and more easy for handling. references: treatment principles in bone reconstruction and limb lengthening of the lower extremity. olesen uk, nygaard t, kold sv, hede a. ugeskr laeger. nov ; ( ) at this moment author has licence agreement with the producer of external fixation devices. all patients were classified into the isolated hip fracture and the concomitant fracture. we analyzed these patients'' characteristics such as age, gender, bone mineral density (bmd), body mass index (bmi), korean version of mini-mental state examination (mmse-k), injury mechanism, and length of hospital stay. results: the most common site of upper extremity fracture was distal radius fracture of patients ( . %), followed by proximal humeral fracture of ( . %). concomitant fractures occurred on the same side in patients ( . %). the mean age of patients with a concomitant fracture was younger than that of patients with an isolated hip fracture (p \ . ). mean preinjury mmse-k was . in isolated hip fracture and . in concomitant fracture patients (p \ . ). mean length of hospital stay was statistically significant different between two groups (p \ . ). according to fracture site of hip, there was no statistically different prevalence of upper extremity fracture in femoral intertrochanteric fracture compared to the neck fracture. conclusions: we found a . % prevalence of concomitant hip and upper extremity fractures. it was found that the younger the age with preserved cognitive ability in elderly patients with a hip fracture, the higher the prevalence of upper extremity fracture. in addition, it is important to keep in mind that patients with a concomitant fracture have a longer hospital stay and difficulty in rehabilitation. on the other hand, the amount of bleeding was ml in group e and ml in group l, and there was no significant difference between the two groups. poor cases on postoperative images were % in group e and % in group l, and the joa hip score was . (groupe) and . (group l). in clinical results is significantly improved in group l. conclusions: the treatment results improved significantly in group l. as the number of experienced cases increased from these results, the reduction accuracy and treatment results improved, so experience was considered important for improving the treatment results of acetabular fractures. the additional value of the weight-bearing and gravity stress radiograph in determining stability of isolated type b ankle fractures introduction: the goal of the current study is to investigate whether the weight-bearing and gravity stress radiographs have additional value in determining stability in isolated type b fibular fractures. this in order to make the important distinction between fractures that need surgical treatment and fractures that can be safely treated conservatively. material and methods: patients with an isolated type b ankle fracture, without medial or posterior fracture, and a medial clear space (mcs) \ mm on the regular mortise radiograph were included. in the emergency room, a gravity radiograph was performed (in accordance with out protocol). within week, an additional mri scan was made. at this moment, in patients a weight-bearing radiograph was performed too. the mcs measurements of these regular mortise, gravity and weight-bearing radiograph were compared with the mri findings. the mri scan was set as reference standard to detect injury of the deltoid ligament in order to determine (in)stability. results: mean mcs on mortise radiograph was . mm (range . - . ); in ( . %) patients the mcs was [ mm and in patients ( . %) the superior clear space (scs) was [ mcs ? mm. in ( . %) patients, the scs [ mcs ? mm. on the gravity stress radiograph, . % of the patients had a mcs [ mm. the weight-bearing radiograph showed a mcs [ mm in ( . %) patients. in ( . %) patients, the mri showed a complete rupture of the deltoid ligament. in ( . %) patients a partial rupture was seen. patients ( . %) received surgical treatment. in all conservatively treated patients, no secondary dislocation occurred and there was no need for postponed surgical treatment. conclusions: the gravity stress view has a tendency to overestimate the mcs. thus, potentially too many stable fractures are incorrectly diagnosed instable and receive unnecessarily surgical treatment (with additional costs and risks). the weight-bearing radiograph, on the contrary, does not overrate the medial injury and can safely be used in the decision making process of treating conservatively and weightbearing (for example by using a brace) introduction: the purpose of this study was to identify the effect of the intravenous iron supplementation on demand of perioperative blood transfusion and post-operative hemoglobin recovery in geriatric hip fractures. material and methods: a retrospective cohort study was performed on patients who underwent surgery with proximal femoral nail for hip fracture and age years old or older between jan and may in a single center. the participants were divided into groups according to preoperative intravenous iron supplementation (iron isomaltoside, monofer Ò , pharmacosmos, holbaek, denmark); group (n = ) with monofer mg before surgery and group (n = ) without monofer. transfusion was preformed when the hgb was less than mg/dl). primary endpoint was incidence of perioperative transfusion. secondary endpoints were various hemoglobin (hgb) levels. results: the average age of the participants were . years old, and average body mass index (bmi) was . . demographic data including age, sex, bmi, comorbidity (charlson comorbidity index) of each group showed no difference. the complications from intravenous iron administration were not occurred. the preoperative hgb was . mg/dl (group . ± . vs, group . ± . , p = . ). the hgb at the postoperative day was . mg/dl (group . ± . vs group . ± . , p = . ). the average hgb at the postoperative month was . mg/dl (group . ± . vs group . ± . , p = . ). transfusion rate was . % ( / ) and the rate showed no difference between groups ( . % vs . %, p = . . the recovery of hgb between postoperative month and preoperative state showed statistically difference (group . vs group -. , p = . ), and iron supplementation group had more recovery. conclusions: intravenous iron supplement before the hip fracture surgery in elderly helped to recover hgb at postoperative month. comminuted subtrochanteric femur fractures-our experiences introduction: subtrochanteric femoral fractures account for approximately % of all the hip fractures and their treatment represents a challenge because of the short proximal fragment and highenergy forces. material and methods: a total of patients with subtrochanteric, highly comminuted fractures, were included in this study, with age range from to years. the mechanism of injury in all patients was high-energy trauma. in each case we applied a long gamma nail (limma lto) without focus opening. results: in all patients, good clinical and radiologic results were accomplished, in addition to early weight-bearing, without shortening of the legs, or consequences on the state of the hip and morbidity in general. conclusions: although the comminuted subtrochanteric femur fractures represent a challenge for the orthopedic surgeons, osteosynthesis using long gamma nail without the focus opening provides outstanding results. introduction: this study analyzed the association between the postoperative reduced position obtained on using short femoral nails (sfns) and the amount of sliding after fixation in unstable trochanteric fractures. material and methods: this retrospective study included patients with unstable trochanteric fractures with posterolateral support deficiency who underwent osteosynthesis with sfns and were followedup for months or longer. the study included men and women with a mean age of . years at the time of fracture. closed or open reduction was performed to achieve anatomical to medial type position on frontal view and anatomical to extramedullary type position on lateral view, followed by fixation with sfns. immediately and extramedullary type in patients immediately after surgery. three months after surgery, the reduced position worsened from the anatomical to intramedullary type in patients. according to the reduced positions at months after surgery, the mean amount of sliding was . mm in patients with intramedullary type, . mm in those with anatomical type, and . mm in those with extramedullary type. the amount was larger in those with intramedullary type than in those with anatomical and extramedullary types. moreover, excessive sliding was observed in patient with intramedullary type. conclusions:to prevent excessive sliding by ensuring anteromedial bony support in unstable trochanteric fractures with posterolateral support deficiency, open reduction should be aggressively performed to overcorrect to the extramedullary type when reduction performed on a traction table results in either anatomical or intramedullary type positioning. in this paper, we report patient previously studied for osteomyelitis caused by high-energy missile trauma, in . that study involved a total of patients with osteomyelits, divided into two groups, according to the treatment protocol applied. the group included patients treated using classic surgical methods, including debridement, curretage, forage, perfusion drainage and sequestration. the group included patients treated using recommended surgical methods and used pmma antibiotic beads. years after, we tried to contact all of the patients, for the purpose of follow-up. however, only patient was available for analysis. among patients we followed-up, were treated using recommended surgical protocol, while the remaining patients were treated using classic surgical methods. we present the patients' general status, as well as the local surgical status and radiographic analysis, years after. we obtained long-term results of both treatment protocols applied. from the group , patients developed chronic recurrent osteomyelitis, while only one patient from the group developed such condition. introduction: the aim of this study was to evaluate the treatment results using anterior subcutaneous internal fixation(infix) for the pelvic fractures and to consider an improvement strategy for the complications. material and methods: from to , pelvic fractures were enrolled. there were two males and females. the average age was years. there were fragility fractures and five high energy fractures. our operative procedure was as below: the connection between screws and rod was just above the fascia of the sartorius muscle. the connection bar was pre-bended before the operation using the initial axial ct scan. we assessed bone union, additional fixation, the distance between the femoral artery and connection rod (dar), the distance of protruded bar lateral to the connection (dpb), and complications. results: bone union achieved in out of cases. there was one nonunion and three early deaths because of medical complications. seventeen out of cases required additional posterior fixations. the average dar was . ( . - . mm) , and the dpb was . ( - ) mm. thirteen out of cases ( . %) had complications. there were seven lateral femoral cutaneous nerve (lfcn) symptoms ( required implant removal (ir)), two infections ( required ir), one hematoma (ir), one irritation (ir), one heterotopic ossification, one loosening (re-operation). there were no femoral vessels and nerve-related symptoms. to release lfcn and surrounding soft tissues decreased the nerve symptoms. conclusions: to connect the screws, and the rod just above the sartorius fascia could avoid major vessels and nerve complications, and also irritations. although this study found a high complication rate of infix, to release the lfcn and around soft tissue could decrease the complications. introduction: several studies have reported that posterior or anterior tilt increases the risk of reoperation in undisplaced femoral neck fractures (garden i/ii) after internal fixation performed using nonangular stable devices such as pins and multiple screws. however, to the best of our knowledge, there is limited research involving angular stable devices. the present study aimed to investigate the clinical outcomes in undisplaced femoral neck fractures after internal fixation using angular stable devices. material and methods: this retrospective study included patients (mean age, . [range, - ] years) who underwent internal fixation using angular stable devices between january and january . undisplaced femoral neck fractures with garden alignment index (gai) b °(posterior tilt angle c °) or gai b °( anterior tilt angle c °) were included (posterior: , anterior: ) in this study. patients were followed up for at least months (mean, . months). we analyzed the preoperative and last-followed gai on lateral radiographs, non-union, and late segmental collapse (lsc). results: among the patients, non-union was identified in ( . %) and lsc was observed in ( . %). the mean preoperative gai was . °(range, °- °), and the mean last-followed gai was . °( °- °). the overall complication (non-union and lsc) rate was . % ( / patients). among patients with gai c °, lsc occurred in ( . %). conclusions: in undisplaced femoral neck fractures, preoperative posterior c °is a risk factor for postoperative complications even when internal fixation is performed using angular stable devices; thus, primary arthroplasty may be considered. case history: the patient is a -year-old female who had undergone lumpectomy at the age of when she was diagnosed with breast cancer. she had antiresorptive drug therapy for bone metastasis, since years after the lumpectomy. she fell down from standing height and was diagnosed as right femoral subtrochanteric fracture. her femur was fixed with short femoral nail. she complained left hip pain at age .she complained left hip pain from july . clinical findings: she could walk with crutch.rom of left hip was normal. investigation/results: breast surgeon took mri and there was metastasis in the proximal part of femur. he thought the cause of pain was this metastasis. however, there was fracture line at the height of lesser trochanter when she visited our department. diagnosis: atypical fracture was strongly suspected, however, fracture line was little higher as normal atypical fracture. therapy and progressions: osteosynthesis with long femoral nail was performed months after first visit to our department because of increasing pain. pathological findings were metastasis and fracture. after surgery, radiation to femur was performed. she can walk without pain by crutch and fracture line is almost disappeared on months after surgery. comments: atypical femoral fractures (affs) are recently observed as a complication of antiresorptive drugs for bone metastasis. however, there were metastasis and atypical fracture in this case. introduction: in the present study we aim to evaluate the articular surface reduction quality by means of postoperative computer tomography (ct), in complex tibial plateau fractures, treated with an illizarov frame. materials and methods: this retrospective case series covers the period from - to - . forty-four patients with a mean age of years (range - years), with a complex intrarticular proximal tibia fracture were included. fracture types iii to vi according to schatzker's classification were included. the majority were closed injuries, apart from cases (a gustilo anderson type a and a type ). all patients were placed on a fracture table. a mini-open reduction of the articular surface was followed by application of a knee spanning illizarov frame. post-operatively all patients were subject to ct of the injured knee. outcomes were measured using the american knee society score. results: mean outpatient follow up was of at least months (range of - months). mean time for fracture consolidation . weeks (ranging from to weeks). according to the degree of postoperative articular surface depression patients were grouped as follows: had under mm, had - mm and over mm of depression. those with less than . mm of collapse had % chances of an excellent result according to akss. on the contrary, those with more than . mm of articular surface collapse had % chances for low scores and functional results. the achievement of a mechanical axis within °of the contralateral limb was positively correlated with good functional results but did not have a correlation with the akss. conclusions: complex tibial plateau fractures may be treated successfully with mini open reduction and the application of an illizarov frame. post-operative ct denotes the exact degree of displacement of the articular surface, which is prognostic regarding outcome. postoperative x-rays may be misleading, since they can underestimate articular surface collapse. introduction: a new trauma center building was constructed in march , and the process from the trauma bay to the operation room is faster. we hypothesized that this process improved the survival rate of trauma patients in need of trauma laparotomy. material and methods: the new trauma center separates the trauma bay from the emergency room, and the trauma team exam patients initially. it also has a separate operation room that is always available for emergency surgery. therefore, the decision to perform laparotomy and time to operation has been shortened. from january to december , trauma patients who underwent emergency laparotomy were included. those younger than years, who had delayed operation, underwent surgical observation, delayed admission by patient, or underwent angiography first were excluded. patients were dichotomized to the before-trauma-center (bc) and after-traumacenter (ac) groups, and their characteristics and clinical outcomes were compared. results: of patients, were included in the bc group and were included in the ac group. the times from admission to operation introduction: acute care is a growing worldwide burden with increasing visits to the emergency department (ed). the acute care system in the netherlands is almost overloaded and costs are increasing. almost % of ed visits have surgical disease. there is no nationwide acute care surgery (acs) model implemented yet, and resources and infrastructure are organized differently in almost every hospital. this study provides an overview of the existing systems nationwide, and basis for a national uniform model. material and methods: an online survey was distributed through the dutch surgical society and sent to all dutch hospitals. after sending a reminder, the survey was closed and results were analyzed. results: thirty-two hospitals ( %) participated in the survey. in % a surgeon (trauma, vascular or gastro-intestinal) was assigned as consultant and responsible for ed admissions, emergencies in-house, and in some cases also emergency surgeries. % of hospitals have an ed observation unit (edou). a dedicated emergency surgery operating room (esor) is available in % ( / available in %), and used efficiently in % primarily due to the following challenges: elective surgery scheduled at esor ( %), necessary stop of esor when elective programs are delayed ( %). in hospitals without an esor, the emergency surgeries are scheduled in between elective surgeries resulting in extending programs into the evening. finally, % of respondents was familiar with acs, with % being positive about exploring options of implementing such a model in our country, and % of the respondents opts for more focus on acs in surgical residency. conclusions: in the netherlands the organization of acute care varies. the main common bottleneck is the logistics around the or. implementation of a dedicated esor and unconditional availability / of this or seem to be the most important factors for optimal efficiency. although there needs to be more focus on acs in general, implementing a uniform model nationwide seems challenging at this moment. trauma team activations (tta) at an european trauma center: cases analyzed s. saar , , e. lipping , h. vospert , r. volmer , h. k. laas , j. lepp , k. g. isand , p. talving , north estonia medical centre, division of acute care surgery, tallinn, estonia, university of tartu, tartu, estonia, north estonia medical centre, tallinn, estonia introduction: the north estonia medical centre (nemc) is the largest trauma center in estonia with evolving capabilities. however, studies scrutinizing trauma team activations (tta) are currently lacking. thus, we initiated an investigation to document tta profile and outcomes. material and methods: all tta patients admitted to the nemc between / and / were retrospectively identified. data collected included demographics, injury severity score (iss), management, hospital length of stay (hlos), and in-hospital outcomes. primary outcome was -day mortality. results: overall, patients were included. mean age was . ± . years and . % were male. penetrating and blunt trauma accounted for . % and . % of the cases, respectively. non-ground level falls were the predominant mechanism of injury constituting . % of the admissions. mean iss was . ± . and . % of the patients were severely injured (iss [ ). blood alcohol level (bal) was positive at . %. a total of . % of the patients had an emergent operation. mean hlos was . ± . days.overall -day mortality and mortality of severely injured patients was . % and . %, respectively. conclusions: the current investigation documents comparable outcomes with established european trauma facilities [ , ] . blunt injury patterns predominate, however, high penetrating trauma incidence for european settings was noted. high rate of positive bal in tta patients warrants national preventive measures. introduction: the acute care surgery (acs) model was initially developed as a dedicated service for the provision of high quality / non-trauma emergency surgical care. after implementation in the united states (us), the model has been adopted in several variations around the world.in this systemic review we investigated which components are essential for a potential uniform acs model, by giving an overview of the current available acs models worldwide and their state of implementation. material and methods: a literature search ( - ) was conducted using pubmed, medline, embase, cochrane library and web of science databases following the prisma guidelines. all relevant data of acs models were extracted from included articles. results: sixty-five articles describing acs models in different countries were included in this review. the majority consist of a dedicated surgical service, providing non-trauma emergency surgical coverage, with daytime on-site attending coverage by an attending surgeon who is cleared from elective duties, and / in-house resident coverage. emergency department coverage and access to an acute care operating room varied widely across countries. critical care is fully embedded in the original us model as part of the acute care chain (acc), while in most other countries it is still a separate unit. while in most european countries acs is not a recognised specialty yet, there is a tendency towards more structured acute care, with training and separation from elective practice. conclusions: acs is gradually implemented worldwide. however, large national and international heterogeneity exists in the structure and components of the model. critical care is still a separate unit and specialty in most systems while it is essential to be part of the acc in order to provide the best peri-operative care of the physiologically deranged patient. universal acceptance of one global acs model seems challenging, however a global consensus on essential components would benefit any healthcare system. introduction: the recent financial crisis in greece is coped mainly with reformations towards cost effectiveness and rationality in the management of public expenses. the goal of the study is to evaluate the cost and time effectiveness in the management of the surgical patients admitted in emergency department (ed). methods: for a period of h/day in consecutive days, surgical cases presented in the ed of a tertiary university hospital of athens were followed. inclusion criteria were need for laboratory tests or imaging examinations or an immediate resuscitative intervention. data recorded regarding demographics, vitals, critical time points, disease and management. physician related data and cost of examinations were also collected. case severity was calculated by early warning score [ ] . results: she average waiting time for each patient was min and the average total time until final decision was : h. blood tests costs reached an average of , € per case and imaging an average of , €. the striking finding was that only one out of patients was of medium clinical risk, while all the others were of low. thus, substantial symptoms and clinical findings were lacking and as the ''tertiary care'' character of the hospital was mandating conclusive diagnosis, exams were ordered. this approach absorbs time and funds putting at risk the very few severe cases which are the target population for the magnitude of the facility. the current study indicates that the use of a tertiary hospital as a primary health care center by the public, is disorganizing the system, and increase the cost in time, funds, and preventable morbidity and mortality. a pre-hospital triage and management of the low severity cases system is pending to be established in our environment and becomes top priority in an era of prolonged financial crash. for years, surgical emergencies in ecuador have been managed without significant standardization. scarce numbers of specialists, lack of a constant presence of full-time teaching faculty versed in emergency surgery and lack of continuity with surgical trainees led to variability in clinical and surgical decision-making. to address these issues, the regional hospital vicente corral moscoso (hvcm) adapted and implemented a model of ''trauma and acute care surgery'' (tacs) to the reality of cuenca, ecuador. a cohort study was carried out, comparing trauma and acute care surgery patients exposed to the ''traditional care model'' before the implementation of the tacs model. variables assessed included: surgical wait times, number of hospital visits, number of surgical interventions, number of surgeries performed per surgeon and inhospital mortality. higher mortality was found in the traditional care model (rr of . , p b . ) compared to the tacs model. we observed a statistically significant decrease in surgical wait time ( . - . h for emergency general surgery, . - . h for trauma, p b . ). lengthof-stay decreased in trauma patients ( - days p b . ). the total number of surgical interventions increased ( , . - , . , p b . ) ; by extension, the total number of surgeries performed per surgeon also increased ( . - . , p b . ) . the implementation of tacs model in a typical resource-restrained, tertiary care hospital in latin america had a positive impact by decreasing surgical waiting time in trauma and emergency surgery patients, and length-of-stay in trauma patients. we also noted a statistically significant decrease in mortality. while cost could not be objectively evaluated with the available data, savings to the overall system and patients can be inferred by decreased mortality, length-ofstay and surgical wait times. to our knowledge, this is the first implementation of an tacs model that has been described in latin america. introduction: traumatic injuries constitute one of our major public health challenges. the most effective means to reduce the impact trauma has on individuals and society is primary injury prevention, reducing the incidence of traumatic events, which relies on detailed knowledge of risk factors. the aim of this study is to facilitate targeted injury prevention through improved data collection and analysis on impairing substances as risk factors for traumatic injuries. material and methods: idart is a national prospective observational study including analyses of the toxicological profile of all patients c year of age admitted via trauma team activation to any norwegian trauma hospital (n ) during a month study period. residual blood from routinely drawn blood samples at trauma admission is analyzed for alcohol, illegal and psychoactive drugs. toxicological data will be linked to clinical data from the national trauma registry. results: the study period started march st, , and during the first months patients were included from trauma hospitals. more than % of the included patients tested positive for psychoactive substances according to preliminary data. data on the prevalence of different psychoactive substances disaggregated by mechanism of injury, demography and geography from the month study period will be presented. conclusions: the idart study will provide a detailed descriptive analysis on the prevalence of alcohol, illicit and medicinal drug use among all patients admitted to a norwegian hospital with suspected severe injury. subgroup analyses will include prevalence of alcohol and other substances in subgroups analyses on patient and injury characteristics and geographical variations. analyses will aim to identify high risk groups according age, gender, circumstances of the injury, geographical location and type of psychoactive substance. the dutch nationwide trauma registry: the value of capturing all acute trauma admissions m. driessen , l. sturms , l. leenen lnaz/umcu, trauma surgery, nijmegen, netherlands introduction: twenty years ago the dutch government decided to reform the trauma care system and designated level regional trauma centers (rtcs). these centers, in collaboration with ambulance services and regional hospitals, have managed to set up regionalized inclusive trauma systems. moreover, they set up the dutch national trauma registry (dntr) as a quality evaluation and epidemiology resource. in this resource all acute hospital admissions were included, in order to measure the hospital and prehospital processes and outcomes. in the current study we demonstrate its current status and compare it with national trauma registries from the uk and germany. material and methods: the dntr includes all injured patients treated at the ed of % of all hospitals in the netherlands within h after the trauma followed by direct admission, transfer to another hospital or death at the ed. a representative descriptive analysis of extracted data from is demonstrated. results: between and a total of , trauma cases have been registered in the dntr. hospital participation has increased from % up to %. in alone, a total of . patient were included, % concerned males, the median age was years. % of all admissions had an iss c , of which % was treated at a rtc. from this cohort, in comparison, only % and % of the dntr patients met tr-dgu or tarn inclusion criteria. particularly children, elderly and patients admitted at non rtcs are not captured in the tr-dgu or tarn. also, part of iss c and fatal cases do not meet tr-dgu or tarn inclusion criteria. conclusions: the dntr has evolved into a comprehensive wellstructured nationwide population-based trauma register, with an annual number of , cases being entered in the database the dtr has grown to be one of the largest trauma databases in europe. the registry enables studies on the injury burden and quality and efficiency of the entire trauma care system encompassing all traumareceiving hospitals. introduction: trauma mortality is not distributed evenly. rural areas have higher incidence rates of trauma mortality than urban areas. the rural northern part of the nordic countries have common challenges with sparsely populated areas, long distances, and an arctic climate. the aim of this study was to compare the cause and rate of fatal injuries in the northernmost area of the nordic countries over a fiveyear period. material and methods: in this retrospective cohort we used the cause of death registries and collated all deaths from to with an external cause of death (icd- , v -y , except y - and t - ). the study area was the three northernmost counties in norway, the four northernmost counties in finland and sweden and the whole of iceland. we used % confidence intervals (ci ) to test for differences between the countries. results: there were deaths in the study area during the -year period. low energy (le) trauma constituted % and high energy (he) trauma % of deaths. northern finland had the highest incidence for both high energy trauma and low energy trauma. iceland had the lowest incidence for high-, and low energy trauma. iceland had the lowest prehospital share of deaths at % and the lowest incidence of injuries occurring in a rural location. the incidence rates for he trauma death was , / . /year in northern finland, , / . /year in iceland, , / . /year in northern norway and , / . /year in northern sweden. conclusions: there were significant and unexpected differences in the epidemiology of trauma death between the countries. the differences suggest that a comparison of the trauma care systems and preventive strategies in the countries is required. the diurnal and seasonal relationships of pedestrian injuries secondary to motor vehicles in young people introduction: there remains a significant morbidity and mortality in young pedestrians that are hit by motor vehicles, even in the era of pedestrian crossings and speed limits. the aim of this study was to compare incidence and injury severity of motor vehicle-related pedestrian trauma according to time of day and season in a young population. we hypothesised that injuries in young people would be more prevalent during dusk and dawn and during autumn and winter. material and methods: data was reviewed from patients in the - year old age group in the trauma audit and research network (tarn) national database, who had been involved as a pedestrian in a motor vehicle accident between and . the incidence of injuries, their severity (using the injury severity score [iss]), hospital transfer time and mortality were analysed according to the hours of daylight, darkness and seasons. results: . % of injuries occurred during time of darkness post sunset, while . % occurred during daylight. the incidence of injuries in motor vehicle accidents, in absolute terms, was highest during - , with a second peak at - . the greatest injury rate (number of injuries/hour) occurred during - and - with respective rates of . and . injuries scoring an iss over occurred . % at - and a further . % until . mortality was greatest during - involving out of the total deaths. autumn was the predominant season and lead to . % of injuries, with a further . % in winter. this demonstrated a clear difference to . % and . % in spring and summer. conclusions: we have identified a relationship between reduced daylight and the frequency and severity of pedestrian trauma in young people suggesting that reduced visibility may play a significant role which could be addressed through a targeted public health approach to implement change. enhancing cost effectiveness in a system in crisis: a , patient study a. tsolakidis , c. christou , p. smyrnis , a. prionas , a. tooulias , g. tsoulfas , v. n. papadopoulos aristotle university of thessaloniki, st department of surgery, papageorgiou general hospital, thessaloniki, thessaloniki, greece introduction: to date, there is no national trauma database in greece. the goal of our study is to record and evaluate trauma management at our university hospital as well as to measure the associated healthcare cost, while laying out the foundations for a national database. material and methods: retrospective study of trauma patients (n = , ) between and . demographic information, injury patterns and severity, outcomes and cost were recorded. results: the proportion of patients that were transferred to the hospital by the national emergency medical services was , %, whereas ( %) of our trauma patients did not meet the us trauma field triage algorithm criteria. over-triage of trauma patients to our facility ranged from . to . %, depending on the criteria used. ( . %) of our patients received operative management and % ( ) of them had postoperative complications. an iss [ was seen in ( %) of our patients and their mortality was , %. the overall non-salary cost for trauma management was . . euros. the cost resulting from the observed over-triage ranged from . to . . euros. furthermore ( . %) of our patients underwent at least one ct scan that did not show any significant traumatic lesion. the cost of hospitalization of these patients was . euros. conclusions: the prehospital triage of trauma patients in the greek national health system is ineffective, with significant over triaging, leading to excessive costs. appropriate use of criteria for diagnostic procedures and algorithms may lead to a, much-needed, reduction of these costs. introduction: in japan, there are emergency and critical care centers nationwide (one center for approximately every , people), and a system is in place to accept local critically ill patients h a day, irrespective of whether their conditions are intrinsic or extrinsic. however, manpower and medical care systems differ depending on the emergency and critical care center, and the establishment of a system for consolidating severe trauma patients has been particularly problematic. material and methods: this study examined cases where the patient had some sign of life when encountered by ambulance teams of the cases of traffic accident deaths that occurred in chiba prefecture between and . thirteen emergency and critical care center representatives in chiba prefecture met to verify each case based on data from the police, fire department, and medical institutions. the cases were classified into ( ) preventable trauma death (ptd) cases, ( ) suspected ptd cases, and ( ) non-life-saving cases; the problems (causes of ptd) in each case were examined. result: there were cases ( %) of ptd and suspected ptd. sixty-eight of these cases were transported to emergency and critical care centers. the most common cause of death was bleeding, accounting for cases and the locations where the problems that caused ptd occurred were outside of the hospital (n = ) and in the hospital (n = ). the problems that occurred in the hospital (including duplications) include circulatory management (n = , %), the treatment plan (n = , %), delay of lifesaving surgery (n = , %), and delay of diagnosis (n = , %). most of these occurred in the initial emergency care room. conclusion: this study clarified that ptd still occurs in relation to bleeding control in the current trauma care system in chiba prefecture. it is vital to establish a national ''trauma center'' and to thoroughly consolidate trauma cases to eradicate ptd. analysis of the impact of the implementation of a trauma team in a trauma center from an upper-middle-income country introduction: trauma teams (tt) improve the care process and the outcomes. a multidisciplinary tt was conformed in september to achieve a rapid response by specialists in emergency medicine, trauma surgery, diagnostic imaging services, and blood bank in a level i trauma university hospital in southwestern colombia. objective: to evaluate the impact of a tt implementation in terms of times of attention and mortality. material and methods: retrospective study. all the patients with the highest level of tt activation treated in the months after the tt implementation were included. the subjects triaged to the trauma center in the months pre tt were taken as controls. four hundred sixty-four patients were included, before the implementation of the tt (btt) and after (att). demographic data, trauma characteristics, times to tomography, and trauma surgery and mortality were recorded. the analysis was made on stata , Ò . categorical variables were described as quantities and proportionscontinuous variables as mean and standard deviation or median and interquartile range (iqr). categorical variables were compared by chi or fisher's test. continuous variables with student's t or wilcoxon-mann-withney. a multiple logistic regression model was created to evaluate the impact on mortality if being treated att, adjusted by age, trauma severity, and physiologic response on admission. results: the time from admission to the ct scan was min (iqr - ) in the btt group and min (iqr - ) in the att group, p &lt; . . the time to trauma surgery was min (iqr - ) in the btt group and min iqr - ) in the att group, p &lt; . . mortality in the btt group was . % and . % in the att group. adjusted or was . ( . - . ) p = . conclusions: the implementation of a multidisciplinary trauma team associated with a reduction of the times to tomography and surgery and with a decrease in mortality risk. no prediction of an unfavourable outcome after surgical treatment of chronic subdural hematoma patients using machine-learning l. riemann , a. younsi , c. habel , j. fischer , a. unterberg , k. zweckberger university hospital heidelberg, neurosurgery, heidelberg, germany introduction: chronic subdural hematomas (csdh) are expected to become the most frequent neurosurgical disease by the year . although often perceived as a ''benign'' condition, considerable rates of mortality and poor outcome have been reported. we therefore evaluated factors associated with an unfavorable outcome after surgical treatment of csdh patients by developing a predictive model using machine-learning. material and methods: consecutive patients treated for csdh with surgical evacuation between and at a single institution were retrospectively analyzed. potential demographical, clinical, imaging and laboratory predictors were assessed and a decision-tree predicting unfavorable outcome (gos - ) was subsequently developed using the classification and regression tree (cart) algorithm. out-of-sample model performance was evaluated using repeated cross-validation (fivefold with repetitions). results: eligible patients were analyzed. median age was (iqr - ) years and % were males. mortality rate was . % and rate of unfavorable outcome was . %. the developed decision-tree to predict unfavorable outcome had splits and included the following clinical variables (in descending order of calculated importance): gcs, comorbidities, hb, and age. after cross-validation, the following model performance metrics were obtained: a model accuracy of . ( . - . ), sensitivity of . ( . - . ), and specificity of . ( . - . ). conclusions: gcs, comorbidities, hb, and age were identified as the most important clinical predictors for an unfavorable outcome in csdh patients after surgery. the developed model was simple and still displayed a high accuracy and very high specificity, the sensitivity was however rather low. our results might help clinicians to better assess the prognosis in patients with csdh. introduction: in most developing countries access to tertiary care neurosurgical setup is uncommon. majority trauma including neurotrauma & medical conditions requiring emergency neurosurgical interventions present to a general surgeon. this study is an attempt to highlight the importance of emergency neurosurgery as a skill amongst general surgeons & also focus on the challenges in managing such cases in austere environments material and methods: this study was a retrospective analysis of progressively collected data of trauma patients with a specific focus on head injuries & emergency neurosurgical interventions for both traumatic & non traumatic indications in a level trauma centre in a semi urban area over a period of years from august to september results: a total of patients of trauma were analysed out of which were head injuries. road traffic accidents accounted for nearly % of head injuries. atypical trauma especially in rural setup e.g. train collision, animal related causes were also seen. males accounted for majority (m:f = . : ). mean age was yrs. patients had imaging findings suggestive of severe head injury. acute sdh was the commonest post traumatic finding and mca territory infarct in non traumatic group. patients underwent emergency neurosurgical intervention with a survival of %. factors associated with poor outcome were delayed presentation (p \ . ), sdh with diffuse axonal injury. alcohol consumption was a significant factor. conclusions: emergency neurosurgery is an essential skill for general surgeons. performing such cases in a low resource environment in absence of modern day facilities for imaging, icp monitoring & powered equipment presents a significant challenge. general surgeons should be able to perform operative interventions with basic handheld instruments. operative management whenever indicated should be done & helps improve outcomes. head trauma in polytraumatized patient. analysis of risk factors and neurological prognosis b. castro , , , m. morote gonzález , , , l. cebolla , , , a. sada , , , l. seisdedos , , , , , j. gil , c. rey valcárcel , , f. j. turégano fuentes , , c. tristan , c. ruiz moreno hgugm, surgery, madrid, spain, hospital, madrid, spain, hospital, madrid, spain, hospitall, madrid, spain, hospital, madrid, spain, hospital, madrid, spain, hospital, madrid, sri lanka introduction: severe trauma is one of the most frequent causes of death and disability and traumatic brain injury (tbi) in polytrauma is the main cause of death and disability in survivors. the aim of this study is to analyze mortality associated to tbi in the last years, prognostic factors associated with it and neurological outcomes in survivors with tbi. methods: retrospective observational study that includes risk factors and functional neurologic evaluation in polytrauma patients attended in gregorio marañon hospital between - . inclusion criteria were severe trauma patients (iss c ) with a tbi and abnormal ct of the head. we analyzed mortality trend in two periods : - and - , and neurological evolution and outcome at discharge with functional scores (ramkin scale and gos) in the second one. results: from to , severe trauma patients were admitted, ( , %) with brain or central nervous system injuries visible on head ct. median age was ' ; . % were men. the global mortality of the cohort has been , %, . % of them for neurological causes. ischemic heart disease, anticoagulation, abnormal pupils or eye opening, the need for surgery, shock, gos, iss, niss, cranial ais are significant associated with higher mortality (p \ , ).the mortality rate due to neurological causes decreases in the second period from , to , %, this descent being statistically significant (p = , ). between and , % patients died from cnsi, and , % of tbi survivors had a vegetative status at discharge, , % had major disability, and , % had a good neurological recovery. conclusions: mortality due to tbi decreased in the last years, but this improvement after tbi was at the expense of a high rate of vegetative status and great disability, showing the need for continuous research in this area. introduction: severe traumatic brain injury (tbi) constitutes one of the most frequent causes of intensive care unit admissions and is a major cause of death and disability among young people. decompressive craniectomy (dc) is a life-saving measure used to relieve intracranial pressure (icp). this procedure is related with low mortality rates and poor functional outcomes. the aim of this study is to analyze the survival rates and prognostic factors related with functional outcomes after dc for severe tbi. material and methods: retrospective, single center study of patients with severe tbi in whom a dc was performed between the years and . demographic features, clinical parameters, radiological findings and clinical outcomes were included in the study. for the statistical analysis we used anova, chi-square, kaplan meyer, cox regression and logistic regression. a p value of less than . was considered to indicate statistical significance. results: the mean initial glasgow coma scale was , ± , and the mean initial motor response (imr) was , ± , . the mean icp after dc was , ± , . the -day survival after dc was %. twenty percent of the patients improve ate least point in the glasgow outcome scale (gos) between and months after surgery. twelve patients improve from unfavorable gos to favorable gos. at -month follow-up, % of the patients has gos [ . younger age, high irm a post-operative icp were the factors significantly associated with a higher chance of outcome improvement. conclusions: dc is useful for the management of refractory intracranial hypertension related to severe tbi, and in selected patients is associated with good functional outcomes. introduction: antiplatelets and anticoagulation, commonly referred to as antithrombotic therapy, are frequently used in patients c years. the use of antiplatelets and anticoagulation are associated with increased incidence of intracranial bleeding ( , ) . there are two research questions addressed in this study: ( ) does preinjury antithrombotic therapy affect survival in elderly patients with tbi? ( ) are direct oral anticoagulants (doacs) associated with better survival than vitamin k antagonists (vka) in tbi patients on anticoagulation? materials andmethods: retrospective cohort study based on data extracted from the oslo tbi registry. included in the study are tbi patients c years admitted to ouh with cerebral-ct showing signs of acute trauma (hemorrhage, fracture, vascular injury) in the time period - . the impact of age, comorbidity, antithrombotic medication and antithrombotic reversal protocol for survival will be explored. results: the patient inclusion is ongoing. preliminary data will be presented at the st ectes in april . the estimated number of tbi patients c years with cerebral-ct showing signs of acute trauma in the study period is * . in this patients group, the expected preinjury use of antiplatelet and anticoagulation medication is * % and * %, respectively. conclusions: the knowledge regarding impact of preinjury antithrombotic therapy on survival in elderly tbi patients is clinically relevant, and may improve patient management in the acute phase of injury. references: introduction: traumatic acute subdural hematoma (asdh), especially the large ones in need of surgical evacuation, is associated with high mortality. contemporary population-based series of surgically treated asdh are sparse. the two main aims of this single-center study from oslo university hospital (ous) were to estimate incidence of surgery for asdh in the population of helse sør-Øst, and estimate in-hospital and -month survival of these patients. treatment of tbi at ous adheres to the brain trauma foundation guidelines, with icp controlled therapy and evacuation of asdh when gcs \ and hematoma volume c cm or midline shift c mm or hematoma width [ mm. the goals of tbi treatment for adults have been to maintain icp \ mmhg and cerebral perfusion pressure (cpp) c mmhg. methods: from . . all patients with traumatic brain injury (tbi) with positive head ct, admitted to ous, living in helse sør-Øst ( . million inhabitants) and having a norwegian social security number, have been included in our approved tbi-quality register. included in the present study are all patients with asdh undergoing evacuation of the hematoma within days of trauma. the following data were extracted from the register; demographic variables, date of injury and trauma mechanism, severity of head injury according to hiss grade, rotterdam ct score, surgical procedures, multitrauma, glasgow outcome scale at discharge and date of death. results: asdh patients were operated in the -year period - , % males, mean age was years ( - ), the most frequent trauma mechanism was falls ( %), % were under influence of ethanol, % had severe tbi and % had multitrauma. the incidence of surgically treated asdh in helse sør-Øst was / . /year. in-hospital and -month mortality was . % and %, respectively. conclusion: the presented data for incidence and mortality will be compared with earlier reports. age-related difference in impacts of coagulopathy in patients with isolated traumatic brain injury: an observational cohort study w. takayama , a. endo , y. otomo tokyo medical and dental university hospital of medicine, trauma and acute critical care, tokyo, japan background: age and trauma-induced coagulopathy (tic) have been reported to be the predictors of poor outcome following traumatic brain injury (tbi). whether the impact of brain injury induced coagulopathy on outcomes have age related differently is unknown. objectives: we evaluated the age-related difference in the impact of tic on outcomes in patients with isolated tbi. methods: a retrospective observational study was conducted in two tertiary emergency critical care medical centers in japan from to . the patients with isolated tbi [head abbreviated injury scale (ais) c , and other ais \ ] were included. we evaluated the impact of coagulopathy (international normalized ratio c . , and/or platelet count \ /l, and/or fibrinogen b mg/dl) on the outcomes [glasgow outcome scale-extended (gos-e) scores, inhospital mortality and ventilation free days (vfd)] in both group using univariate and multivariate models. furthermore, we visualized the impact of coagulopathy on gos-e according to age, by using a generalized additive model. results: of the patients studied, they were divided based on their age: non-elderly group (n = , - years) and elderly group (n = , age c years). although, in the multivariate model, age and coagulopathy were significantly associated with lower gos-e, in-hospital mortality and shorter vfd in the non-elderly group, significant impact of coagulopathy was not observed for all the outcomes in the elderly group. the correlation between coagulopathy and lower gos-e decreased with age after round years old. conclusions: in patients with isolated tbi, impact of coagulopathy on functional and survival outcomes was lower in geriatric patients. no difference in mortality between isolated tbi and polytrauma with tbi: it is all about the brain introduction: despite improvements in trauma and critical care mortality caused by traumatic brain injury (tbi) remains high. [ ] as polytrauma is naturally associated with increased mortality, this study compared mortality rates in isolated tbi (itbi) patients and polytrauma patients with tbi admitted to icu. material and methods: a -year retrospective cohort study included both consecutive trauma patients with itbi with ais head c (ais of other body regions b ) and polytrauma patients with ais head c admitted to a level-i trauma center icu. patients \ years of age, injury caused by asphyxiation, drowning, burns and transfers from and to other hospitals were excluded. patient demographics, shock and resuscitation parameters, denver multiple organ failure scores and acute respiratory distress syndrome (ards) data were collected. [ ] data is shown as medians with interquartile ranges. p-values \ . were statistically significant. results: a total of patients were included. the median age was ( - ) years, ( %) patients were male, median iss was ( - ). seventy-nine ( %) of all patients died. polytrauma patients developed more often ards ( % vs % p = . ) but had similar mods rates ( % vs % p = . ). polytrauma patients stayed longer on the ventilator ( vs. days p b . ), longer in icu ( vs. days p b . ) and longer in hospital ( vs. days p b . ). there was no distinction in in-hospital mortality of itbi and polytrauma patients ( % vs. % p = . ). tbi contributed to all deaths in itbi patients and all but three deaths ( %) in polytrauma patients. conclusions: tbi was the main cause of death in both groups. there was no difference in mortality rates between polytrauma patients with tbi and itbi patients, even though polytrauma patients were more severely injured. references: [ ] dewan mc et al. estimating the global incidence of traumatic brain injury. j neurosurg. ; ( ): - . no significant relationships or conflict of interests. how modeling the brain ventricles could help brain trauma understanding ( ). in pathological cases as in hydrocephalus, or in brain trauma, it is likely that each patient's ventricle structure has an impact on the way they behave. for instance, a shock wave may turn out differently according to the ventricle's shape. this can explain why for a same shock, the clinical translation is not the same. the aim of the study is to implement a finite element model of the cranio-cerebral system and to analyse the impact of a trauma simulation. material and methods: this is amonocentricretrospective study from . the database contains ct scans of healthy patients. we used itk-snap software to segment the ventricles and matlab to implement the model. results: the mean volume of the total ventricles is ml (sd = ). the median is ml (table ) .to identify the correlation between the parameters acquired we performed a pearson test. we found multiple significant correlations and one of the most relevant one is between the ventricular volume and the width of the third ventricle ( table ). showing that the total ventricular volume is statistically correlated to the width of the third ventricle is clinically interesting. we could potentially simplify our analysis of the ventricular system in head trauma by measuring less coordinates and yet come up to an accurate prognosis. the ventricle volumes are used as neuroimaging marker of brain changes in health and brain trauma. to our knowledge, it is the first time they are studied in vivo on ct-scan. this study and the existing correlations are relevant for the configuration of the finite element model on going. it can surely help the comprehension of the interaction between the structural parts of the cranio-cerebral system during brain trauma. (excitatory-glutamate, and inhibitory-c-aminobutyric acid, gaba), is crucial for the normal cerebral functioning. gaba concentrations vary in different cerebral zones [ ] responsible for different cerebral tasks. in this study, [gaba] is measured in the posterior cingulate cortex (pcc) of children with acute mtbi. material and methods: acute mtbi patients (\ h since injury, . ± . y.o) and healthy controls ( . ± . y.o). mri scanner philips achieva t was used. standard mri protocol for tbi revealed no pathological lesions in brain of any subject. magnetic resonance spectroscopy (mega-press [ ] ) was applied to obtain gaba signal without macromolecules. spectroscopy voxel is demonstrated on fig. . intensities of gaba, glutamate ? glutamine, creatine and water signals were calculated in gannet program [ ] . absolute concentrations were calculated. mann-whitney was used to reveal the statistical significance of between-group differences. results: typical gaba spectrum processing in gannet is demonstrated on fig. . no changes in glx were found. the values of [gaba] in pcc are demonstrated on fig. : the increase in gaba is not statistically significant. conclusions: this is the first study of [gaba] in pcc of children with acute mtbi. the result of current work disagrees with our previous study, where gaba was increased (p \ . ) in the anterior cingulate cortex of children with mtbi [ ] . this indicates to a necessity of further data collecting in order to reveal any [gaba] alterations in various cerebral loci. this would help to identify the causes of an inhibition/excitation imbalance and to predict possible dysfunctions of cns following mtbi. results: tnaa and naag concentrations along with stable naa concentration were found to be reduced in patient group. reduced asp and elevated mi concentrations were also found. the main finding of the study is that tnaa signal reduction in wm after mild traumatic brain injury is associated with the drop of the naag concentration rather than of naa one, as it was thought previously. this highlights the importance of separation of these signals at least for wm studies to avoid misinterpretations of the results. naag plays an important role in its selective activation of the mglur receptors, thus providing neuroprotective and neuroreparative function immediately after mtbi. it might have potential for the development of new therapy strategy for patients with injuries of various severity. introduction: traumatic brain injury (tbi) is globally recognized as a major health and socioeconomic issue. however, reported numbers vary and often represent subgroups. the number of hospital-admitted tbi has an important impact on hospital resources. thus, the monitoring of hospitalized tbi patients is needed. in , oslo neurosurgical tbi registry was established and includes patients admitted to oslo university hospital (ouh) with traumatic intracranial injury identified by neuroimaging. the aim is to introduce the registry; describe the patient group and volume. material and methods: descriptive study from oslo neurosurgical tbi registry. results: patients from south-east region were included in - (population million). mean age was years (sd ), % were males. most frequent cause of injury was falls ( %), increasing with age. % was influenced by alcohol at time of injury. preinjury antithrombotic therapy was common ( %). most of the patients had multiple pathologies on ct caput, e.g. simultaneous cranial fracture, sdh, tsah and brain contusion (four most frequent). accompanying injuries were found in %. % was transported to ouh directly form accident scene. % was classified as severe tbi upon arrival ouh, % was intubated, and trauma team was activated in %. median annual and monthly numbers of cases were (range - ) and (range - ), respectively. no clear change in case load between years and months, except a slight decline in march. admission rate peaked during the weekend. patients were continuously admitted throughout day and night, [ % between : and : . conclusions: patients included in the registry were older than those included in previous tbi studies. the numbers of cases admitted were stable across the months and years. however, the majority of patients were admitted during weekends and nights; thus handled by duty staff. relationship between brain-body temperature difference and neurologic outcomes in patients with severe head trauma introduction: brain is one of the most vulnerable organ to temperature. the association between core body temperature(ct) and neurologic outcomes in patients with post-cardiac arrest, severe head trauma and stroke has been reported. there were few reports comparing brain temperature(bt) with ct and peripheral temperature(pt). we investigated the association of differences among bt, ct and pt with neurologic outcomes in patients with severe head trauma. material and methods: we retrospectively reviewed data for patients with severe head trauma who underwent monitoring intracranial cerebral pressure(icp), bt, ct and pt simultaneously between january and december . results: we evaluated patients with a median age of years (range - years). glasgow outcome scale(gos) at discharge were as follows: good recovery(gr) , severely disabled(sd) , vegetative state(vs) , death(d) . table showed the average values of icp, cerebral perfusion pressure(cpp), bt, ct, pt, differences between each temperature (bt-ct, ct-pt, bt-pt) and gos in each patients. there was remarkable difference between bt and ct in the dead patient, whereas less differences were found in the other alive patients. we found greater difference between bt/ct and pt in the vs patients than gr patients. conclusions: greater differences between bt/ct and pt can be related to poorer neurologic outcomes introduction: minor head traumas are difficult to assess even with guidelines, hence head cts are often requested. as head cts are increasingly accessible, the demand on the radiology department often exceeds its capacity. there has been an increase in head cts at the oslo emergency department (oed), norway. the scandinavian guidelines for initial management of head injuries in adults (sg) is standard practice in the oed when assessing patients with head trauma.the aim of this study is to assess the number of patients with traumatic brain injury, evaluate guideline compliance and false negative initial reports by junior radiologists. material and methods: a consecutive cohort of patients from jan-june who received a head ct at oed due to minor head trauma was assessed. data was gathered from the ct request form, radiology report and ct images. the data points analyzed were: type of trauma, gcs, anticoagulants, loss of consciousness, nausea and vomiting, positive traumatic ct findings, and number of head cts within a year period. results: intracranial bleeds were reported in ( %) patients, ( . %) required neurosurgical intervention. skull fractures were reported in ( . %) patients, however no intracranial bleeds were present. it was impossible to assess guideline compliance because % of the referrals lacked adequate clinical information. ten bleeds were missed, however no further action was needed. % received more than head cts in years conclusions: head injury guidelines can improve clinical practice and reduce unnecessary ct scans; thus minimizing radiation exposure. based on the low number of positive findings, we hypothesize that sg compliance can be improved at oed. compliance was not assessable for nearly half of the patients, due to vital clinical factors missing. implementation of a standardized ct referral form based on the sg and educating junior ed doctors may decrease the number of unnecessary head cts. introduction: to date, there is no ideal allograft that provides local antibiotic release. along with this, existing fillers are expensive material, which complicates their application in practice. all this leads to the need to look for new ways to solve this problem. material and methods: gentamicin was used as an antibacterial drug because of its wide spectrum of action and thermal stability. for the study, staphylococcus aureus attc was used as a microbial strain. the antibiotic release from the studied materials was determined by equilibrium dialysis over the entire observation period. gentamicin antibiotic concentration was determined by hplc. results: an allograft impregnated with an antibiotic, prepared according to the marburg system in the area of the subcortical part of the bone, suppresses the staphylococcus aureus attc strain twice as much as perossal. when comparing bone allografts impregnated in various ways, the longest release time showed a perforated allograft.a bone graft impregnated with an antibiotic by incubation showed a % longer release time compared to perossal granules (p \ . ).when in vitro incubation of the antibiotic gentamicin with the drug ''perossal'', the dissociation rate is more than % in the first two days. when the antibiotic gentamicin with a bone allograft is incubated in vitro on the second day, dissociation into the extracellular space makes up more than % of the drug from the previously bound (p \ . ), which also indicates a longer release time from the bone allograft. conclusions: in vitro, a bone allograft impregnated with an antibiotic is able to reversibly bind the antibiotic gentamicin and gradually release it over a period of days. the use of a bone allograft impregnated with an antibiotic suppresses the growth zones of staphylococcus aureus strains. references: rudenko a., impregnation of the bone allograft: comparison of heads coloring. european journal of trauma and emergency surgery (suppl) p. acute appendicitis and pregnancy: from incidence to modern management: literature review and proposal for consensus estes experts guidelines a. l. bubuianu , a. mihailescu , g. pokusevski tameside general hospital, general/emergency surgery, ashtonunder-lyne, united kingdom introduction: acute abdominal pathology during pregnancy has historically been a challenging decision for the emergency surgeon, that had to deal with patients at same time. acute appendicitis has probably the highest prevalence of all. early involvement of the gynaecological team was considered paramount and the ongoing debate laparoscopic versus open intervention, has been more recently challenged by case reports where antibiotics alone have been a successful strategy. material and methods: literature review has been conducted by the investigating team, using the following search algorithm: reviewers screened pubmed portal to conduct a thorough search of the most important medical databases, cochrane's library, medline and embase. case reports and low quality case series have been excluded from the literature review. results: there is currently no general consensus in regards to operative strategy in acute appendicitis during pregnancy, but most authors described safety of laparoscopic intervention in the first trimesters and favoured open approach in a mother closer to term. the antibiotic treatment alone can only be considered in presumed early appendicitis, where there are no features of pending perforation, presence of phlebolith or established peritonitis and should be done under the close monitoring of experienced general surgeons. conclusions: an expert consensus is required in first instance, (set of questions submitted to audience at end of presentation for their expert opinion) regarding optimal treatment strategy in acute appendicitis during pregnancy, followed by a multicenter prospective randomised control trial, which we are hopeful to engage with help of numerous european hospitals where estes members activate. introduction: deep tissue pressure injuries (dtpi) are complex and difficult to treat. the higher prevalence is observed in paraplegic and elderly populations. primary closure of large, stage- dtpis is rarely feasible and flap closure is customarily applied. presented is a technique using tension relief system (trs; topclosureÒ tension relief system) and regulated oxygen and irrigation negative pressure wound therapy (roi-npt; vcareaÒ) to facilitate simple primary wound closure of dtpis. methods: large, stage- dtpis were closed by a limited surgical procedure entailing conservative debridement, en-bloc primary wound closure based on the application of trs and roi-npt. results: details of the closure of consecutive large dtpis in patients is presented. immediate primary closure was achieved in cases, while three others were closed over - days. surgery time ranged between . and h and hospitalization between and days. following a median follow-up of months (range - months), all wounds healed with one late recurrence. post-operative wound infection observed in one patient was successfully treated with systemic antibiotics. minor skin damage inflicted by the tension sutures at the anchoring sites healed spontaneously. gradual return to partial loading of the operated area was enabled within - weeks and full weight-bearing was achieved within - weeks. introduction: chronic pain is a disabling condition affecting - % of trauma patients. considering the burden of chronic pain, interest in interventions to prevent this disorder after trauma has grown. a descriptive review of literature was undertaken to assess the evidence on these interventions. material and methods: medline, cinahl and cochrane library databases were searched to identify interventional studies published up to august . websites of injury, critical care and pain organizations were also consulted to retrieve relevant guidelines. the literature search used combinations of medical subject headings and keyword under the themes of pain, trauma, surgery and preventive interventions. results: many knowledge syntheses relevant to the population of trauma published between and were found. low to moderate level of evidence was reported for pharmacological interventions such as the administration of ketamine, neuropathic pain medication and multimodal analgesia. local or regional nerve block in the presence of factures was associated with a high level of evidence. very low to low evidence was described for nonpharmacological interventions including cryotherapy and early mobilization. finally, psychological interventions were associated with a low to moderate level of evidence and multimodal pain management interventions (pharmacological and non-pharmacological) with a high level of evidence. conclusions: research is still needed to define the role of interventions to prevent chronic pain in trauma patients. thus far, multimodal pain management interventions involving multidisciplinary team management appear to be the most promising. implementing such interventions could reduce the negative consequences associated with chronic pain. introduction: chronic use of opioids has been documented % of trauma patients. accordingly, the tapering opioids prescription program in trauma (topp-trauma) was developed. the aim of this study was to assess the feasibility of topp-trauma and explore the efficacy of topp-trauma in reducing opioid use. material and methods: a -arm pilot rct was conducted in patients presenting a high risk for chronic opioid use. we aimed to recruit participants to receive either topp-trauma or an educational pamphlet. topp-trauma comprised educational and counseling sessions. the feasibility assessment of topp-trauma was based on the ability to provide its components. the morphine equivalent dose (med) per day as well as pain intensity and pain interference with activities were measured at and weeks following discharge. results: preliminary findings based on data collected in participants showed that counseling sessions were most frequently needed to completely taper opioids. sessions attendance reached %. nearly % of eligible patients accepted to participate and an attrition rate of % was found. even though the experimental group consumed a higher med h prior to hospital discharge compared to the control group ( . vs . ), its med/day intake was lower at weeks ( . vs . ) and weeks ( vs . ). these self-reported data were validated by the total med delivered by participants'' pharmacy at both time points ( . vs . at weeks; . vs . at weeks). minimal mean score differences were observed in both groups with regard to pain intensity and interference with activities. conclusions: data collected until now provided evidence on the feasibility of topp-trauma and on the program potential efficacy. challenges that will require to be addressed in future rct include the acceptance to take part in the study and participants' drop out. introduction: head preserving surgical treatment for ao-type b fractures with little to no dislocation consists of three canullated screws or a dynamic hip screw (dhs). there is a new alternative: the femoral neck system (fns). the fns has some advantages over dhs. the anti-rotation screw provides extra rotational stability because of the diverging design. furthermore, the incision is smaller in fns and only one locking screw is necessary for plate fixation. we present the first results of this new surgical fixation of femoral neck fractures with fns. material and methods: during the period of november until october , all patients with femoral neck fractures treated with fns, were included in this prospective single center cohort study. patient characteristics, fracture classification (ao, garden, pauwel), perioperative parameters and postoperative complications were registered. patients were allowed to mobilize based on the principle of permissive weightbearing. follow up was planned after weeks and weeks. primary outcome measure was cut-out rate within months. results: twenty-four patients with a femoral neck fracture (ao-type b) were surgically treated with fns. median age was , (range - ). median operation time was mins (range - ). mean duration of in hospital stay was days (range - days). twentytwo ( , %) patients completed the regular follow up of weeks. one patient ( %) had a reoperation due to a cut-out. during follow up one patient developed a wound-infection ( %) which was treated with intravenous antibiotics conclusions: femoral neck system as surgical treatment for femoral neck fractures shows promising first results. low cut-out rate, limited operation time, low mortality and short duration of in-hospital stay make this device a possible alternative for dhs of canullated screws. definitive conclusions should be made after studying long term results in larger cohorts. references: none. new personalized approach to enteroatmospheric fistulas using d bioprinting device introduction: enteroatmospheric fistula is a challenge for surgeons. it presents a great clinical variability. this diversity means that, despite having tried multiple devices and techniques to achieve local control of the intestinal effluvium over the rest of the wound, there is currently no technique that can solve this problem in all patients. d printing is a novel therapy that allows the customization of the devices according to the needs of each patient. the aim of this study is to describe the technique of manufacturing a custom device designed by bioscanner imaging and manufactured using a d printer for use in the management of enteroatmospheric fistula. we describe our initial results. materials and methods: we present four patients with enteroatmospheric fistula. the intestinal segment involved, the dimensions of the wound, the intestinal debit and the size of the exposed intestinal surface are substantially. all require an average of - daily cures by the nurse. after obtaining images of each fistula with a bioscanner, a personalized device was designed and made by a d printer. the polycaprolactone device was placed including inside the fistulous orifices and surrounding it with npwt in order to accelerate the healing of the wound to ostomize the fistula or achieve its definitive closure. results: four devices with different designs have been manufactured. the wound remained isolated from the intestinal contents after placement, favouring the granulation of the surrounding tissue with npwt and thus avoiding contamination of the wound. the system remained without leaks for an average of h, reducing the need for daily cures, improving patient comfort and avoiding complications. conclusions: the use of a manufacturing model using d bioprosthesis printing in order to create a personalized device that fits the characteristics of the patient's wound is feasible and offers promising results in the management of enteroatmospheric fistulas. new approaches in bone tissue engineering: innovative scaffold design for principle unlimited size bone substitutes introduction: in bone tissue engineering (bte), autologous boneregenerative cells are combined with a scaffold for large bone defect treatment. microporous, polylactic acid scaffolds showed good healing results in bone defects in small animals. transfer to large animal models, however, is challenging and not easily achieved simply by upscaling the design. increasing diffusion distances has a negative impact on cell survival and nutrition supply. this can lead to cell death and ultimately implant failure.this approach focuses on scaffold architectures, that meet all the requirements for a modern bone substitute. biological-functional, porous subunits in a loadbearing, compression-resistant frame structure characterise the innovative design. an open, macro-and microporous internal architecture provides optimal conditions for oxygen and nutrient supply in the inner areas of the implant by diffusion. material and methods: during the design process, prototypes (temple (figure a) , grid (figure b) , onion (figure c)) were dprinted (fused filament fabrication) using polylactic acid (pla). -after incubation with saos- (sarcoma osteogenic) cells for days (measurements on days , , and ), cell morphology, distribution and survival (fluorescence microscopy, ldh-based cytotoxicity assay), metabolic activity (mtt test) and osteogenic gene expression were determined. results: all designs not only showed cell colonization, but cells also sustained their ability to differentiate (already after days) and to divide. the open, hierarchical-structured design, with its innovative porous structure, provides a good basis for cell settlement and proliferation. the modular design allows easy upscaling and offers potential solutions to previous limitations scaffold developement in bone tissue engineering. references: the value of d reconstructions in determining post-operative reduction in acetabular fractures: a pilot study introduction: in patients with acetabular fractures, the reconstructed three-dimensional ( d) model of the contralateral acetabulum could be used as a mirrored template for the anatomic configuration of the affected joint. this has not been validated. material and methods: computer tomography (ct)-scans of twenty patients with unaffected acetabula were used. the symmetry of the generated d models was evaluated through; ( ) mirroring of the acetabulum; ( ) initial rough matching; ( ) automatic optimisation of the matching via surface-based matching; ( ) calculation of distances between surfaces by evaluating the euclidean (straight-line) error distance between the closest points between left and right. the percentages of surface-points of the left and right acetabulum with a distance smaller than . , . , . and . mm were calculated and evaluated, in relation to matta's criteria, for acetabular fracture reductions. the analysis was performed using the mirrored left acetabulum matched onto the right original structure (left mirrored to right original; ''lm ro'') and the right mirrored to left original (rm lo). to determine the inter-observer agreement the procedure was repeated by a second assessor for the first ten patients. results: patients had a mean ± sd age of . ± . years, % was male. the mean distance deviation was less than . mm in all comparisons. the calculated distances in . % of the surface points of the left and right acetabulum were below the tolerance threshold of . mm, based on matta's anatomical reduction critera (table ). absolute differences between assessors were\ . mm per patient with an overall moderate agreement of %. conclusions: d reconstructed models of healthy left and right acetabula are highly similar and could potentially be used as mirrored duplicates. the next step will be to investigate these results in patients with reduced acetabular fractures. : matta, j. ( ).j bone joint sur am. : - pr minimally invasive plate osteosynthesis technique for distal humeral fracture: a cadaveric study v. hofmann , c. deininger , t. freude , f. wichlas university hospital salzburg, orthopedics and traumatology, salzburg, austria introduction: in our study we want to evaluate the feasibility of minimally invasive plate osteosynthesis (mipo) technique for distal humeral fracture using anatomically precontoured double plate osteosynthesis. material and methods: eight elbows from four thiel fixed cadavers were included. on unfractued cadavers we tested the minimally invasive approach with two separate incisions, one at the lateral and one at the medial epicondylus. the preformed plates were inserted directly into the bone on sides and fixed with percutaneous screws. then we created an ao type a and c fracture. the reduction was performed under x-ray control and stabilized with k-wires. then we also inserted the plates in mipo technique. in the case of an intraarticular fracture, an olecranon osteotomy was additional performed in a minimal invasive way to control the distal humeral joint surface. after finishing reduction and fixation the approach were extended to control the fracture alignement, position of the plates and to expose the ulnar nerve. results: the plate position was satisfactory and we could not detect any major soft tissue damage or ulnar nerve injury by using the minimally invasive plate osteosynthesis technique. in the extraarticular fractures, reduction was achieved with k-wires and was acceptable in all cases. the intra-articular fractures were controlled by an additional olecranon osteotomy using the mipo technique with a good view on the joint surface of the distal humerus. conclusions: the findings of the present study show that mipo technique in distal humerus fracture is feasible and save especially for ao type a fractures. in ao type c fractures the olecranon osteotomy provided enough visibility to evaluate the distal humeral joint surface. the surgical technique is demanding, and care must be taken not to injure the ulnar nerve. never the less it is an effective surgical treatment method and an alternative option to open techniques. correlation between pelvic incidence and acetabular orientation in anteversion and inclination-an analysis based on a d statistical model of the pelvic ring introduction: the pelvic ring is a complex bony structure with a central role for the human''s mobility building the connecting part between the upper body and the lower extremities. pelvic incidence and acetabular orientation are two important parameters used in the description of pelvic anatomy and are of central importance for understanding the biomechanical interaction of spine, pelvis and hip joints. the objective of the study was the analysis of a potential correlation between pelvic incidence and acetabular orientation. material and methods: a d statistical model of the pelvic ring consisting of individual ct scans of european adults without bony pathologies was used to analyse pelvic incidence and acetabular orientation in anteversion and inclination. an additional analysis on the correlation between those parameters was performed using the software spss. results: a slight positive correlation between pelvic incidence and acetabular anteversion could be shown (r = . ; p = . ) as well as a strong positive correlation between anteversion and inclination (r = . ; p \ . ). pelvic incidence and acetabular inclination showed none statistically significant correlation (r = . ; p = . ). conclusions: the results of the study might contribute to a better understanding of the biomechanical interaction between the axial skeleton and the lower extremities and deliver valuable information concerning preoperative planning in orthopaedic and trauma surgery of the lumbar spine, the pelvis and the hip joints like for example reconstructive surgery after trauma, operative treatment of congenital or acquired deformities or total joint arthroplasty. references: boulay et al., ''pelvic incidence: a predictive factor for three-dimensional acetabular orientation-a preliminarystudy. '' anat res int. ; : . doi: . / . epub . introduction: the majority of distal clavicle fractures (dcfs) are displaced fractures and are prone to delayed-or non-union. there are several options for surgical reconstruction, open reduction and fixation or hook plate, but in patients with a comminuted or small fracture they are known to have a high complication and failure rate, and secondary surgery for removal is often necessary. we hypothesize that resection of the distal fracture fragment and subsequent stabilization with the lockdown device, is an alternative for selected patients with dcfs. methods: eleven patients with a comminuted dcf were treated with a lockdown device. data on pain and range of motion were documented and the constant shoulder score (css), oxford shoulder score (oss) and nottingham clavicle score (ncs) were assessed at one year follow-up. results: eight patients underwent surgery within weeks, compared to patients where the surgery was delayed ([ weeks) due to persisting pain and delayed-union. none of the patients had postoperative complications. in months after treatment, patients were complaint-free. one patient had hardware removal due to pain at the site of the screw head. four patients were assessed after one year follow-up. the mean pain score was . . the mean flexion , °, abduction , °, exorotation °and extension °. the css had a mean of . , oss . and the ncs a mean of . conclusions: all patients had a good short-term clinical outcome and hardware complications did not occur. we are the first to describe the use of the lockdown device in dcfs. this device is not dependent on fracture healing and secondary surgery is not necessary, therefore it can be an alternative in the treatment of dcfs. a larger series and longer follow-up is necessary to confirm this conclusion. in this ongoing study, the remainder seven patients will be included and presented at the estes. moore type i tibial head fractures are one of the most challenging fractures to treat. material and methods: we performed the following approaches on eight thiel fixed cadavers: the anterolateral (with an osteotomy of the tuberculum gerdyi, a subcapital fibula-osteotomy and an osteotomy of the tuberositas tibia), the medial approach (with submeniscal arthrotomy and a dissection of the medial collateral ligament) and the posterior approach with a submeniscal athrotomy. the reachable borders of the articular joint surface have been marked by a k-wire. the visual joint surface has also been radiographically documented by inserting k-wires into the tibia head. finally the results have been photo documented on the exarticulated joints. results: the reachable areas of the articular surface have been defined and documented. the combination of the subcapital fibulaosteotomy and the submeniscal arthrotomy showed the most increase in accessibility to the articular surface in the dorsal part. an additional osteotomy of the tuberculum gerdyi increased the vision on the entire lateral and anterior articular surface. the submeniscal arthrotomy, at the medial approach, has not a good view on the surface. the posterior approach showed only a limited view on the lateral and medial articular surface at the dorsal part. none of the surgical approaches sufficiently visualizes the intercondylar region. conclusions: a fracture-specific approach strategy is critical for the preoperative planning of complex tibia-head fractures. subcapital fibula osteotomy is the most efficient surgical approach to reach the posterior and lateral articular surface. for the anterior articular surface, the best overview was achieved by an osteotomy of the tuberculum gerdyi. it was not possible to see and control the intercondylar region with any approach. introduction: osteosarcoma (os) is the most common bone carcinoma in humans. at the time of the first diagnosis are already in about % metastases present. the current treatment strategies include above all radical surgical resection and chemotherapy. in the search for alternative therapy methods. treatment with cold atmospheric plasma (cap) shows promising prospects. at the cellular level, this leads to various cellular mechanisms and finally to induction of anticancerogenic effects such as growth inhibition, apoptosis, and changes in the cell-cell interactions. the impact of cap on the integrity of the cell membrane of os cells, however, is unknown. material and methods: suspended cells from two human osteosarcoma cell lines (u -os, mnng) were treated for s, s, and s with cap. cell proliferation was determined after h, , , , and h using casy cell counter. dye loss assay was performed by using fluorescein diacetate (fda). this was followed by indirect treatment with cap for s. in the cell-free supernatant was determined by tecan multireader the dye emission. flow cytometry assay was used after cap treatments and incubation with fda. the mean fda fluorescence intensity of individual cells in the flow cytometer was measured. results: cell kinetics showed significant inhibition of cell proliferation in both cell lines after cap treatment. the assays for determination of the dye level showed a significantly increased membrane permeability of both cell lines after cap treatment. the significant effect on the membrane integrity correlated with treatment duration. conclusions: this confirms a modulating influence of cap on the functionality of the cell membrane and may support the anti-proliferative effect of the cap treatment. thus, cap is a promising therapy option, especially for chemotherapy-resistant entities introduction: osteosarcoma (os) is the most common bone cancer in humans. standard therapy includes radical surgical resection and chemotherapy, but due to strong toxic effects, new treatment options are urgently needed. currently, there is a discussion about expanding the oncological therapy spectrum and treat with cold atmospheric plasma (cap). it is a reactive ionized gas rich in radicals, photons, and electromagnetic rays. its biological effects are primarily mediated by reactive oxygen and nitrogen species (rons). due to its low temperature, cap is suited for medical applications. in vitro studies have shown the antitumoral effect of cap also for pancreatic cancer, melanoma, ovarian, breast, and colon cancer. material and methods: human os cell lines u -os and mnng/ hos were used. proliferation assay. the growth of cap-treated cells was examined using a casy cell counter. caspase / assay. following cap treatment, the activities of caspase- and caspase- were measured using a specific substrate peptide coupled with a fluorescent dye (cellevent tm ). single-cell gel electrophoresis comet assay. dna damage after cap treatment was identified using alkaline microgel electrophoresis. dna migration was measured using comet score software. the percentage of tail dna was used to indicate the relative fluorescence intensity of the head and tail. tunel assay. after cap treatment tunel analysis was performed. results: the results revealed that the cap treatment of os cell lines leads to significant inhibition of cell growth. subsequently, the activation of caspases and the induction of apoptotic dna fragmentation was demonstrated. the treatment of os cells with cap leads to an induction of apoptosis and a reduction of cell growth. introduction: extra peritoneal packing (epp) is a quick and highly effective method to control pelvic hemorrhage. we hypothesized that this procedure may be as safely and efficiently performed in the emergency room (er) as in the operating room (or). methods: retrospective study of patients who underwent epp in the er or or in two trauma centers in israel between - . material and methods: retrospective study of patients who underwent epp in the er or or in two trauma centers in israel between - . results: patients were included in our study, in the er-epp group and in the or-epp group. the mean injury severity score (iss) was . ± . . following epp, hemodynamic stability was successfully achieved in of patients ( . %). a raise in the mean arterial pressure (map) with a median of mmhg (mean . ± . , p = . ) was documented. all patients who did not achieve hemodynamic stability after epp had multiple sources of bleeding or fatal head injury and eventually succumbed. the overall mortality rate was . % ( / ) with no difference between the or and er-epp groups. patients who underwent epp in the er showed higher change in map (p = . ). no differences were found between er and or epp in the amount of transfused blood products, surgical site infections and length of stay in the hospital. however, patients who underwent er epp were more prone to develop deep vein thrombosis (dvt): % ( / ) vs % ( / ) in er and or-epp groups respectively (p = . ). conclusions: epp is equally effective when performed in the er or or with similar surgical site infection rates but higher incidence of dvt. level of evidence: retrospective cohort study, level iv. introduction: application of supraacetabular schanz screws is usually performed under image intensifier guidance. the aim of this study was to perform it without imaging, with the hypothesis that, respecting anatomical landmarks, pre-and intraoperative fluoroscopy can be avoided. material and methods: insertion of the supra-acetabular schanz screws was performed in human adult cadavers. with cadavers placed in supine position, the anterior superior iliac spine (asis) was palpated. starting from this landmark, cm were measured in a distal and cm in the medial direction. at this point, a cm long oblique skin incision was performed. through this approach, mm schanz screws were drilled bilaterally into the supra-acetabular corridor with an angulation of °to distal as well as °to medial. combined obturator oblique-outlet views (cooo) were taken bilaterally to prove the screw position. six of the specimens underwent a d-ctscan. images were evaluated concerning correct screw positioning. skin and subcutaneous tissues were removed in the ilioinguinal region and possible lesions to the lateral femoral cutaneous nerve (lfcn) or to the joint capsule were evaluated. results: during radiographic evaluation of the cooo-scans ( specimens) and the d-scans ( specimens), the schanz screws were placed inside the supra-acetabular corridor in all specimens ( / ). during dissections, no intracapsular screw placements or lfcn lesions were found. conclusions: using our technique, all schanz screws could be sufficiently inserted without intraprocedural x-ray imaging. references: . karaharju, e. and p. slätis, external fixation of double vertical pelvic fractures with a trapezoid compression frame. inhury, . : p. - . . mears, d. and f. fu, external fixation in pelvic fractures. orthop clin north am, . : p. - . . mears, d. and f. fu, modern concepts of external skeletal fixation of the pelvis. clin orthop, . : p. - . pr epidemiology of self-inflicted major trauma r. stoner , n. misra , l. mason aintree university hospital, liverpool, united kingdom introduction: in the united kingdom, severely injured patients are taken directly to a major trauma centre (mtc). whilst deliberate self harm (dsh) is a known mechanism for this, there is limited prior research. - % of major trauma is thought to be self inflicted , . our aim was to describe the epidemiology of presentation to our mtc resulting from dsh. material and methods: retrospective review of patient records in our mtc for adult trauma team activations between / / and / / . data was collected on patient demographics, location type, injury severity score (iss), mechanism of injury and mortality. results: episodes of dsh made up . % of all trauma cases, involving patients; . % re-attended. z-scores show no change in incidence over time, but significant variability month by month, with / months [ sd from mean. mean patient age years (range - ). . % were male. . % came from residential location and . % from prison. most common mechanism was penetrating trauma ( . %). in-hospital mortality was % ( . % in males vs . % in females, chi p = . ). conclusions: this is the largest review of self inflicted trauma cases in a uk mtc, with a similar incidence to prior studies. there was no observed correlation with season or trend over time. mortality was higher in male patients, in keeping with national statistics on suicide, whilst dsh in females was linked to less severe injury; severity is related to mechanism of injury. injury from self stabbing/cutting was most common in patients from residential locations, whilst hanging was more common in prisoners. this study identifies preventable risk factors for major self inflicted injury. introduction: the distribution of trauma deaths was classically described following a trimodal pattern. during the last decade improvements in trauma care as damage control resuscitation (dcr) have minimized resuscitation injury. we hypothesized that the implementation of dcr in severely injured trauma patients is associated with less mortality and modifies mortality pattern. material and methods: we performed a -year ( - ) retrospective cohort analysis of all severely injured trauma patients (niss c ) who underwent surgery at our level trauma center. since , dcr was implemented including damage control surgery, minimizing crystalloids and increasing the use of blood products. our patients were stratified into two phases: pre-dcr ( - ( ) and post-dcr ( . results: a total of patients were identified. there were patients ( . %) in the pre-dcr group and patients ( . %) in the post-dcr group. mean age ( . vs . , non significant (ns)), mechanism of injury (blunt trauma: . % vs . %, ns) and shock on admission ( . % vs . %, ns) were similar between groups. there is a significant reduction in the rate of overall mortality ( . % vs . %, p \ . ). while early deaths from traumatic brain injury ( . % vs . %, ns) and hemorrhage ( . % vs . %, ns) are alike, mortality secondary to multisystem organ failure (msof) is lessened ( . vs . %, p \ . ). conclusions: dcr has helped in reducing overall mortality and mortality due to msof in our severely injured trauma patients. introduction: the mangled extremity severity score (mess) was constructed as an objective quantification criterion for limb trauma. a mess of or greater than was proposed as a cut-off point for primary limb amputation. opinions concerning the predictive value of the mess vary broadly in the literature. the aim of this study was to evaluate the applicability of the mess in a contemporary civilian central european cohort. material and methods: all patients treated for extremity injuries with arterial reconstruction at two centres between january and december were assessed. the mangled extremity severity score (mess) and the amputation rate were determined. results: seventy-one patients met the inclusion criteria and could be evaluated for trauma mechanism and injury patterns. the mean mess was ). seventy-three percent of all patients ( / ) had a mess b and % ( / ) of c . eight patients ( %) underwent secondary amputation. patients with a mess c showed a higher, but statistically not significant secondary amputation rate ( . %; / ) than those with a mess b ( . %; / ; p = . ). the area under the roc curve was . (ci . ; . ). conclusions: based on these results, the mess seems to be an inappropriate predictor for amputation in civilian settings in central europe possibly due to therapeutic advances in the treatment of orthopaedic, vascular, neurologic and soft tissue traumas. introduction: in polytrauma victims the acute respiratory distress syndrome (ards) is a major cause of morbidity and mortality. it presents a complex pathophysiology that is characterized by pulmonary activated coagulation and reduced fibrinolysis. due to the fact that the pulmonary endothelium is considered a key modulator of ards and that tpa in plasma is predominantly synthesized and secreted by vascular endothelial cells, we hypothesized that the time courses of serum tissue-type plasminogen activator (tpa) and its main inhibitor, the plasminogen activator inhibitor type- (pai- ), might indicate a clinical approach to preventing ards in polytrauma victims. material and methods: twenty-eight consecutive polytraumatized patients with concomitant thoracic trauma, age c years, iss c , who were directly admitted to our level i trauma center, were evaluated. blood samples were taken initially and on day , , , , , , and during hospitalization. luminex multi-analyte-technology was used for analysis of tpa and pai- antigen levels. results: both levels were particularly high at admission. although they significantly declined within three and seven days, respectively, they remained elevated throughout three weeks. throughout this observation period mean tpa antigen levels were higher in polytrauma victims suffering ards than in those without ards, whereas mean pai- levels were higher in polytrauma victims sustaining pneumonia than in those without pneumonia. noteworthy, in each patient, who developed ards, the tpa antigen level raised up to the onset of the syndrome and declined afterwards. conclusions: the development of ards has to be expected in a polytrauma victims if the tpa antigen level continues to rise after admission. potentially, in patients with a low risk of excessive bleeding the onset of the syndrome might be prevented by the timely administration of recombinant profibrinolytic proteins. motocross is a dangerous business: small bowell perforation case report case history: a year-old male, previously healthy, was admitted to the ed after being involved in a motorcross accident. he suffered blunt abdominal trauma. clinical findings: at admission, patient presented pale but haemodinamically stable. physical examination was unremarkable except for an evident abdominal wall hematoma and abdominal guarding over the left quadrants. investigation/results: abdominal ultrasound showed an intestinal loop with decreased peristalsis with a small amount of liquid adjacent (fig ) . due to the patient's haemodynamic stability, ct scan was performed (fig . ) which showed liquid in the left flank and iliac fossa, but without an identifiable intrabdominal lesion. diagnosis: the patient was admitted to the operating theatre with acute abdomen. therapy and progressions: intraoperatively fecal peritonitis was evident from a cm-hole on the antimesenteric border of the jejunum, the enterotomy was closed and profuse lavage was done; the abdominal wall closed without drainage. the patient went through an empirical antibiotic cycle. liquids per os were started on the first postoperative day and the patient progressed without issues. he was discharged at the th postoperative day. the remaining follow-up was uneventful. comments: small bowel perforation after blunt abdominal trauma is rare. sbmi has a high morbidity and mortality that increase with delayed diagnosis; however, clinical and radiographic signs of perforation are often absent, like in the case presented. ct is considered the gold-standard. in our specific situation, the small bowel perforation did not produce any pneumoperitoneum in a young patient with very good physiologic status that kept him hemodynamically stable. the prognosis of pelvic injury is closely related to the severity of vascular injury rather than the complexity of bony fracture y. wu , c. hsieh , c. fu chang gung memorial hospital, trauma and emergency surgery department, taoyuan city, taiwan introduction: pelvic injuries are among the most dangerous and deadly trauma. although complex pelvic fractures are often associated with vascular injuries, it is still unclear regarding the impact of the severity of vascular injury to the outcome of patients. we hypothesized that, in addition to the complexity of bony fracture, the severity of pelvic vascular injury plays a more decisive role to the patients'' outcome. material and methods:medical records of patients with pelvic fracture in a single trauma center between jan and dec were retrospectively reviewed. those who had an abbreviated injury scale (ais) c other than pelvis were excluded. based on ct results, the type of pelvic fracture was classified according to young-burgess classification, and the severity of vascular injury were recorded as minor (fracture with or without hematoma) or severe (hematoma with contrast pooling or extravasation). the patient demographics, clinical parameters, and outcome measures were compared between the groups. results: among the patients, severe vascular injury were noted in patients. patients with severe vascular injuries had significantly increased amount of red blood cell transfusion (rbct) ( . vs. . units, p = . ), longer icu stay (is) ( . vs. . days, p = . ) and total hospital stay (hs) ( . vs. . days, p = . ) compared to minor vascular injuries. on the other hand, those with complicated pelvic fracture (lc type ii/iii, apc type ii/iii, vs and combined type) had similar amount of rbct and is compared to that of simple pelvic fracture (lc type i, apc type i) except a longer hs ( . vs. . days, p = . ). conclusions: our results indicated that the severity of vascular injury is more closely correlated to the outcome of patients with pelvic fractures than the type of bony fracture does. in addition to the type of bony fracture, the grade of vascular injury should be considered as an important part of pelvic injury classification. associated abdominal injuries do not influence reduction quality in operatively treated pelvic fractures-a multicenter cohort study from the german pelvic registry results: . patients with pelvic injuries were treated during this period. . % had a concomitant abdominal trauma. the mean age was . ± . years. comparing the two groups, patients with a combination of pelvic and abdominal trauma were significantly younger ( . ± . vs. . ± . years; p \ . ). both, complication rates ( . % vs. . %; p \ . ) and mortality ( . % vs. . %;p \ . ) were significantly higher. in the subgroup of acetabular fractures, the time until definitive surgery of the pelvis was significantly longer in the group with the combined injury ( . ± . vs. . ± . days; p \ . ) . the grade of successful anatomic reduction did not differ between the two groups. conclusions: patients with a pelvic injury have a concomitant abdominal trauma in about % of the cases. the clinical course is significantly prolonged in patients with a combined injury, with increased rates of morbidity and mortality. however, the quality of the postoperative results is not influenced by a concomitant abdominal injury. a. martins rangel , r. pozzi , j. alfredo cavalcante padilha , s. sardinha , f. eduardo silva , d. teixeira rangel heat, trauma center, são gonçalo, brazil f.f.c., male, years old, was admitted to the trauma center about h after a stabbing wound in the neck. upon examination the patient was mechanically ventilated and hemodynamically stable, with an exposed sectioned trachea, which had a tracheostomy tube applied. the penetrating injury itself was mostly allocated in zone ii. he had a ct angiography and was referred to the or for surgical treatment. the cervicotomy found that both the external and internal right jugular veins had been injured alongside the sternocleidomastoid, sternohyoid and homohyoid muscles, the thyroid cartilage, just above the vocal cords, which had exposed the anterior larynx and the epiglottis the right anterior jugular vein and smaller tributaries of the right internal jugular vein, were ligated; a tracheostomy was performed and the thyroid cartilage and anterior laryngopharyngeal wall were reconstructed with the epiglottis implantation, sternoid, homohyoid and sternocleidomastoid muscle sutures, after which the platysma was closed but not the skin, left to secondary healing. patient was extubated within h, discharged from icu on the fifth postoperative day. thickened oral diet was introduced on the th day, and by the th day he was discharged without the tracheostomy tube, with a normal diet. comments: the cervical region is an area susceptible to serious injury due to the presence of vital structures, with massive hemorrhage, airway obstruction, cervical spine injuries and cerebral ischemia as the leading causes of death. initial management of penetrating injuries follows the principles of trauma care with airway control initially. references: bhatt nr-penetrating neck injury from a screwdriver: can the no zone approach be applied to zone i injuries? bmj yan wang-penetrating neck trauma caused by a rebar-a case report. medicine ( ) introduction: annually, approximately , people decease as a result of a fall in the netherlands, according to the statistics netherlands. the aim of this study is to evaluate the demographic parameters, fall characteristics and resulting injury patterns of this group in the region of amsterdam. methods: all patients deceased as a result of injury due to a fall in the period july st until july st in the region of amsterdam were included. data were collected from the database (formatus) of the department of forensic medicine (public health service amsterdam). results: during the study period , patients deceased after a fall. the mean age was years ( - years) and % was male. a psychiatric disease was diagnosed or suspected in % of the population of which cognitive impairment, including dementia, was encountered in most of the cases ( %). the majority of the falls happened at home ( %) or at nursing facilities. a minority ( . %) was work related. over % of the falls was from standing position, . % was not from standing position of which . % regarded falls from stairs, the majority was male. multitrauma patients accounted for . % of the population. from the remaining , patients, . % sustained one or more injuries to the pelvis or extremities. central nervous system (cns) injuries were described in . % of the patients. mortality was in . % of the cases due to primary cns injury, . % was due to complications of which clinical deterioration ( . %) and infection ( . %) were the most common. conclusions: in the region of amsterdam the majority of deaths due to a fall regards the geriatric population. fall from standing position and mortality due to complications, mainly clinical deterioration, accounted for the majority of deaths. intervention to prevent falls and thereby complications need more awareness to reduce mortality. results from a multidisciplinary blunt splenic injury protocol introduction: the majority of splenic injuries are currently managed non-operatively. failure of non-operative management includes grade iv or v splenic injury or vascular abnormalities that are suitable for embolization. the primary indication for operative management of blunt splenic injury is hemodynamic instability. in our center, the last twenty splenic injuries, admitted during two years, were not managed according to published guidelines. ten patients ( %) underwent splenectomy, being unstable only of them ( % of the whole sample). material and methods: staff from anesthesiology, interventional radiology and trauma surgery came up with a joint protocol. grade iii splenic injury non-operatively management, including fluid responsiveness (achieving shock index (ht/bp) below . after a bolus of colloids) and, focus placed only on hemodynamic stability instead of on vascular abnormalities are our principal modifications regarding already published protocols. results: seventeen patients with blunt spleen trauma were admitted after starting up our protocol. six ( grade iii, grade ii and grade i) splenic injuries were successfully managed non-operatively. prophylatic embolization was performed in five patients: were grade iv spleen trauma and were grade iii spleen trauma with vascular abnormalities. one grade iii splenic trauma was embolized due to a pseudoaneurysm detected in ct scan performed h post injury. five grade v spleen trauma required urgent surgery. of them presented with shock index [ . . conclusions: our multidisciplinary protocol has helped in improving outcomes in blunt splenic injuries. we have achieved an almost full compliance to our protocol. case history: -year old male experienced severe blunt trauma after a bus accident. clinical findings: he is found alert (gcs = ), hemodynamically stable and with a patent airway. he presented catastrophic lower left limb where tourniquet was applied. gram of tranexamic acid (txa) and ml of crystalloids were administered. he was intubated in the site of injury and transfered to our center, being always hemodynamically stable. on hospital admission he was normotensive (bp = / mmhg, sinus rithm ppm), shock index \ . . he suffered uneventfully amputation of the limb with no need for blood products transfusion. his past medical history was only pertinent for hypertension. investigation/results: following urgent damage control surgery, ct scan was performed where acute bilateral pulmonary embolism was diagnosed. diagnosis: asymptomatic acute bilateral pulmonary embolism therapy and progressions: during icu stay, the patient kept hemodynamically stable. endotracheal tube is removed one day later and he is successfully transfered to the ward three days later. comments: hypercoagulability can occur after severe tissue injury, that is likely related to tissue factor exposure and impaired endothelial release of tissue plasminogen activator (tpa). in contrast, when shock and hypoperfusion occur, activation of the protein c pathway and endothelial tpa release induce a shift from a procoagulant to a hypocoagulable and hyperfibrinolytic state with a high risk of bleeding. it can be inferred that a patient presenting with severe tissue injury without shock is at high risk of perioperative thrombosis and txa might not be administered. ( ) . it signifies high energy force, representative of severe overall trauma. study reported mortality of blunt pelvic trauma to reach . - % ( ) . injury severity score (iss), hypotension, head injury, posterior fracture & haemorrhage have been implicated ( ) . however, there is a paucity of data in developing countries. this study identifies the problem burden, management outcomes and factors predicting mortality. material and methods: patients had pelvic trauma, retrospectively from jan to dec and prospectively from may to april . patients was included after excluding less than years and coagulation disorder results: majority were males ( . %),with a mean age of . . mechanism was rti ( . %) followed by fall from height ( %), railway accidents ( . %). mean iss & rts was . and . respectively. associated injury were long bone fractures ( . %), chest injuries ( . %).head injury ( . %). lateral compression ( . %), was the most common followed by anteroposterior compression ( %) & combined ( . %).majority underwent operative intervention ( . %) for pelvis or associated injury. the mortality rate was . % secondary to haemorrhagic shock ( . %) and sepsis ( . %). the factors were male gender, age, iss, rts, head injury, unstable pelvis. however, no association with haemoglobin, long bone fracture, and massive transfusion protocol was found conclusions: our study showed a mortality of . % which is comparing with previous study introduction: the number of patients admitted to oslo university hospital (ouh) due to bicycle trauma is increasing. we aimed to identify possible predictors of serious and fatal bicycle injury. material and methods: the ouh trauma registry was searched for patients treated for bicycle trauma between and . data extraction included putative predictors of serious and fatal injuries, defined as iss c and death within days, respectively. univariate analyses were performed and reported as odds ratios (or). p \ . was regarded as statistically significant. results: bicyclists were admitted, % were males, median age was years (range - ). injury mechanisms were single bicycle crash in %, collision with a motorized vehicle in %, bicycle vs. bicycle in % and others in %. serious injuries were seen in % and . % died. predictors for serious and fatal bicycle trauma are presented in figure . conclusions: we identified age c , high comorbidity and loss of consciousness (gcs b ) as predictors for both serious and fatal injury after bicycle trauma. single bicycle crash was the most common cause of serious bicycle injury in our trauma center. diagnosis, investigation and results: all case reports represent polytrauma patients with clinical worsening and admission to the icu, with subsequent development of acute respiratory distress syndrome (ards) refractory to primary measures. therapy and progressions: different mechanisms led to the development of ards in the different cases. on a primary approach, standard measures such as curarization, recruitment maneuvers, prone positioning and peep increase were applied whenever possible. an absence of improvement led to an almost inevitable need of extracorporeal membrane oxygenation (ecmo) rescue therapy. all patients responded positively to this treatment without major complications and were eventually discharged from the icu. comments: ards is a major cause of respiratory failure in polytrauma patients. among the many therapeutic options, ecmo emerges as a powerful tool as rescue therapy in respiratory failure refractory to all other measures, being the present case reports corroborative examples of its efficiency. introduction: nowadays when cities are improving fast and significantly, including transportation system, even more we encounter with high energy trauma . still the most vulnerable on the roads are pedestrians. material and methods: the analysis of the data collected prospectively from january to october was performed including the mechanism and diagnosis of polytrauma, patient demographics and the main outcomes. results: in total, patients were assessed according to the polytrauma protocol. the median age of the cohort was years (iqr - ), male patients, . % vs. . % females, p = . . the most frequent mechanism was a pedestrian struck by a vehicle in . % cases, and falling from a height of over m in . %. of those patients who had musculoskeletal injuries, in . % the trauma mechanism was a fall from a height and in . % pedestrians were struck by a vehicle, . % of patients who fell from a height and . % of those struck by a vehicle suffered visceral injuries. the most common cause of neurotrauma was a fall from a height in . %, and pedestrians involved in car accidents in . %. from the whole cohort, patients were not saved, resulting in a . % mortality rate. most patients ( ) who died had iss [ . the mortality reached . % among pedestrians struck by a vehicle and . % among patients who fell from a height of over m. conclusions: the most common mechanism in the cohort was a pedestrian struck by a vehicle, followed by falling from a height, with a predominant involvement of male patients. similarly, the most frequent cause of musculoskeletal injuries and visceral injuries was falling from a height and pedestrians struck by a vehicle, demonstrating an important direction for polytrauma prevention. introduction: recent reviews of uk trauma data show altering demographics. patients are increasingly older and sustain lower energy injuries, with falls \ m being the most common ( ) . material and methods: data collected over years in a major trauma centre was used to calculate injury specific admission rates, case fatality rates and injury specific mortality attribution. data on patient age, footwear, lighting, alcohol intoxication and previous admissions were collected in falls \ m resulting in mortality. results: patients sustaining falls \ m represented % of admissions and % of mortalities. all falls represented % of admissions and % of mortalities. case fatality of falls of \ m and [ m was . % and . %. all fall case fatality was . %. this was significantly higher than the case fatality of stabbings ( . %) and rtas ( . %). in falls \ m causing fatality, mean patient age was . years. % of patients aged - were under the influence of alcohol when falling, with % aged - , but only % patients aged - . % aged - who died when falling were wearing slippers. this increased to % in those aged - , and % aged - . % of falls occurred under daylight/full light. % of patients aged - who died after falling had been admitted to hospital within the last year, although this increased to % in those aged - , and % aged - . conclusions: falls were the most common cause for hospital admission, had the highest case fatality of injury mechanisms and caused the most patient mortality. alcohol intoxication was associated with falls in younger patients who died after falling, but this was less common in older patients. wearing slippers was less common in the young but significantly associated with fatal falls in older patients. these results offer a range of therapeutic targets when developing fall prevention strategies. introduction: the treatment of splenic lesions is determined by the hemodynamic situation, the degree of injury and the presence of bleeding. arterial embolization has expanded the indications of the conservative treatment. retrospective observational study on splenic traumatism and its therapeutic options. material and methods: a total of patients with splenic injury have been treated at our centre between and . patients were hemodynamically stable: were embolized and received a conservative treatment. patients were hemodynamically unstable: had a good response to the resuscitation treatment so they were embolized, but there was one patient who deceased because of other causes. from these patients, patients received splenectomy. results: the main objective of this study is to review the management of the trauma patient with splenic injury. of the total of patients with splenic trauma, average iss of , underwent splenic embolization, underwent urgent splenectomy and were treated with conservative treatment. the embolized, were hemodynamic unstable at arrival but responded to the fluid therapy, had a splenic lesion grade iv, a grade iii, grade ii and another a grade i. the success rate of embolization was % in the embolized patients. patient died, only one of them in the embolization group and was not related to the splenic trauma nor embolization, were in the urgent splenectomy group due to severity of trauma, died before receiving any treatment and in the conservative treatment group due to other complications. conclusions: patients who respond to volume or are hemodinamically with high-grade lesions, arterial embolization would be less aggressive treatment options with excellent results. haukeland university hospital, surgical unit/ regional traumacenter, bergen, norway, norwegian university of science and technology, trondheim, norway, haukeland university hospital, physical and rehabilitation medicine, bergen, norway, university of bergen, bergen, norway, st olavs hospital, physical and rehabilitation medicine, trondheim, norway introduction: during the past decades acute trauma care has improved through the development of highly specialized trauma centres and teams. since patients are considerable young when being affected, trauma may lead to life-long physical, cognitive and emotional constraints interfering with an independent self-determined life ( , ) . in , a revised national plan for the treatment of trauma patients in norway was published ( ) . the plan emphasizes the importance of rehabilitation and the need for early interdisciplinary rehabilitation. this study will examine in which extent patients receive rehabilitation in early phase after trauma as recommended in the norwegian national plan. in addition we will examine what follow-up patients receive after trauma, quality of life, functional level and use of health care and next-of kin resources. material and methods: patients admitted to regional trauma center in mid-or western norway in with niss c are recruited to participate. data will be collected from national trauma register, the norwegian patient register, the municipal patient and user register, data from statistics norway, the electronic patient record (epj) and the patient/relatives questionnaire. discussion: the results will be useful in the preparation of patient courses that comply with strong recommendations in the national trauma plan, ensuring equal treatment and raising awarness about rehabilitation for trauma patients. introduction: diaphragmatic lesions involve wounds and rupture of the diaphragm, through penetrating wounds or thoraco-abdominal trauma. their incidence is - %. the diagnosis may be late, despite the technical advances made by medical imaging. the choice of surgical approach and technique is still controversial. mortality is usually related to the associated injuries. the present paper analyzes the incidence of diaphragmatic lesions that occur in thoraco-abdominal trauma, their epidemiology, diagnosis and treatment. material and methods: we performed a retrospective study over a -year period ( - ) , in the surgical units of the emergency county hospital of braila, including all patients diagnosed with diaphragmatic lesions. results: during the study period, patients had thoracic-abdominal trauma. there were cases of blunt trauma and thoracic-abdominal trauma. our study involved cases of diaphragmatic injuries ( . %), by road accident and by white weapon. the sex ratio was : . the average age was years. chest radiography was a contributory preoperative diagnosis in cases. the diaphragmatic wound was on the left side in cases, and its average size was cm. the surgical procedure involved the reduction in the abdomen of the herniated viscera and the monoplane suture of the diaphragm by nonabsorbable ''x'' points in all cases. chest aspiration was the rule. there was only one death in a complex polytrauma case. case history: we report the one case which performed tae, angioplasty, thoracotomy, laparotomy and preperitoneal pelvic packing (ppp) in the hybrid emergency room (h-er). the patient was male in the s, who was riding on his motorcycle and fell from a m height. clinical findings: he was in shock state. diagnosis: we scanned cect and diagnosed subdural hematoma, traumatic subarachnoid hematoma, lt hemopneumothorax, lung contusion, multiple costal bone fracture, intercostal artery injury, splenic injury (gradeiii), pelvic bone fracture. therapy: we inserted the drainage tube to the hemopneumothorax and did the tae for the pelvic bone fracture and splenic injury. after tae, he was in still shock state. the bleeding volume from the lt drainage tube increased, so trauma surgeons did the emergency thoracotomy and thoracic endovascular aortic repair (tevar) for intercostal artery injury. we suspected he also had abdominal compartment syndrome due to recanalization of tae, and they performed the emergency laparotomy and did ppp for the pelvic bone fracture. comments: we install an ivr-ct system in our trauma resuscitation room in october . we named it h-er, as it enables us to do all examinations (sonography, ct and fluoroscopy) and treatments (ir, operation) required for trauma in a single room. we have to perform prompt diagnosis and treatment, especially in cases of severe polytrauma cases. a retrospective study proved that the h-er had shortened the time of ct initiation and emergency procedure and that lead to improve mortality ). h-er is a novel trauma resuscitation room to do all treatments required in the only one room for severe traumatic patients introduction: according to the previous advanced trauma life support (atls) guidance, the early assessment of trauma patients with haemorrhage were classified upon the vital signs. recently, national trauma registry analyses suggested to extend the assessment criteria with the base deficit (bd), referring to the metabolic status. our objective was to investigate the relevance of bd and to explore new prognostic factors in the early assessment of the severely injured. material and methods: our study included patients registered between . . and . . on our emergency ward for whom the trauma team was activated. they were grouped into severity groups (i-iv) according to either the vital signs (classical) or the extended criteria with bd. the data were extracted from medical documentations of the early phase of treatment. as primary outcome, we compared the -h mortality rate of the patient groups. we studied the need for massive transfusion and intensive care unit care as secondary outcomes. results: according to the classical assessment, % of the patients were assigned to group i (lowest risk for haemorrhagic shock) and % to group ii. the remaining % were grouped into groups iii and iv (higher risk). with taking bd into consideration, % were reassigned to a higher risk group; however, this change affected only groups i and ii. the -h mortality changed only in group i ( . % vs . %; p = . ). bd did not affect the need for massive transfusion. in groups i and ii, . % of the patients, in groups iii-iv % needed intensive care unit treatment. conclusions: bd is an effective prognostic factor in the early assessment of trauma patients. however, compared to the vital signbased evaluation, it provides extra informaton only in less severe cases. according to our findings, it may help to assess the need for the administration of blood products. grants: nkfi k ; ginop- . . - - - ; efop- . . - - - . complejo hospitalario de jaén, servicio de cirugía general y del aparato digestivo, jaén, spain, complejo hospitalario de jaén, servicio de anestesiología y reanimación, jaén, spain case history: years old male, with history of hypertension and dyslipidemia, suffered a backhoe accident and was admitted in a regional hospital. on initial assesment he presented contusion and two laceration wounds in left chest and in lumbar region. body ct informed subcutaneous emphysema and left rib fractures from th to th, left hemidiaphragm edema, laminar left pneumothorax and contusive lung. posterior lumbar hematoma and no intra-abdominal free fluid. laceration wounds were partially sutured, with drainages through the wounds clinical findings: he was transferred to our emergency department, presenting dyspnea, tachycardia, sweating, painful luq and left hemithorax worsening with breathing investigation/results: reviewed by our radiologist, tc images showed herniation of abdominal organs into the chest diagnosis: traumatic hernia in left costophrenic recess. multiple rib fractures therapy and progressions: the hernia contents (left colonic flexure and omentum) were reduced and defect closed with primary repair in emergency surgery. rib fractures treated by osteosynthesis.on th pod left renal artery dissection and renal infarction were evidence in a new ct. comments: diaphragmatic injuries are caused by blunt or penetrating thoraco-abdominal trauma. potentially life-threatening due to the herniation of abdominal organs and severe associated lesions. clinical suspicion is important as prompt diagnosis and treatment are necessary for good outcomes. in our case, the initial clinical assessment was incorrect and the transfer put the patient in danger as an emergency surgery should have been performed before transfer. this enhances the importance of a correct initial management of polytrauma patients. introduction: the fractures of the calcaneus account for about - % of all fractures of the human skeleton. the majority of these fractures ( %) are intra-articular and surgical intervention is a widely accepted way of treatment material and methods: the aim of this study was to evaluate the results of open reduction and internal fixation for di-afc.in a period of years ( - ) patients ( patients with bilateral fractures) with age range from to years old, were treated surgically using the lateral extensile approach. follow-up was - years. the results were evaluated based on x-ray parameters (calcaneal morphology, bohler''s and gissane''s angles), active range of motion, footwear problems and time needed to return to work. the sf- health survey was used for outcome assessment. results: fracture mean healing time was , weeks. the outcome was excellent in cases, good in cases and poor in cases. the complications were malposition of fixation in patients, superficial wound slough in patients, reflex sympathetic dystrophy in patients, deep infection in patients who were treated with antibiotics and metalwork removal following union of the fracture. one patient resulted in metal breakage with consequent pseudarthrosis. finally one patient developed chronic osteomyelitis and is under treatment. the treatment with open reduction and internal fixation for di-afc is indicated, provided that the restoration of calcaneal shape, alignment and height is achieved. long term functional results with mild pain, few alterations in activities of daily living or work, and essentially no footwear problems, can be expected from a properly performed open reduction and internal fixation. extraperitoneal rectal injury in emodinamically unstable patient treated after dcs with external traction applied in an endorectal balloon r. somigli hospital, general and emergency surgery, pistoia, italy case history: a -year-old man was crushed between two vehicles while he was working. he arrived in er hemodynamically unstable, so he underwent to emergency surgery. clinical findings: at rectal examination there was evidence of almost complete antero-lateral anorectal laceration. at abdominal examination there was evidence of anorectal full-thickness laceration and urethra full laceration. investigation/results: no diagnostic was required in preop because of patient instabilty. diagnosis: pelvic fracture with hemodynamic instability, severe rectal injury and complete prostatic urethra transection. therapy and progressions: el, lateral colostomy, pelvic paking, cistostomy and hip external binder. damage control surgery was performed. on pod second look was carried out and an almost complete extraperitoneal rectal injury was found during pelvic depaking. properitoneum was drained and a baloon probe was introduced in the rectum to allow the proximal rectal flap to advance to the distal rectum. stomal washes were performed with no rectal leak and rectal baloon traction mantained for days. radiological and endoscopic check haven't shown any leak and a good mucosal reconstruction. mri no sphincteral anatonical defects. waiting for emg before stoma reversal. comments: the optimal managment for extraperitoneal rectal injuries remains controversial. an approach with lateral colostomy and conservative treatment of rectal lacerations with rectal trac-tion baloon, could represent a safe treatment alternative in those cases with sphincter preservation, with a lower risk of complication. exploring differences between iss and niss scores for -day mortality in adult and elderly trauma patients in a norwegian national trauma cohort m. introduction: injury severity score (iss) and new injury severity score (niss) with a threshold over is commonly used to define severe injury and to define the study population in trauma registrybased studies for both adult and elderly trauma patients ( ) . for elderly patients (c years) this might be unreasonably high and might lead to exclusion of significantly injured elderly with increased risk of mortality. the aim of this study was to assess whether there were significant differences in -days mortality between adults and elderly trauma patients for different frequently used iss and niss thresholds material and methods: the norwegian trauma registry was interrogated to identify all adult (c years) trauma patients included in the registry from january through december . data were dichotomized to age groups ''adult'' and ''elderly'' ( - and c respectively) with -days mortality as primary endpoint. mortality rates were assessed for iss and niss thresholds of [ , [ and [ . we applied descriptive statistics and chi-squared test for comparisons. results: patients with available information about age, -days mortality and iss and niss scores were included in the analysis, of which patients were - years old and patients were c years. adult and elderly patients died, giving overall mortality rates of . % and . % respectively. for iss and niss [ there was a significantly higher -days mortality in elderly trauma patients ( . % and . % respectively) than adult patients ( . and . % respectively) (p \ , ), as for all other iss and niss thresholds tested. conclusions: this study demonstrates that elderly trauma patients has a significantly higher mortality risk than adult trauma patients at all iss or niss-thresholds analysed. this group has a significant mortality even at iss and niss above . introduction: the trauma tertiary survey (tts) is a widely accepted tool in the prevention of missed injury. existing literature on its effectiveness focusses on multitrauma patients. this study investigates the yield of the tertiary survey in trauma who are admitted for tts, without having any significant injury. material and methods: a single center retrospective cohort study was performed in a level ii trauma center. trauma patients without any clinically significant injury at the primary and secondary survey were included. the primary outcome was missed injury found during tts (type ). secondary outcomes were missed injury found after tts but during admission (type ), mortality and hospital length of stay [ days. results: from included patients, patients ( . %) had a type missed injury. alcohol consumption was associated with an increased risk for type missed injuries (odds ratio = . , % ci: . - . ) . a type missed injury was only found once, it concerned the only case of trauma related mortility. out of nonoperated patients, ( . %) were admitted for more than two days. these patients were significantly older ( vs. years, p \ . ) and had a higher asa classification, - vs. - ( . % vs. . %, p \ . ). conclusions: tts showed missed injuries in only . % of trauma patients who had no clinical significant injury found during primary and secondary survey. high costs of admission, together with a low yield found for this study's population the cost benefit of hospitalizing these patients is for discussion. future research should therefore focus on the identification of predictors of a positive tertiary survey. references: . advanced trauma life supportÒ student course manual. . keijzers, et al., the effect of tertiary surveys on missed injuries in trauma: a systematic review. . enderson et al., the tertiary trauma survey: a prospective study of missed injury. the -h rule in the emergency department and its association with surgical mortality in one public hospital in israel: retrospective study i. ashkenazi hillel yaffe medical center, hadera, israel introduction: in order to improve patient treatment the -h rule in the emergency department (ed) was introduced in many countries as well as in israel. within four h, patients attending the ed must be seen, treated, and a decision must be reached whether these patients are to be admitted or discharged. though a popular performancebased measure, whether the -h rule in ed is associated with a decrease in mortality is controversial. the primary objective of this study was to evaluate the association between time in the ed and surgical mortality in one public hospital in israel. material and methods: included in this retrospective study were patients admitted to the ed of hymc during . patients dying on the first day were excluded. . results: included in this study were , patients. of these, , ( . %) patients were hospitalized and the rest were discharged. overall, patients died. general surgery accounted for , patients of which died ( . % of hospital deaths; . % of all surgical patients; . % of patients hospitalized in general surgery). internal medicine together with general surgery and orthopedic surgery accounted for . %, . % and . % of the mortalities observed in patients with decisions made within - h, in patients with decisions made beyond h and in all the patients respectively. forty-five patients with decisions made within h died compared to with decisions made beyond h. these represent . % and . % of all surgical patients in the ed (whether hospitalized or discharged) and . % and . % of those hospitalized. conclusions: general surgery is the second largest contributor to hospital morality. in both absolute terms and relative terms, mortality was not increased by delays in decisions made beyond h. the adoption of this performance-based measure should be questioned. introduction: trauma is an important cause of mortality [ , ] . researchers are looking for optimal death/survival predictive models in trauma population. one way is to validate traumatic scores for different medical systems [ ] . the aim of our study was to validate the new injury severy score (niss) in severe trauma ( introduction: the international classification of diseases-based injury severity score (iciss) has been proposed as a reliable tool to measure trauma system performance especially in countries where a trauma registry has not been yet established. the purpose of this study is to assess the predictive capability for in-hospital mortality of iciss with international and adjusted survival risk ratios (srrs) in greek trauma population. material and methods: this single center, retrospective cohort study was conducted in a greek tertiary care hospital between january to december . the trauma population was defined as hospitalized patients with a principal hospital discharge diagnosis in the range icd- s -t . duplicated injury icd codes, readmissions, transfer to another hospital and missing data were excluded. the primary outcome was in-hospital mortality. adjusted srrs was calculated from patients with multiple injuries and the following two iciss scores were evaluated: multiplicative-injury (iciss) and singleworst-injury (swi). the models were assessed in terms of their discrimination, measured by receiver operating curve (roc) analysis and calibration measured using calibration curves. results: a total of patients were included in the study. median age was ± years and mortality rate was , %. based on international srrs, the area under the curve was , ( % ci . - . ) for iciss-multiplicative and , ( % ci . - . ) for iciss-worst injury. both modes had statistically significant better performance with adjusted greek srrs (aur = , % ci . - . and aur = , % ci . - . , respectively). conclusions: this analysis has demonstrated the validity iciss model for in-hospital mortality prediction in greek trauma population. further research is warranted to confirm the performance of iciss using a sufficiently sized sample to define national srrs. introduction: the occurrence of intra-abdominal abscesses is the most serious post-operative infective complication after appendectomy. a significant amount of research has been conducted in an attempt to identify those patients at greatest risk. pct is initially described as an early, sensitive and specific marker for sepsis associated with bacterial infection. we hypothesize that pct could serve as a predictor of the development of intraabdominal abscess and postoperative infective complication material and methods: the present study is a prospective, single centre, observational cohort study involving patients undergoing emergency appendectomy. all patients admitted to the acute care surgery ward for appendicitis were screened for study eligibility. pct poc samples will be obtained preoperatively (t ) and post procedure (t ) at h (t ), h (t ), and days (t ) post procedure. the primary objective of this study was to assess the diagnostic accuracy of point-of-care testing for pct in identifying post appendectomy abscess. the secondary objective was to determine the diagnostic accuracy in identifying any infective complication conclusions: we expect the incidence of abscess and infective complication to be increased in the pct elevated group compared with the control group. previous investigations indicate the overall morbidity related to infective complication is approximately - % of patient undergoing laparoscopic appendectomy. our pilot study revealed that the incidence could be as high as % in patients with prolonged elevated pct levels. introduction: hand trauma is a common cause for attendance to the accident and emergency (a&e), accounting for nearly - % of all patients . it is essential that accurate treatment and management is done as the implications of mismanagement are long term, which may lead to disability, loss of work and income, livelihood, and even psychological issues . the presence of a specialised hand surgeon is essential for management of these injuries , but in the a&e setting it is not always possible to have such specialised care and there is a need for an efficient triage system. materials and methods: we did an audit in our department and found a delay in the referral of patients from a&e to our trauma clinic, which was quite expected due to a high patient inflow. we devised a trauma pathway for the a&e, known as the d-system which outlines for them till what day from trauma is a particular hand patient safe to be sent to the hand clinic or who needs an urgent referral to a higher trauma centre, based on urgency of need of intervention. the pathway is in the form of a simple flowchart, which is easy to understand even for junior members of the team. we intend to do another audit after implementation of the pathway to assess change in practice. conclusion: it is essential to have simplified pathways for non-specialist areas in order to streamline treatment and offer the best care, in the limited availability of resources, especially at smaller hospitals. our aim is to develop one such system and assess it's effective in delivering better care. introduction: a quantitative method for measuring trauma severity has many potential applications. the intent of this study was to evaluate the accuracy of the mgap score and its components in prediction of in-hospital mortality versus the accuracy of the revised trauma score rts at a trauma center. material and methods: this study included patients with trauma. data regarding age, mechanism of injury, systolic blood pressure, glasgow coma score and respiratory rate were collected at trauma center of alberto torres hospital. mgap and rts scores were calculated, and their accuracy to predict survival/death outcome. results the study included patients, ranging in age from to years, % male. from the total sample, patients who suffered from penetrating trauma and patients who suffered from blunt trauma were observed. in the comparison of the scores, rts and mgap, there was no significant superiority in any of them for predicting the outcome -which in our study was hospital discharge or death -even when compared by trauma mechanism. the gcs proved to be a very sensitive criterion in both scores, especially in patients with traumatic brain injury, totaling patients in our statistical analysis, of which , % had a negative outcome. rts was slightly superior than mgap in patients classified by the score as high chance of mortality, with % versus % of assertiveness. conclusions: up to the moment, there is no evidence to support the superiority of one of the analyzed scores as a predictor of mortality in the patients evaluated. although the rts score is more widely used in trauma centers, the application of the mgap score is more feasible in pre or in-hospital care of polytrauma patients, since it does not use respiratory rate in its parameters. validation of d-dimer for screening for venous thromboembolism in pelvic and lower extremity trauma patients t. uehara , , t. noda , t. yumoto , n. kobayashi , a. nakao , t. ozaki okayama university, emergency healthcare and disaster medicine, okayama, japan, okayama university, orthopaedic surgery, okayama, japan, okayama university, musculoskeletal traumatology, okayama, japan, okayama university, emergency and critical care medicine, okayama, japan, okayama saidaiji hospital, okayama, japan introduction: venous thromboembolism (vte) is a life-threatening complication after major trauma patients. we previously reported that the patients with higher injury severity score (iss) and lower extremity trauma had high risk for vte. additionally, high d-dimer levels (cut-off d-dimer value, . lg/ml) on day were useful for screening for vte in major trauma patients. we validated d-dimer levels for vte screening for patients with pelvic and lower extremity trauma. material and methods: a retrospective study was undertaken between april and august at the okayama university hospital. patients with pelvic or lower extremity trauma were included (median iss, ). we collected following data; age, sex, iss, the number of operation times, value of d-dimer in screening, incidence of vte and use of anticoagulants. results: eleven patients showed high d-dimer levels in screening, furthermore, six patients were diagnosed vte using contrast-enhanced computed tomography. symptomatic pulmonary embolism was not occurred. patients with vte had undergone orthopaedic surgeries two or more times. fourteen patients received therapeutically or prophylactic anticoagulation therapies. conclusions: measurements of d-dimer levels after pelvic or lower extremity trauma patients were useful for screening of incidence of vte. direct oral anticoagulants were convenient for treatment to vte. trauma patients often needed several times of surgeries, heparin was also useful in perioperative period. introduction: early assessment of the clinical status of severely injured patients is crucial for guiding surgical treatment. several scales are available to differentiate between risk categories. we compared four established scoring systems in regard to their predictive abilities for early versus late in-hospital complications. methods: database from a level i trauma center. the following four scales were tested: the clinical grading scale (cgs; covers acidosis, shock, coagulation, and soft tissue injuries), the modified clinical grading scale (mcgs), the polytrauma grading score (ptgs), and the early appropriate care protocol (eac; covers acid-base changes). admission values were selected from each scale and the following endpoints were compared: mortality, pneumonia, sepsis, death from hemorrhagic shock, and multiple organ failure. results: in total, severely injured patients were included (mean age, . ± years; mean iss, . ± . points; incidence of pneumonia, . %; incidence of sepsis, . %; death from hem. shock, . %; death from multiple organ failure (mof), . %; mortality rate, . %). istinct differences in the prediction of complications, including mortality, for these scores (or ranging from . to . ). the ptgs demonstrated the highest predictive value for any late complication (or = . ), sepsis (or = . , p = . ), or pneumonia (or = . , p = . ). the eac demonstrated good prediction for hemorrhage-induced early mortality (or = . , p \ . ), but did not predict late complications (sepsis, or = . and p = . ; pneumonia, or = . and p = . ) cgs and mcgs are not comparable and should not be used interchangeably (krippendorff a = . ). conclusion: our data show that prediction of complications is more precise after using values that covers different physiological systems (coagulation, hemorrhage, acid-base changes, and soft tissue damage) when compared with using values of only one physiological system (e.g., acidosis). none of the authors have any conflicts of interest to declare. mortality rate related to trauma mechanisms in trauma center at alberto torres hospital from january to july r. p. pereira , r. adriana martins , j. a. c. padilha , f. e. silva , , d. rangel alberto torres hospital, trauma center, são gonçalo, brazil, federal university of rio de janeiro, niterói, brazil introduction: to demonstrate the healthcare services of the trauma center of rio de janeiro based on epidemiological data and on the specificity of the type of initial care delivered to multiple trauma patients, comparing the mortality rate at the second peak of death with the worldwide literature. materials/methods: retrospective study extracted from ''ct heat'' database. polytraumatized patients of both sexes were included and the mortality rate was calculated taking into account the second peak of death from trauma, gender, age and primary mechanisms of injury. discussion: the data collected show % mortality in the second peak, with firearm projectiles ( %) followed by traffic accident and fall as the primary causes of death. conclusion: because of the structural and health care profile of this trauma center, it was possible to reach the desirable mortality rate according to the worldwide literature (less than %). introduction: trauma patients are sometimes in critical condition upon arrival and need aggressive treatments to survive. despite all efforts many end up dying. it seems necessary to try to identify those patients with a very high risk of death to avoid futile treatments. the aim of our study was to develop a simple clinical tool to predict mortality in trauma patients that can be easily calculated in the ed. material and methods: we analyzed data from all trauma patients arriving at a spanish trauma hospital from june to june . patient demographics, physiologic trauma scores, vital signs, and glasgow coma scale (gcs) were recorded. our primary outcome was mortality. logistic regression analysis (lra) was performed using three variables (age, shock index (si), and gcs) to determine the appropriate weights for predicting mortality. using them, we constructed a simple score to calculate mortality. results: patients were studied. the mortality rate was . %. our score was constructed using weights derived from lra for age [ y ( points), si [ ( points) , and gcs conclusions: our score is easy and quick to calculate and could be a useful tool to predict mortality using early available parameters upon arrival in the ed. acknowledging the ethics involved in this topic, this score could sort out patients with a very high risk of death and in whom aggressive therapeutic measures could be limited early or withdrawn in agreement with family members references: haider a, et al ( ) ( ) ( ) states the average cost for an a&e attendance and non-elective inpatient stay is £ and £ , respectively highlighting the importance for schemes to reduce hospital admissions. assess impact of ambulatory care, surgical emergency assessment unit (seau) and ''emergency surgeon of the week'' (esw) on hospital admissions for surgical referrals (gp/ a&e). material and methods: retrospective analysis of prospectively collected data of hospital admissions from the patient centre database before and after implementation of seau (in november ) and esw (in november ), including the units'' activities. emergency general surgeon followed : (monday-thursday, - ) rota based at seau. results: since ( months), seau has reviewed (new ; follow ups ) patients. surgical admissions (sa) pre and post implementation seau were * and */month respectively, a drop by %. esw helped a further drop by another % to */month. % of new referrals were admitted and overall % of all patients reviewed were admitted. juniors (st /st ) and seniors (st - /staff grades/consultants) admitted % and % of the referrals respectively. uss and ct were performed in dedicated seau slots. % attending seau were likely to recommend the unit to friends or relatives. conclusions: in the face of unprecedented demand for hospital beds (more so in the winter), ''emergency surgeon of the week'' based at seau could be the answer to relieving the capacity, financial pressures and providing high quality safe patient care for our already strained nhs. surgical emergencies, an educational and medico-economic challenge introduction: surgical emergencies are a frequent reason for consultation in the emergency department and are responsible for significant morbidity and mortality. our study aims to present the number of patients admitted for a surgical emergency in a french level trauma-center and the volume of patients operated in emergency depending on the different specialties. method: we conducted a retrospective, single-center study of the hospital emergency department (uas) of the university hospital center of nice between january and december . we studied the volume represented by surgical emergencies according to the different specialties. results: the emergency department welcomed , patients, of which , surgical emergencies patients accounted for % of the total activity; patients were operated on urgently, which represents % of all surgical procedures in our hospital. conclusion: surgical emergencies are an important part of the activity of our hospitals. an academic definition is difficult to achieve. a regional organization is needed for the management and optimal care of these patients. the creation of regional centers, as for the trauma centers, seems indispensable, especially for the most serious patients, allowing both a better medico-economic and educational management of surgical emergencies. introduction: every new admission to the icu prompts a handover from the referring department to the icu staff. this step in the patient pathway provides an opportunity for information to be lost and for patient care to be compromised. mortality rates in intensive care have fallen over the last years, however, % of patients admitted to an icu will die during their admission ( ) . communication errors contribute to approximately two-thirds of notable clinical incidents; over half of these are related to a handover ( ) . nice have concluded that structured handovers can result in reduced mortality, reduced length of hospital stay and improvements in senior clinical staff and nurse satisfaction ( ) . material and methods: a checklist was created to review to score the handover. this was created with doctors and nurses and is relevant for handovers between all staff members. information was gathered prospectively by directly observing handovers on the icu. results: there is a notable discrepancy in the quality of handovers of new patients. this is true of handovers between doctors, nurses and a combination of the two. % (n = ) of patients weren't handed over to a doctor. the most commonly missed pieces of information were details of the patient's weight ( %, n = ), their height ( %, n = ), whether the patient has previously been admitted to an icu ( %, n = ) and whether the patient has any allergies ( %, n = ). conclusions: the handover of new patients to the icu is often unstructured and important information is missed. this can be said for all staff members and grades, and for handovers from all hospital departments. introduction: bowel resection for acute mesenteric ischaemia (ami) in elderly is associated with significant morbidity and mortality, and increasing age and frailty are associated with increased risk. this study aims to assess the short-term outcomes for elderly patients undergoing surgery for ami, and to assess the accuracy of surgical risk calculators in this population, to determine their utility in preoperative discussions. introduction: intertrochanteric femoral fracture of the super-elderly is often difficult to treat because good surgery does not always lead to good functional prognosis. we investigated the factors affecting the functional prognosis in patients with intertrochanteric fracture over years old. material and methods: cases of intertrochanteric fracture over years old who had undergone surgical treatment at our hospital between december and september were examined. nine men and women, age at injury ranged from to years, with a median of years. the average postoperative follow-up period was . months. for these cases, the mobility was classified into independent walking, assisted walking (cane, walker), wheelchair, bedridden, and the transition of pre-and postoperative mobility was analyzed. the significance test was performed using the mann-whitney u test, and p \ . was considered significant. results: by fracture type, when jensen classifications i and ii were stable, iii, iv, and v were unstable, mobility of unstable type was significantly reduced (p = . ). when the waiting period for surgery is divided by the median of days, there was no difference in mobility reduction between groups of less than days and groups of more than days (p = . ). although there was no significant difference in the presence or absence of preoperative rehabilitation intervention (p = . ), there was a tendency for less decline in mobility when preoperative rehabilitation intervention was performed. conclusions: in the treatment of this fracture, early surgical treatment after injury is recommended, but in the case of very elderly people, waiting is often required due to existing diseases and poor general condition . this study suggests the importance of preoperative rehabilitation intervention during the waiting period for surgery to prevent disuse disorders. references: . kelly-pettersson et al. waiting time to surgery is correlated with an increased risk of serious adverse events during hospital stay in patients with hip-fracture: a cohort study international journal of nursing studies ( ) - . older patients with traumatic shock exhibited lower pulse pressure compared with younger patients; an analysis of nationwide trauma data base in japan introduction: the study purpose was to assess the effect of age on the relationship between pulse pressure (pp) and systolic blood pressure (sbp) in patients with traumatic shock. material and methods: in this retrospective cohort study using nationwide trauma data base in japan from april to may , trauma patients years of age and older with sbp \ mmhg were selected. patients with severe traumatic brain injury (the abbreviated injury scale on head [ ) and cardiac arrest (hr = and sbp \ mmhg) were excluded. linear regression analysis assessed association between pp and sbp interacted by age group dichotomized as \ or c years old. results: during the study period, patients were included. the linear regression analysis indicated the significant association between pp and sbp in overall population (ec, estimated coefficient = . %ci [ . , . ], p \ . ). association between pp and sbp was significantly interacted by the age group (ec = . %ci [ . , . ] introduction: high rates of trauma in south africa (sa) predominantly affect the youth, yet the geriatric population is not exempt. in addition to inherent challenges of age, elderly trauma patients are further compromised by resource constraints. we aimed to assess injuries and outcomes in elderly patients admitted to a tertiary trauma unit in sa. material and methods: a retrospective record review was done of all patients years and older, admitted to the trauma unit over an -month period. injury severity score (iss), mechanism of injury (moi), in-hospital complications and length of hospital stay were documented. results: patients (mean age: years; % female) were included with mean iss of . the most frequent mois included nontraumatic falls ( %), falls from height ( %), motor-vehicle collisions ( %), pedestrian vehicle collisions ( %), and blunt injuries ( %, % intentionally inflicted). eighty patients ( %) experienced at least one in-hospital complication. the mortality rate was %. the mean length of hospital stay was days. conclusions: despite the known vulnerablities of the elderly, the mortality rate and isss of this cohort were relativley low. however, when compared to first world literature, intentionally inflicted injuries and certain preventable mois (e.g. fall from height and pedestrian vehicle collisions) were common, [ ] [ ] introduction: the majority of new colorectal cancer is diagnosed in people [ years, yet the elderly are less likely to undergo curative surgery. chronological age is poorly correlated with post-operative outcomes and is not an acceptable measure of risk. conversely, frailty is a strong predictor of poor outcomes following surgery and presents an opportunity for patient optimisation. the aim of this systematic review is to assess the available evidence between frailty and outcomes in patients of all ages undergoing surgical resections for colorectal cancer. material and methods: pubmed was searched for articles reporting outcomes for patients deemed frail undergoing elective or emergency colorectal cancer resection up until august . the primary outcome was mortality ( and day). secondary outcomes; length of stay, readmission, reoperation & post-operative complications. results: studies identified, studies were deemed eligible for inclusion. study types, frailty assessments & outcomes measured were variable. despite this heterogeneity, categorisation of ''frailty'' was associated with higher rates of post-operative mortality, complications, readmission, and length of stay. conclusions: based on current evidence, frailty is a strong predictor of poor clinical outcomes in patients undergoing surgery for colorectal cancer. standardisation of frailty assessment and measure of outcomes is needed for more robust analysis. accurate risk stratification of patients will allow us to make informed treatment decisions and identify patients who may benefit from prehabilitation and intensive tailored post-operative care. introduction: pneumatosis intestinalis (pi) and hepatic portal venous gas (hpvg) are two radiological findings associated with a broad range of medical conditions. pi can be primary ( % of cases),usually with a benign course, or secondary ( % of cases),which results from obstructive or ischemic gastrointestinal diseases. only a minority of pi is associated to free abdominal air. in literature there is no consensus on radiological and biochemical markers of favourable outcome nor on treatment options-medical or surgical. we tried to identify prognostic markers in a series admitted to a single university hospital. material and methods: the medical records of patients with pi and/or hpvg admitted to ospedale maggiore policlinico (milan, italy) in the period - were collected the ct scan were reviewed by a single radiologist. results: mean age was . ± years ( - ). pi was primary in , % of the patients (n = ), and secondary in , % (n = ). at ct, pi was associated to portal gas in patients ( %) ( dead, alive) and to free air in patients ( %) ( dead, alive). linear or rounded gas collections were equally distributed in primary and secondary pi. the colon was involved in patients ( %), followed by the small intestine in ( , %),and the stomach (n = ). in patients serum lactate was [ , and died. leucocytosis (wbc [ , /mm ) was present in patients ( alive).four patients had peritonitis and abdominal tenderness. laparotomy was performed in primary (alive) and secondary pi ( deaths).in two patients it was diagnostic; in and associated to right or left colectomy, in to ileal resection and in to other procedures.surgery was judged futile in patients; all died a few hours after emergency department access. conclusions: we could not found any relationship between clinical, biochemical and radiological findings and outcome of pi. mesenteric and portal gas is a ominous finding, but did not reach significant value. successful transcatheter arterial embolization for a giant pseudoaneurysm of gluteal muscle due to ground level fall in elderly woman with direct oral anticoagulants t. kadoya , r. nakama , k. arakawa , t. ogura , k. kase saiseikai utsunomiya hospital, department of emergency medicine and critical care medicine, utsunomiya, japan, saiseikai utsunomiya hospital, department of radiology, utsunomiya, japan case history: a 's year-old woman using apixaban fell on the ground and was transferred to previous hospital. magnetic resonance imaging was taken and she was diagnosed as gluteal hematoma. she was treated conservatively but hemoglobin (hb) level was gradually decreased. although she was administered red blood cell as needed, anemia progressed. contrast-enhanced ct showed expanding hematoma of gluteal muscle. she transferred our hospital for advanced treatment including surgery on th day on hospital. clinical findings: vital signs were stable on arrival at our hospital. extensive subcutaneous hematoma was found in the right thigh and gluteal lesion. investigation/results: laboratory test showed that hb . g/dl and normal coagulation status. contrast-enhanced ct showed a giant pseudoaneurysm inside the gluteal muscle. therapy and progressions: angiography showed a giant aneurysm of peripheral branch of internal iliac artery. we performed transcatheter arterial embolization (tae) for it by gelatin sponge. after tae, there was no complication and progressive anemia was stopped. she was transferred to another hospital for rehabilitation six days after tae. comments: increase use of direct oral anticoagulants in elderly people could induce severe hemorrhagic trauma by minimal mechanism. tae is minimal invasive and safety procedure for such trauma case. introduction: the number of elderly people will increase during the next few decades. more importantly, the number of people aged or above are projected to increase % in developed countries. in spain, people over age were . % of the population in , and this will increase to . % in . that has implications in the health services and in the management of trauma patients. material and methods: we did a retrospective cohort analysis of trauma patients c y.o. admitted to our level i trauma center during the time-period of - . demographic data, icu care, and mortality were assessed. results: trauma patients c y.o. were admitted during that period. this is a % increase compared with the number of patients admitted during the previous decade ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . mean age was . ± . years, and median new injury severity score (niss) was (interquartile range to ). % were male. the mechanism of injury was % falls, and % pedestrian runovers. patients were admitted to icu, with median niss of and mortality rate of %. among severely injured trauma patients (niss c ) the hospital mortality rate of those c years was %, much higher than in the age group of - years ( %), with a significant difference (p \ . ). no differences mortality rates between - years and youngers with the same niss. conclusions: the geriatric trauma patient population is on the rise worldwide. this should be taken into account in our trauma centres in order to be able to adapt and try to improve trauma care in these patients. introduction: frailty is a geriatric syndrome which has been considered as a risk factor in the elderly, increasing adverse events in terms of global health, as hospitalization, increase of falls, need of institutionalization, and mortality. the aim of this study is to evaluate relationship between frailty, and the presence of major complications in the postoperative course of patients older than years undergoing emergency surgery. material and methods: prospective, longitudinal, cohort study, using four different scales of frailty as a predictor of risk for short-term adverse events, for patients during the postoperative course of emergency surgery (may -september ). the sample is categorized according to four frailty scales (clinical frailty scale, frail score, trst and share-fi) . we analyze the variables regarding diagnoses, clinical examination at admission, surgical procedures, and postoperative outcomes during the first days. clavien-dindo classification was used in order to graduate the severity of complications. results: patients were included with a mean age of , years (sd , ) . , % of the simple are women. frailty prevalence ranges, according to the frailty scales, from , % to %. median hospital stay was days ( iqr , ) . all four frailty scales show statistical differences to predict major complication in our simple. trst and frail scales show the strongest measure of association (or , and , , respectively). the frail phenotype, is also related to an increased of mortality, and frail scale is the frailty scale with largest or (or = , ).only frail show association with longer hospital stay ([ days), and reoperation rate. conclusions: frailty represents a predictive marker of major complications and mortality, for patients older than years undergoing emergency surgery. frail score, shows the strongest relationship with mortality and complications. introduction: age has been identified as a predictor of trauma mortality [ ] and it is known that even low energy trauma may cause severe injuries in the elderly [ ] . the aim of this study was to explore how the elderly trauma patients, and the care thereof, differ from the younger ones in a swedish context. material and methods: the swedish trauma registry (swetrau) was used. consecutive recorded trauma cases that presented at one level ii trauma hospital during december -august were included (n = ). patients were stratified into groups; those c and those results: in the c years group, sex distribution was more even (female . vs . %, p \ . ), physical status according to pretrauma asa classification was higher (mean . vs . , p \ . ) and the trauma mechanism was predominantly low-energy (falls from no height) as opposed to the conclusions: the trauma among elderly swedish patients are more often of low energy compared to the younger population. in spite of this, the elderly are more severely injured, require more surgical interventions, and their short term mortality is increased -fold. measures aimed at prevention of low energy trauma of the elderly are therefore much needed. introduction: there are intramedullary or extramedullary methods in internal fixation od trochanteric fractures. seldynamisalbe internal fixator with two sliding screws (sif), as an extramedullary method, and gamma nail (gn), as an intramedullary method, are in routine trochanteric fractures treatment at our institution for last two decades. material and methods: health related quality of life and hip function were assesed at least two years after surgery, in the series of patients with a surgically treated ao a or a fracture type. there were two groups of patients: group treated by sif and group treated by gn. examination had been performed using sf- test, with its physical component score (pcs) and mental component score (mcs), and harris hip score (hhs) tests. results: in sif group, mean pcs was , , mean mcs was , and mean hhs was , . in gn group mean pcs was , , mean mcs was , and mean hhs was , . there was no significant difference regarding all these parameters between the groups of patients (p [ , ). there was correlation between all evaluated parameters, both in groups of patients particularly and in all patients (p \ , we identified undertriage in , % ( / ). falls from own height ( - m) was found in patients with iss [ , / ( %) of them was found to have been undertiaged (p . ). we found an association between gcs \ and undertriage (p = . ). % ( / ) falls between - m and % ( / ) of these without trauma team. falls between - m , % ( / ) without trauma team. all with fall [ m had trauma team. mortality was % ( / ), no association between height of fall and mortality (p . ). undertriage was not associated with increased mortality (p = , ). median age in mortality group was years versus years in surviving group (p \ . ). in univariate analysis there was association between prehospital bp \ (p . ), gcs \ (p \ , ), iss (p \ . ), prehospital rr [ , rts \ (p \ . ) asa score [ (p \ . ) and mortality. conclusions: we found significant undertriage in the geriatric trauma population with fall injuries. gcs \ and low energy falls was associated with undertriage but not with mortality. laparoscopic direct repair of an incarcerated spigelian hernia c. bergamini , v. iacopini , r. de vincenti , a. bottari , g. alemanno , p. prosperi aou-careggi, emergency surgery, firenze, italy spigelian hernia occurs through a defect in the anterior abdominal wall adjacent to the semilunar line. it is in itself very rare and more over it is difficult to diagnose clinically. it has been estimated that it constitutes . % of abdominal wall hernias. the majority of patients present with symptomatic incarceration of preperitoneal fat or intraabdominal viscera. radiographic studies are beneficial in confirming the diagnosis. the high rate of incarceration with or without strangulation mandates operative repair once the diagnosis is confirmed. the spigelian hernia has been repaired by both conventional and laparoscopic approach. laparoscopic management of spigelian hernia is well established. most of the authors have managed it by transperitoneal approach either by a direct repair or by placing the mesh in intraperitoneal position or raising the peritoneal flap and placing the mesh in extraperitoneal space. there have also been case reports of management of spigelian hernia by total extraperitoneal approach. we present the case of an obese eighty-four y.o patient. complaining for a sudden onset abdominal pain in the right low quadrant, mimicking an appendicitis. the ct scan demonstrated a typical picture of a spigelian hernia containing an intestinal loop. the loop showed classical signs of parietal wall ischemia. the video describes the surgical laparoscopic approach of this case which was able to confirm the diagnosis e to reduce the loop into the abdomen. the loop initially appeared diffusely ischemic, but after the intra-abdominal reduction some signs of vitality started to be noticed. however, they were incomplete; thus the loop was resected. the hernia defect was successively repaired in a direct way because of the small caliber (\ cm of diameter) and the possible contamination coming from the intestinal resection. post-operative course was particularly benign and the patient was discharged on the seventh post-operative day in good health. introduction: trauma audit & research network (tarn) data shows older persons falling from standing height and sustaining significant injury has become the commonest trauma presentation in england and wales . we aimed to assess whether frailty predicts poor outcomes in the elderly. material and methods: retrospective database review of tarn eligible patients [ years old admitted in a week period with documented rockwood clinical frailty score . age, injury severity score (iss), length of stay (los) and mortality were noted. the inhospital mortality group was sub-analysed. logistic regression was performed (stata v ), odds ratios and % ci reported. results: older age was associated with higher cfs in the patients studied. increasing cfs was associated with increased overall mortality (cfs - vs cfs - or . ; % ci . - . ), decreased likelihood of pre-hospital trauma alert and increased length of stay (cfs - stayed days more than cfs - ). all deaths had cfs [ and head or chest injury. adjusting for age and cfs those with chest injury were . times more likely to die (or . %ci . - . ). mortality in those with rib fracture was times higher in cfs - vs cfs - (or . %ci . - . ). conclusions: increasing age and cfs (especially - ) are associated with poor outcomes in elderly trauma, thus cfs is a useful prognostic tool in severely injured elderly patients. chest injuries are a major cause of mortality in this group, especially with increasing frailty. major trauma centres must develop practice management guidelines to appropriately manage these patients. introduction: major trauma causes activation of the complement system, which plays a key role in development of systemic inflammatory response syndrome and multiple organ failure. complement is known to be activated early after trauma , but the relationship between outcome and the extent of complement activation during the first critical hours after injury is unknown. we hypothesized that complement activation in the first hours after trauma displays a highly dynamic pattern which is associated with outcome. material and methods: complement activation was assessed by plasma terminal c b- complement complex (tcc) using elisa in a prospective cohort of trauma patients. samples were obtained at admission, after , , and h, and daily in the intensive care unit. the extent of complement activation was assessed as area under the concentration curves - h after injury (tcc-auc - ). the relative contribution of complement activation, base excess (be) and new injury severity score (niss) to outcome was analyzed by multivariable analyses. results: niss and be were associated with tcc-auc - in bivariate analyses (spearmans rho (p) was respectively , (p = . ) and - . (p = . )). in multivariable analyses, niss and initial tcc alone predicted % of the variability in ventilatorfree days (vfds), whereas initial tcc and tcc-auc - predicted %. tcc auc - alone contributed with % to the model. tcc-auc - was also significantly higher in patients deceased at day ( . ; . - . (median; quartiles) vs. . ; . - . , p = . introduction: massive transfusion protocols [mtp] have been widely adopted for the care of bleeding trauma patients but their actual effectiveness is unclear. this study aims to conduct an updated meta-analysis to evaluate the effect of implementing an mtp on the mortality of trauma patients. material and methods: medline, pubmed, google scholar and cochrane library databases were systematically searched for relevant articles published from january , to july , using a combination of key words and additional manual searching of reference lists. three reviewers independently screened the articles for potential inclusion. eligible articles were original articles in english, included trauma patients and compared mortality outcomes before and after institutional implementation of a mtp. primary outcomes were h and overall mortality. results: nineteen studies met inclusion criteria, analyzing outcomes from , trauma patients. there was a wide range of outcome and process indicators utilized by the different authors. mtps significantly reduced over-all mortality, pre-mtp- . % and post-mtp . % [or . ( . - . )] for trauma patients. -h mortality was not significantly reduced [or . ( . - . )]. conclusions: the institution of an mtp has a significant over-all mortality reduction for trauma patients. we encourage that researchers use standard nomenclature and indicators, provide more details regarding protocols and patient populations and incorporate advances in the management of bleeding trauma patients in all future mtp studies. introduction: when resuscitating patients with hemorrhagic shock following trauma, fluid volume restriction and permissive hypotension prior to bleeding control are emphasized with good outcomes for penetrating trauma patients. however, evidence that these concepts apply well to the management of blunt trauma is lacking. this study aimed to assess the impact of vasopressor use in patients with blunt trauma in severe hemorrhagic shock. material and methods: in this single-center retrospective study, we reviewed records of blunt trauma patients with hemorrhagic shock and included patients with a probability of survival \ . . patient's characteristics, examinations, severity and administrated therapies were compared between survivors and non-survivors. data are described with median ( - % interquartile range) or number. results: thirty patients were included and median injury severity score in survivors vs non-survivors was ( - ) vs ( - ) (p = . ), with no significant difference in probability of survival. despite no significant difference in injury severity, non-survivors were administered vasopressors significantly earlier after admission and at significantly higher doses. total blood transfusion amount administered within h after admission was significantly higher in survivors ( [ conclusions: vasopressor administration and high-dose use for hemorrhagic shock following severe blunt trauma are significantly associated with increased mortality. although the transfused volume of blood products tends to be increased, early termination of vasopressor should be considered. all authors have no significant relationships with regard to this study. early amplitudes of citrated functional fibrinogen in thromboelastography to predict massive transfusion introduction: this study aims to evaluate the role of early amplitudes of the thromboelastography measure of citrated functional fibrinogen (cff) to predict massive transfusion (mtx) defined as transfusion of c of any blood products within an hour of arrival to a major trauma centre. material and methods: trauma patients c years requiring activation of the major haemorrhage protocol with teg performed on a tegÒ s hemostasis analyser were eligible for inclusion. exclusion criteria were arrival [ h after injury, pregnancy, bleeding disorder or anticoagulant use. patient demographics and transfusion requirements were obtained from medical notes. teg manager was accessed to extract amplitudes at min (a ), min (a ) introduction: hyperfibrinolysis, remains a significant characteristic of acute traumatic coagulopathy induced mortality. s a , a cell surface protein, when shed creats an occult hyperfibrinolytic subtype. annexin a (a ), a multicompartment protein that co-localizes with s a and contains a tissue plasminogen activator (tpa) binding site has been shown to enhance tpa activity -fold and thus behaves as marker of hyperfibrinolysis. we hypothesize that increased concentrations of a in blood will enhance tpa fibrinolysis. material and methods: blood was collected from ( ) healthy volunteers. recombinant a in concentrations , , , , , lg/ ml was added blood and then combined with tpa ng/ml. samples were assessed using thromboelastography (teg). blood samples were collected from trauma activations from -current at a single, urban, level- trauma center. samples were assessed using a combination of rapid, citrated native and tpa challenge teg. a levels were established via proteomic analysis. results: a - (lg/ml) significantly increased tpa mediated ly % vs tpa alone (a ? tpa [ - ] median . % vs tpa . % p \ . ). a without tpa had no significant effect on ly % and was similar to the lysis of control (a lg/ml . % vs control . % p = . ). a - (lg/ml) significantly increased r time from control and tpa alone (control normalized to vs a median . -fold increase in minutes p \ . and tpa . -fold decrease vs a median . -fold increase p \ . ). rapid teg for patient vs patient in our ongoing study was . % vs . % and . % and . % respectively on tpa challenge teg. proteomic analysis of a relative activity found a . -fold a activity in patient compared to patient . conclusions: exogenous cell free a significantly increases tpa mediated fibrinolysis measured by teg. preliminary data from our ongoing trauma study evaluating a levels and hyperfibrinolysis coincide with our in vitro study. introduction: massive transfusion protocol can be activated to mobilize the blood products resource in a timely and effective manner. blood products, however, are still wasted or overused. we aimed to study what proportion of patients who met the abc criteria for massive transfusion received or more units packed rbc (prbc). material and methods: a retrospective study all level i trauma patients admitted with arrival systolic blood pressure of or less (july to may ) was recruited. transfusion was complied with stts. all clinical and laboratory findings, and management procedures were populated from the data registry. results: of admitted trauma patients met the inclusion criteria. of patients who where admitted with hypotension, of patients ( . %), who met the abc criteria for receiving or more prbc were stabilized with or units. in other words, stts enabled us to save units of prbc. arrival data, i.e. blood pressure (cut of point: mmhg and p value: . ), shock index (cut of point: . and p value: . ) and pulse rate (cut of point: beat/min and p value: . ) were significantly different in patients prescribed or more units prbc. after initial resuscitation, blood pressure (cut of point: mmhg and p value: . shock index cut of point: . and p value: . ), pulse rate(cut of poinan beat/min and p value: . ) presence of pelvic fracture, positive fast,and base deficit [ were significantly different in the group received or more units prbc. conclusions: massive transfusion protocol with abc criteria may lead to wasted or overused blood products.consideration of dcr continuation strategy complied with stts along with the findings of this study has resulted in a refined protocol characterized by more effective and efficient blood product resource allocation. references: -chang r, holcomb jb. optimal fluid therapy for traumatic hemorrhagic shock. critical care clinics. jan ; ( ) case history: we present the clinical case of a female patient of years old who had been taking aspirin. mechanism of injury: a fall from her own height, resulting in head trauma. clinical findings: dysphonia and stridor, having underwent an immediate orotracheal intubation. investigation/results: she had a head ct done that was normal; and a cervical column and neck ct that showed a large retropharyngeal hematoma, without an associated vertebrae fracture. diagnosis: large retropharyngeal hematoma. therapy and progressions: she was admitted to the intensive care unit for mechanical ventilation. on nd day, she underwent a surgical tracheostomy. on th day, underwent weaning from mechanical ventilation. on h day, was transferred to the ent ward, had the tracheostomy tube removed and was discharged from hospital. comments: a hematoma in this potential space may constitute an immediately life threatening emergency due to airway compromise. in , thomas et al found only cases described in the literature since . the most common cause is the blunt cervical trauma (in % of the cases). other causes are the cervical hyperextension injury, cervical vertebrae fracture, cough, sneeze, strain, blunt head trauma, swallow a foreign body, retropharyngeal infection, carotid artery aneurism, internal jugular vein puncture, metastatic disease, coagulopathy, anticoagulants, etc. in the setting of trauma, the mechanism of injury generally permits explaining the presenting injuries. in this case, the clinical severity expressed by the patient seemed to be disproportional to the resultant injury. however, the presence of haemorrhage contributing factors associated with the existence of fascial spaces in the neck, should warn us of the possibility of formation of deep cervical hematomas that may cause an occult airway obstruction. case history: a -year-old male with a personal history of consumption of alcohol, cannabis, smoked cocaine and heroin. he was found in decubitus position and in a situation of cardiac arrest. the last time he was seen in his baseline situation was h before. after performing cpr and administration of naloxone and flumacenyl, sinus rhythm was achieved. clinical findings: h after admission, increased tension was observed in left leg, thigh and gluteal region. absence dorsalis pedis, tibialis posterior and popliteal pulse was observed in a doppler examination. investigation/results: intracompartmental pressure measurement revealed a result of mmhg in the deep posterior compartment and mmhg in the superficial (diastolic bp mmhg). at admission k levels were . meq/l, creatinine . mg/dl and ck u/l. diagnosis: opioid-related compartment syndrome. therapy and progressions: urgent fasciotomies of the leg and thigh were performed h after diagnosis with a posteromedial and anterolateral approach in the first case and with a lateral approach in the latter. herniation and signs of poor viability in all the compartments were observed. after the surgery, he persisted with anuria and a ck peak of , u/ l, which was next normalized. debridements were performedfor the next days. subsequently, after the isolation of p. stutzeri and mucor in the wound and the absence of signs of vitality, a supracondylar amputation was performed. after, hemodynamic status improved. weeks after the amputation it was possible to withdraw hemodialysis, which he had required since admission. comments: opioid misuse is a topic of growing interest. recent works have reported a worse prognosis in the case of opioid-related compartment syndrome. a high level of suspicion is necessary to make a prompt diagnose in these patients. introduction: the pelvic binder is a mechanical device designed to compress instable pelvic ring fractures and minimize dead space in order to limit blood loss. it is generally recommended to apply a pelvic binder if an unstable injury is suspected and the patient presents with a ''c-problem''. the effectiveness remains questionable though. material and methods: a total of trauma patients between and were retrospectively evaluated regarding instable pelvic injury. patients were admitted with a pelvic binder applied. the correct application was evaluated using ct scout. four groups were generated: group with correct pelvic binder application, group with incorrect placement, group with no pelvic binder at time of admission, group with pelvic binder applied in er. total outcome was determined based upon iss, age, preclinical time, time to ct, shock index, hemoglobin at admission, survival rate, administration of blood products as well as total hospital and icu days. results: % of all pelvic binders were applied incorrectly. patients ( %) suffered an instable pelvic fracture. patient survival was not influenced by the preclinical application of a pelvic binder ( % group vs. , % group , p = , ). no significant statistical difference was found for total icu days , vs. , , p = , ; total hospital days , vs. , , p = , ; rbc transfusion , vs. , , p = , ; iss , vs. , , p = , . conclusions: the correct application of a pelvic binder seems to pose problems preclinically. while the need to minimize blood loss is crucial, our collective did not benefit from this device. additionally, survival rates of the patients that suffered an instable pelvic fracture were unaffected. the iss remains the strongest predictor of total patient survival in pelvic trauma. trauma resuscitation times in a level trauma center in the netherlands: a prospective overview introduction: in trauma, time is considered to be an important factor influencing patient's outcome. in the first hour after injury, appropriate care has the greatest effect on trauma patient's survival. previous research showed that measuring in-hospital trauma resuscitation times, contributes to insights and improvement of the resuscitation process. however, despite developments of atls guidelines, no recent empirical knowledge regarding resuscitation times exists. the aim of this study is to examine in-hospital trauma resuscitation times in a level trauma center in the netherlands. material and methods: a prospective study was performed in level trauma center amsterdam umc location vumc, between may and august . trauma patients, aged c , presented during daytime at the trauma resuscitation room were included. information regarding patient's characteristics, trauma-and injury type, handover duration, duration till start of diagnostics and intervention, total resuscitation time, patient's disposition and survival were compared. results: in total, patients were analyzed. motorized traffic accident ( %) and blunt injury ( %) were the most common mechanism-and injury types. median prehospital to in-hospital handover time was . min (iqr . ) . median duration till start of diagnostics and intervention were . (iqr . ) and . min (iqr . ) respectively. median total resuscitation time showed to be . min (iqr . background: terrorist attacks and civilian mass casualty events are frequent, and some countries have implemented tourniquets for uncontrollable extremity bleeding in civilian settings. we summarized current knowledge on the use of pre-hospital tourniquets in civilian settings to assess whether their use increases the survival rate in civilian patients with life-threatening hemorrhages from the extremities. methods: using the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines, we searched medline (ovid), embase (ovid), cochrane library, and epistemonikos in january . all types of studies that examined the topic in a pre-hospital setting published after january , , were included. the protocol was registered in prospero (crd ). results: among screened records, studies were identified as relevant. due to a lack of relevant civilian studies, military studies were also included. the studies were highly heterogeneous, with low quality of evidence. most studies reported increased survival in the tourniquet group, but few had relevant comparators, and the survival benefit was difficult to estimate. most studies reported a reduced need for blood transfusion, with few and mainly transient adverse effects from tourniquet use. conclusion: the data suggest that the use of commercial tourniquets in a civilian setting to control life-threatening extremity hemorrhage is probably associated with improved survival, reduced need for blood transfusion, and few and transient adverse effects. the effect of venous infusion by emergency medical service personnel on the prognosis of severe traffic accident patients: a nation-wide study in japan y. katayama , t. kitamura , , t. hirose , y. nakagawa , t. shimazu osaka university graduate school of medicine, department of traumatology and acute critical medicine, suita, japan, osaka university graduate school of medicine, environmental and population science, suita, japan introduction: in japan, the law of paramedic was revised in , and it became possible for paramedic in japan to secure an infusion route before cardiac arrest for severe patients. however, the effect of this treatment on the prognosis of severe trauma patients has not been assessed. we assessed this effect on the prognosis of severe traffic accident patients with using population-based ambulance record and nation-wide hospital-based trauma registry in japan. material and methods: this study was a retrospective observational study and the study periods was years between january to december . we linked the nation-wide hospital based trauma registry (jtdb) and the population-based ambulance record in japan in case. in this study, we included the traffic accident patients with iss score more than and excluded cardiopulmonary arrest patients on the arrival of ems on the scene and missing data cases. the main outcome was cardiopulmonary arrest on hospital arrival. mcnemar's test and conditional logistic regression analysis were used to assess the association between the securing a infusion route by ems personnel and the primary outcome after one-to-one propensity score matching for securing a infusion route or not. results: traffic accident patients were eligible for analysis and patients were dripped by ems personnel. after one-to-one propensity score matching, the proportion of cardiopulmonary arrest on hospital arrival were . % ( / ) in patients dripped by ems personnel and . % ( / ) in patients not dripped by ems personnel, respectively (p = . ). the adjusted odds ratio for securing a infusion route was . [ % confidence interval; . - . , p = . ]. conclusions: in this study, there was no association between the securing a infusion route and outcome of traffic accident patients. the association between trauma patient characteristics and adverse laboratory values: which patient characteristics are most predictive? introduction: in more than countries worldwide, laboratory testing is protocol driven since when it was included in the practice guideline of the advanced trauma life support course (atls). however, it is not clear yet which patient characteristics are associated with unfortunate laboratory values. the aim was to create an overview of the characteristics that were associated with adverse laboratory values. material and methods: this cohort study was performed at amsterdam umc, location amc (level trauma center), including patients during a period of years. data concerning age, gender, asa scores, injury severity scores (iss), glasgow coma scores (gcs), mechanism of injury, type of injury (blunt or penetrating) and the presence of helicopter emergency medical services (hems) were obtained. the hematology panel included hemoglobin, hematocrit, mcv, leucocyte and thrombocyte values. the coagulation panel included inr, pt, aptt, fibrinogen and d-dimer values. other panels include arterial blood gas, kidney and liver panels. the association between trauma patient characteristics and laboratory values were determined by using binary and multinomial logistic regression. results: a total of patients were included, consisting of predominantly men ( %) with a mean age of years old. an increase in age and iss was correlated with abnormal laboratory values (p = . ). additionally, male gender, iss [ , blunt trauma and the absence of hems was associated with a deviation in laboratory values (p \ . ). other patient characteristics did not show a significant correlation with adverse laboratory values. case history: a -year-old man presented with a classic case of pituitary apoplexy with a history of headache, nausea and vomiting. clinical findings: he was found to have a sellar and suprasellar mass with internal cystic and hemorrhagic component consistent with a pituitary macroadenoma. investigation/results: he underwent transsphenoidal sugery for a pituitary macroadenoma. because the tumor was invaded to left cavernous sinus, we left small portion of the tumor. eighth day after surgery, he underwent gamma-knife surgery (gks) for residual tumor. after two weeks, he complained of left ptosis. we considered the rd nerve palsy to be a post-radiation reaction at first. after months, the symptoms had been continuous and mri showed increased size of cystic lesion involving left cavernous sinus. diagnosis: ct angiogram demonstrated a saccular aneurysm at left distal ica. endovascular coil embolization was performed. therapy and progressions: after months of the intervention, the rd nerve palsy was partly improving. comments: our case report emphasizes the necessity of cerebrovascular imaging before surgery for pa. mr angiography/ct angiography is not currently obligatory in patients with pituitary adenoma, but in cases with the symptoms of displacement of the neuro-vascular structures it can be of great value. even in patients without such presentations, it may be helpful to evaluate the vascular involvement. case history: a -year-old boy during the preparation for a fishing session was pierced to the left orbitary region by a high-speed spearfishing steel. clinical findings: upon arrival the patient was conscious and responsive with a gcs of , he followed commands appropriately and there were no motor of sensory deficits. investigation/results: plain skull radiographs showed the spear crossing the skull from the left orbit to the posterior part of the parietal bone. diagnosis: the patient was immediately intubated to prevent involuntary movement of the foreign body. ct scan showed the fracture of the left orbitary roof where a centimetres long metallic object crosses the cerebral parenchyma of the left hemisphere and perforates the left parietal skull. therapy and progressions: under direct visualization via transorbital approach the foreign body was removed together with bone fragments, hemostasis was done and orbitary roof repaired. serial cranial ct scan showed progressive reduction of frontal and parietal hematoma. the movement of the eye improved after a few days, normalizing with the regression of periorbital edema. upon discharge at th postoperative day the patient had a gcs score of , no motor deficit and minimal visual loss. comments: penetrating injury of the skull and brain are relatively uncommon events, representing about , % of all head injuries. orbital roof is relatively thinner part of the skull that can provide easy access to projectile objects, which can penetrate into cranial cavity and damaging the brain parenchyma. the principles of treatment are removal of bone fragments and foreign body, control of persistent bleeding and intracranial hypertension, prevention of infection though debridement of all contaminated and necrotic tissue and at the same time preservation of as much nervous tissue as possible. multitraumapatients whith severe head injury (ais ‡ ) are more quickly carried out ct scan on than a patient without severe head injury v. giil-jensen , k. andersen , t. k. helle haukeland univercity hospital, sugical department, bergen, norway, haukeland univercity hospital, ambulance service, bergen, norway introduction: trauma patients who are prone to severe head injuries during trauma may profit from obtaining a rapid clarification of the injury magnitude when using ct examination. in the case of a delayed ct examination, the consequence of the head injury could be more extensive. in this study, we wanted to see if those with severe head injury (ais c ) received a faster ct survey than those who had no severe head injury. material and methods: retrospective registry study of severely injured patients (iss [ ) which had been hospitalized as a trauma patient at haukeland university hospital in the period - . in the study, we have excluded all patients entered as multitrauma but who have iss \ and all patients who have not defined ct time. it turns out that over half of the patients lacked the registration of accurate time for the ct survey in the national trauma register. the number is still considered large enough to find a result. results: patients were received as multitraumatic at haukeland university hospital during the period. of these, was severely injured. of these, patients had severe head injuries and they again had head injuries as the only serious injury (ais c ). median time from arrival receipt to start ct, for this group was min. in the control group that was severely injured but without severe head injury is the same time min. there was patients in the control group. conclusions: for the patients in this study who had severe injuries (n: ), the median time from the arrival in the emergency department to the ct starts was min shorter for severe head injuries than for the group without severe head injuries. introduction: the patients with severe traumatic brain injury (stbi) who needs surgical intervention often experience acute traumatic coagulopathy (atc). earlier transfusion with high blood product ratios (plasma, platelets, and red blood cells via : : ratio) is recommended for severely injured patients. however, recommended blood product ratio for stbi is still controversial. material and methods: we retrospectively reviewed successive adult stbi who underwent surgical treatment in our hospital between january and december . we have transfused plasma aggressively to maintain blood fibrinogen above - mg/dl. we evaluated the total amount of transfusion and mortality. we exclude cases administered fibrinogen concentrate. results: patients were enrolled. the amount of transfusion for h is rbc . units, ffp . units, pc . units . stbi with severe other trauma needs higher ratio of plasma. discussion: tissue injury of stbi causes severe coagulopathy and : : transfusion was thought to be insufficient for stbi in order to maintain fibrinogen. we agressively transfused plasma but we achieved fibrinogen value above only in % of stbi with severe other trauma. agressive plasma transfusion had limitation for hyperfibrinolysis so we expect other product, for example fibrinogen concentrate. introduction: traumatic brain injury (tbi) remains a leading cause of hospital admission and mortality amongst trauma patients. intracranial hemorrhage (ich) can occur with tbi and presents a severe complication. low complication tolerance in developed countries and uncertainty on actual risk cause excessive diagnostics and hospitalization, considered unnecessary and expensive. methods: tbi cases indicated for cranial computer tomography (ct) according to international guidelines, at our level i trauma center between - were retrospectively included. multivariate logistic regression was performed for ich, progression and mortality predictors. results: tbi patients (m: . ; age at trauma: . ± . ), were included. ct was performed in . %, skull fracture diagnosed in . %, ich in . %, ich progression in . %. in patients \ a, chronic alcohol consumption (p = . ) and neurocranial fracture (p \ . ) were significant ich risk factors in a multivariate analysis. in patients between - a, chronic alcohol consumption (p \ . ) and skull fracture (p \ . ) revealed as significant ich predictors. in patients [ a, age (p = . ), anticoagulation (p = . ) and neurocranial fracture (p \ . ) were significant risk factors for ich, age (p = . ) was an independent risk factor for mortality. late onset ich only occurred in cases with at least of factors: age [ , anticoagulation, neurocranial fracture. overall hospitalization might have been reduced by . % via low risk cases. conclusions: triggered by decreasing error tolerance, international guidelines for mild tbi focus on safety maximization. repeated ct in initially ich negative cases should only be considered in high risk patients. non-ich cases aged \ years do not gain safety from observation or hospitalization. recommendations from our data might, without impact on patient safety, reduce costs by unnecessary hospitalization and diagnostics. references: to be added by the authors. evaluation of low-value clinical practices in acute trauma care: a multi-center retrospective study l. moore , k. soltana , j. clément , a. turgeon , î mercier , r. krouchev , p. a. tardif , s. bouderba , a. belcaid université laval, social and preventive medicine, québec, canada, chu de québec-université-laval, québec, canada, université-laval, québec, canada, introduction: low-value clinical practices have been identified as one of the most important areas of excess healthcare spending and are associated with adverse health outcomes. the objectives of this study were to estimate the frequency low-value practices in injury care and assess inter-hospital variations. material and methods: we identified low-value clinical practices from internationally recognized clinical guidelines. we conducted a population-based retrospective cohort study using data from an inclusive canadian trauma system ( - ) to calculate frequencies and assessed inter-hospital variations with intra-class correlation coefficients (icc). results: we identified low-value practices of which could be measured and validated using trauma registry data. the three lowvalue clinical practices with the highest absolute and relative frequencies were pelvic x-rays in hemodynamically stable patients with a negative physical exam for pelvic injury ( . %), head ct in adults with minor head injury who were negative on a validated clinical decision rule ( . %) and chest x-ray in hemodynamically stable patients with a normal physical exam ( . %). we observed high inter-hospital variation for surgical management of penetrating zone ii neck injury without hard signs (icc = %), and moderate variation for head ct in adults with minor head injury who were negative on a validated clinical decision rule (icc = . %). conclusions: we have developed and validated algorithms to evaluate nine potentially low-value clinical practices using trauma registry data. highest frequencies were observed for imaging in the emergency department and the highest inter-hospital variation was observed for inappropriate surgical management. these data can be used to advance the agenda on low-value care for injury admissions. dysfunction of functional connectivity between default mode network and cerebellar structures in patients with mtbi in acute stage. rsfmri and dti study introduction: mild traumatic brain injury (mtbi) occupies one of the first places in children injuries. among all brain networks at the resting state, the default mode network (dmn) is the most widely studied network. the aim of this study is to examine functional connectivity in normal-appearing cortex in acute period of mtbi using rsfmri. material and methods: mr negative participants were studied in age from to years (mean age- . years). group of patients consisted of children with mild traumatic brain injury in acute stage. age-matched healthy volunteers comprised control group. all studies were performed at phillips achieva . t mri scanner using -channel head coil. fmri data were processed using functional connectivity toolbox conn. seed-based analysis was performed in order to reveal disturbances in functional connectivity. statistical processing was performed using statistica . results: dti analysis didn't show any changes in values of apparent diffusion coefficient (adc) and fractional anisotropy (fa) between two groups (see fig. ). no statistically significant differences in correlation strength between dmn parts were observed in two groups (see fig. ). intergroup seed-based analysis revealed statistically significant (p \ , ) difference in neural correlations between dmn parts and vermis (cerebellum structural part): positive link in control group and negative link in group of patients. conclusions: one of the most common symptoms of mtbi is dizziness as a result of impaired movements coordination. vermis as an essential cerebellum part plays an important role in the vestibuloocular system which is involved in the learning of basic motor skills in the brain. vermis aids in the synchronization of eye and motor functions in order for the visual field and the motor skills to function together.our results show that mtbi appears to be a possible reason of connectivity malfunction in normal-appearing vermis. references: predictors of developing post-traumatic endophthalmitis introduction: h magnetic resonance spectroscopy ( h mrs) allows to study structural and metabolic brain disorders in various pathological conditions in vivo. non-invasive method determines its advantage for use in children in serious condition with acute cerebral injuries. this determined the purpose of the study: to identify criteria of irreversible brain damage based on the h mr spectra analysis in comatose children with acute traumatic brain injury (tbi) or anoxia. material and methods: patients ( months- years) were examined in the acute period of severe cerebral injury (gcs score - ): six were in acute and subacute period of severe tbi, one patient was examined on the seventh day after drowning, and one-a day after acute cerebral blood flow (hemorrhage). all patients died in - days after the study. control group included healthy children aged from to years. single voxel h mrs and d h mrs was performed on t scanner. h spectroscopic voxel (te/tr = / ms, voi = cm , nsa = ) was oriented on mri intact areas: cortex of frontal, parietal and occipital lobes (fig. ) , thalamic nuclei (fig. ) , cerebellum, brainstem (fig. ) . for d h mrs a spin-echo point-resolved spectroscopy (press) sequence was used (te/tr = / ms) with the spectroscopic voi of cm on frontal lobes. results: in all spectra lactate (lac) signal, dominating all other signals, was detected. n-acetylaspartate (naa) was reduced by % and creatine/phosphocreatine (cr)-by %. conclusions: h mrs is a non-invasive prognostic method in patients with acute cerebral brain damage in coma. the cause of patients' death is the shift of cerebral glucose metabolism to an anaerobic type, as evidenced by the accumulation of lac. disturbance of energy metabolism causes a decrease of cr and a decrease in the neuronal marker naa. the combination of these patterns in acute cerebral injury, regardless of etiology indicates irreversible brain tissue damage. introduction: scalds and contact burns are the most common burn injuries both in children and adults. data are conflicting regarding which type of burns are more severe. we compared scalds, contact, and flame/fire burns at our burn center to determine which type were more likely to result in full thickness injuries and prolonged length of stay (los). material and methods: we conducted a structured retrospective medical record review of all patient admissions to a regional burn unit over a -year period between and . data included demographic, clinical, and specific burn characteristics. the association between patient predictor variables and outcomes (full thickness burns, los) was explored using chi-square and stepwise logistic regression. results: there were , patients with either scald (n = , %), fire/flame (n = , %) or contact burns (n = , %). burn depth was not available for cases ( %). mean (sd) age was ( ), % were male. mean (sd) total body surface area (tbsa) was ( )%. % of burns contained areas of full thickness injury. patients with scalds were younger than those with contact or fire burns ( ± vs. ± vs. ± years respectively, p \ . ). the percentage of burns that were full thickness by etiology were contacts ( %), fire/flame ( %) and scalds ( %); p \ . . after adjusting for age, location, and tbsa, scalds were less likely to result in full thickness injuries than contact burns (odds ratio . , %%ci, . - . ). adjusting for multiple testing, univariate analysis (as well as the multivariate analysis) showed no difference in % rd degree burns between fire and contact burns, but scalds were significantly lower than each of those. los for scalds ( ± ) and contact burns ( ± ) was significantly shorter than for fire/flame ( ± days, p \ . ). conclusions: while less common, contact and flame burns were more likely to result in full thickness injuries than scalds. references: epidemiology, treatment, costs, and long-term outcomes of patients with fireworks-related injuries (rocket); a multicenter prospective observational case series introduction: the aim of this study is to provide detailed information about the patient and injury characteristics, medical and societal costs, and clinical and functional outcome in patients with injuries resulting from fireworks. material and methods: a multicenter, prospective, observational case series performed in the southwest netherlands trauma region, which reflects % of the netherlands and includes a level i trauma center, a burn center, and an eye hospital. all patients with any injury from consumer fireworks, treated at a dutch hospital between december , and january , , were eligible for inclusion. exclusion criteria were unknown contact information or insufficient understanding of dutch or english language. the primary outcome measure was injury characteristics. secondary outcome measures included treatment, direct medical and indirect societal costs, and clinical and functional outcome until one year after trauma. results: out of patients agreed to participate in this study. the majority was male (n = ; %), % were children \ years, and % were bystanders. injuries were located to the upper extremity or eyes and were mostly burns (n = ; %) of partial thickness (n = ; %). fifteen ( %) patients were admitted and ( %) patients needed surgery. the mean total costs per patient were € , ( % ci € , to € , ). patient-reported quality of life and functional outcome was not significantly different during follow-up compared with pre-trauma. conclusion: the most common injuries afflicted by consumer fireworks were burns, mostly located to the upper extremity, and eye injuries. fireworks can result in severe injuries, for which ( %) patients needed hospital admission and ( %) patients needed surgical treatment. although some injuries resulted in permanent disability, year after trauma it in general did not have major or longlasting impact on patients'' self-reported quality of life or functional abilities. persistent inflammation, immunosuppression and catabolism syndrome after polytrauma: a rare syndrome with major consequences. l. hesselink , r. spijkerman , r. hoepelman , l. koenderman , l. leenen , f. hietbrink umc utrecht, trauma surgery, utrecht, netherlands, wilhelmina children's hospital, center for translational immunology, utrecht, netherlands introduction: more severely injured patients survive the critical first phase after trauma nowadays. a substantial portion of these patients require long-term critical care support and suffer from recurrent infections. this clinical condition fits in a syndrome referred to as ''persistent inflammation, immunosuppression and catabolism syndrome'' (pics). the aim of this study was to investigate the incidence of pics and clinical outcomes of trauma patients with pics in a level one trauma center. material and methods: all trauma patients c years admitted to the intensive care unit (icu) for c days between and , were included. patients with isolated neurological injuries were excluded. pics patients were identified by icu stay c days, c infectious complications and increased catabolism. infectious complications included infections during hospitalization and readmissions due to an infection. increased catabolism was defined as weight loss [ %, a body mass index. results: of the , polytrauma patients, patients had an icu stay c days. after exclusion of patients with isolated neurological injuries, patients were included. of these patients, developed pics. pics patients sustained infectious complications on average (compared to in the non-pics group, p \ . ) and . % of the pics patients developed sepsis. also, pics patients had a longer hospital stay (mean of days versus days, p \ . ) and sustained more surgical procedures (mean of versus per patient, p \ . ). infectious readmissions occurred until years after the initial trauma. conclusions: patients who develop pics experience long-term inflammatory complications that lead to frequent readmissions and surgical procedures. therefore, despite its low incidence, this clinical condition forms a burden on patients and a substantial financial burden on society. hyperbilirubinemia as a risk factor of the trauma icu patient introduction: hyperbilirubinemia is common in the intensive care unit (icu). hyperbilirubinemia has been considered as a risk factor of the icu patient. hyperbilirubinemia can have various causes. the hyperbilirubinemia has never been studied for the trauma icu patient. the aim of this study is to elucidate the incidence and effects of the hyperbilirubinemia for the trauma icu patient. material and methods: retrospective review of the trauma icu patients from . . to . . . initial bilirubin serum level, h bilirubin level, day bilirubin level, highest bilirubin level, overall morbidity and mortality and other clinical variables were identified and evaluated. the patients who have highest bilirubin level c . mg/dl were defined as hyperbilirubinemia group. results: a total patients were enrolled in this study. hyperbilirubinemia above serum bilirubin c . mg/dl were appeared in patients. the mortality of the hyperbilirubinemia group was higher than the other group ( . % vs . %, p = . ). the icu stay of the hyperbilirubinemia group was longer than the other group ( . day vs . day, p = . ). the hyperbilirubinemia group had more incidences of pneumonia, acute kidney injury, and sepsis than the other group ( . % vs %, p = . / . % vs . %, p = . / % vs %, p \ . ). conclusions: the hyperbilirubinemia is a risk factor of the trauma icu. if the hyperbilirubinemia is appeared, the cause of the hyperbilirubinemia should be evaluated and make an effort to correct hyperbilirubinemia for the each cause of the hyperbilirubinemia. case history: we present the clinical case of a male patient of years old. injury mechanism: a firework burst on his right forearm. clinical findings: injury: a large area of carbonization of the muscles of the flexor compartment. signs and symptoms: intense pain in the hand and forearm with local oedema and tension. diagnosis: deep burn of the forearm. therapy and progressions: surgical debridement and fasciotomy of this compartment; followed by deferred and progressive primary closure by means of rubber bands that were tightened as the oedema diminuished-shoelace technique. evolution: discharged from hospital on the th pos op day; follow-up at rd and th month without functional impairment, with a good healing evolution. comments: deep burns that reach the subfascial planes of the limbs, increase the pressure in the muscular compartments, and may progress to a compartment syndrome. there is no specific cutoff value of pressure for this diagnosis; consequently, the final decision to proceed with a fasciotomy relies on the clinical experience. surgical debridement and fasciotomy may result in large wounds, sometimes difficult to close. grafts and flaps result in another wounds and carry a risk of pain, infection, scar shrinking and necrosis. the diagnosis of a limb compartment syndrome is almost always a clinical one and requires a high index of suspicion so as to the fasciotomy is done in time. the shoelace technique is a simple, reproducible and cost-effective method of deferred closure of a large wound, preserving functionality and resulting in a good final cosmesis. references: johnson ls et al, management of extremity fasciotomy sites prospective randomized evaluation of two techniques, am j surg. . the use of propranolol in the management of acute thermal burn injury: evaluation of the effect of fixed dosages in african patients c. jac-okereke , i. onah , esut teaching hospital, surgery, enugu, nigeria, national orthopaedic hospital, enugu, nigeria introduction: propranolol has been shown to improve outcomes in burn patients. its effects are achieved at doses that reduce the heart rate by - %. africans have a different propranolol pharmacogenetic profile as compared to other races. there is paucity of literary works on the use of propranolol in africans with burns. in our study, we explored the effectiveness of fixed dosages of propranolol in nigerian patients. material and methods: this was a prospective comparative study of adult burn patients; two test groups received propranolol mg/day and mg/day respectively. the average daily pulse rate prior to and after the administration of propranolol were compared. results: patients in the control group had no effective reduction in their pulse rate. patients who received propranolol at a dose of mg/day had a reduction c %. no adverse events were observed. conclusion: it is important to establish the effective dosage of propranolol in burn patients of african-descent and explore its potential benefits in their treatment. although we cannot draw strong case history: the authors present in their paper three cases of blunt abdominal injury caused by seat belt in car accident. in the first two cases there was no diagnostic problem thanks to clear clinical finding. in the third case there was no clinical correlation and even repeated auxiliary examinations did not indicate the need for surgical intervention of the abdominal cavity. clinical findings: case no. -male y. old, haemodynamic stability, thoracic an abdominal pain, fast positivity, on ct free fluid in abdominal cavity, small spleen laceration, positivity of peritoneal symptomatology. case no. -male y. old, haemodynamic stability, bilateral hypogastric pain without peritoneal symptomatology, fast with small perihepatic fluid, on ct fluido-pneumoperitoneum. case no. -female y. old, haemodynamic stability, thoracic pain, massive oedema on the right side of the neck and supraclavicular area, without abdominal symptomatology. fast with small subhepatal fluid collection- mm, ct scan with large neck haematoma and fracture of st rib, apical pneumothorax- mm. intraabdominal only subhepatal fluid stripe- mm, suspected of small hepatic laceration. after days the clinical status rapidly changed, during h peritoneal symptomatology occured. on control ct scan fluido-pneumoperitoneum was detected. investigation/results: all patients underwent surgical procedure diagnosis: bowel mesenteric injury therapy and progressions: the first patient underwent ileo-caecal and hartmann resection, by the second patient was small intestine and col. sigmoideum resection needed, and the last one underwent ileal resection and npwt. comments: despite the current diagnostic methods blunt abdominal injuries, unlike the penetrating ones, can present a certain diagnostic problem especially when they are accompanied by other serious conditions such as manifest chest injuries. introduction: patients with hypertension and peritonitis must undergo a laparotomy. in isolated parenchymal lesions of the liver, the spleen or kidneys interventional or conservative approaches are more frequently used. to miss a hollow viscus organ lesion, that would need an operative procedure, is a constant fear. it is the aim of this study to identify significant predictors of the simultaneous presence of a hollow viscus lesion in patients with parenchymal organ lesions. material and methods: data of over ' inpatients of a levelone-trauma centre between and were analysed. only hemodynamically stable patients with a splenic-, liver-, or kidney injury (independent of grade) after blunt abdominal trauma were included. significant predictors were detected in bi-and multivariant analysis. results: of the patients with an average age of ± years % (n = ) had a splenic-, % (n = ) a liver-and % (n = ) a kidney rupture. the total iss was ± points. in patients ( %) a hollow viscus injury could be found (stomach n = , small bowl n = , colon n = , rectum n = ). injuries of the thorax ( %), the extremities ( %), the head ( %), the vertebra column ( %) and the pelvis ( %) were diagnosed as concomitant injuries. due to multivariant analysis neither age, gender, heart frequency at admission, gcs, base excess, the coagulation parameters, the hemoglobin value nor the separate injury regions could be identified to be predictive factors for the presence of a hollow viscus lesion. conclusions: clinical parameters taken at admission are not useful to predict hollow viscus injuries. the ct-scan is currently seen to be the best possible imaging modality, but it can be false negative, especially within the first min after trauma. repetitive clinical examination is necessary. in doubt a diagnostic laparoscopy or even laparotomy has to be performed. introduction: a heavy abdominal trauma is associated with a high morbidity and mortality. it is the aim of this study to show injury patterns in the abdomen and concomitant injuries in polytraumatized patients as well as to identify risk factors of the decease. material and methods: data of over ' inpatients of a level-one trauma centre between and were retrospectively analysed. only patients with a relevant abdominal trauma (ais abdomen c ) were included. the ais score was determined either with a contrast enhanced computed tomography or intraoperatively. significant risk factors were detected in bi-and multivariate analysis. results: patients with an averaga age of ± years were included. % (n = ) had an ais abdomen of , % (n = ) of and % (n = ) of . the overall iss was ± points. the mechanism of injury was mainly blunt ( %). a splenic rupture was present in % (n = ), a liver rupture in % (n = ) and a kidney rupture in % (n = ). hollow viscus injuries were present in % (small bowl n = , colon n = , stomach n = , rectum n = , bladder n = ). concomitant injuries were determined in % of the patients. of these % were diagnosed a thoracic injury, % injuries at the extremities, % head injuries. % spinal injuries and % pelvic injuries. the mortality was % (n = ). a liver rupture (p = . , or . ), pelvic injuries (p = . , or . ), age (p = . , or . ), hypotension (systolic blood pressure \ mmhg) (p = . , or . ) and a low gcs at admission (p \ . , or . ) were determined to be significant risk factors. conclusions: in our trauma department life threatening abdominal traumata are treated about every days. lethal abdominal injuries were mostly associated with serious liver ruptures or pelvic injuries. due to our experience we recommend the use of an early ct-scan as thereby the injury severity can be fast and precisely assessed. case history: a yo female was tranferred to our icu on day of a severe acute necrotizing alchoolic pancreatitis with mof. crrt with cytosorb was immediately started. on day after onset (dao ) an acs with a new organ failure (lung) showed up. open abdomen (oa) and tac with mesh-mediated/npwt got a temporary improvement. clinical findings: on dao (oa ), reopening of the mesh entailed a sudden fascial retraction of cm. a new larger mesh was positioned. on dao (oa ) the fascial defect measured both on ct slices and in or was cm. provision of a longterm oa was done. therapy and progressions: a new fascial traction device (fas-ciotensÒ, germany) was positioned on dao (oa ), with a continuous traction weight of - kg. revision was scheduled any - days, according to clinical needs, including combined anterior and retroperitoneal necrosectomy. progressive traction allowed to get a cm fascial gap under traction on dao (oa ). anterior cst was thus performed and fascia primarily closed. completion of necrosectomy was done through the bilateral lumbar incisions and npwt. comments: early fascial closure is a goal in oa. mesh-mediated traction/npwt is the most effective strategy, but primary fascial closure is sometimes impossible. the duration of oa is a key point. fasciotensÒ allowed to overcome the failure of mesh-mediated option and avoided fascia retraction in a longterm oa. it was quickly managed by the nurse staff, allowed a easier access to the abdomen and a proper positioning of the protective film. its effectiveness in this demanding case makes it an interesting option for shortening fascial closure in septic oa too. background: small bowel obstruction (sbo) caused by intra-abdominal adhesions is one of the main surgical emergencies. in most of the time, adhesions are created by previous abdominal surgeries. without any severity signs, the medical treatment is first proposed to avoid superfluous surgery. we noticed that the failure of medical treatment is frequently seen in patients previously operated of appendicectomy. the purpose of this study is to determine the eventual relation between a previous appendicectomy and failure of medical treatment in sbo. methods: we conducted a retrospective data collection using a diagnostic code for bowel obstruction in patients who have consulted in emergency from . . to . . at the salengro university hospital in lille. using the administrative database, patients were identified. we excluded all children, patients with wrong diagnosis and those whose outcome was not known. finally, patients with sbo on intra-abdominal adhesions confirmed on ct-scan were reviewed. the patients were separated in two groups. the group (g ) included patients who required surgical management during hospitalization ( patients) and group (g ) patients with successful medical treatment ( patients). we compared the rate of previous appendectomy in these two groups using a pearson's chi-squared test. in a second step, we tried to find out if there were others factor associated with failure of medical management. results: there was significant difference between the two groups with a higher rate of appendectomy in the surgical management group g (p = . ). this difference was even more pronounced if appendectomy was the only surgical history. in the subgroup analysis of patients with previous appendicectomy, the laparoscopic approach or laparotomy didn't influence the outcome of the management of the sbo. conclusion: this study shows the difference between the two groups of sbo, with more surgery sanction in the group of patients previously operated of appendicectomy. perhaps because this surgery involves the very distal part of the small bowel and decrease the efficiency of a proximal nasogastric aspiration. these results should not change the initial management of sbo by medical treatment in absence of severity signs. however, knowing this data, we have to consider that a history of appendicectomy is a risk factor of failure of medical treatment in this situation. introduction: diaphragmatic injuries are a rare consequence of closed thoraco-abdominal trauma that could be difficult to detect due to paucity of clinical signs and frequent erroneous interpretation of radiological images. the overall incidence of diaphragmatic injury is , - , % in blunt trauma. if the injury is not recognized it could lead to considerable risk of late morbidity and mortality. this study reviews our years experience in the management of this patients. material and methods: a retrospective review of trauma registry of our tertiary referral centre was performed. preoperative, intraoperative and postoperative data were analysed to assess determinants of mortality, morbidity and effect of therapeutic delay by univariate analysis models. penetrating injuries were excluded from the study. results: over years patients with diaphragmatic injury due to blunt trauma were identified: had a simple laceration of the diaphragm without hernia, had acute and chronic diaphragmatic hernia. the mean patient age was years (range -- years). overall mortality was %. the site of injury was the left diaphragm in cases, the right diaphragm in cases and bilateral in case.the hernia content was stomach ( ), colon ( ), spleen ( ), liver ( ), omentum ( ) and multiorgan ( ). all acute patients were managed with primary suture repair via laparotomy except for two patients that required additional thoracotomy; chronic patients were treated laparoscopically in cases ( , %), in which a synthetic or a biosynthetic mesh was used to reinforce the suture. higher morbidity and mortality was seen in multiple associated injuries, head injuries associated, right diaprhagm injury, age [ years and treatment delay [ h. conclusions: delayed treatment of diaphragmatic injuries could be dramatic: it is importnat not to misinterpreter the radiological findings and to reassess the patient mantaining a high level of suspicion of these injuries. trauma opposing vector forces resulting in distal avulsion of internal oblique muscle: a case report p. spada , p. fransvea , g. altieri , m. di grezia , v. cozza , g. pepe , a. la greca , g. sganga fondazione policlinico universitario agostino gemelli irccs, catholic university of rome, division of emergency surgery, roma, italy case history: abdominal muscle injuries after blunt trauma are rare but increasingly recognized. here we report a case of blunt trauma resulting in a complete disinsertion of the distal part of the internal oblique muscle. case report: y.o. male, was involved in a roll over motor vehicle accident. primary survey was carried out according to atlsÒ approach with good response. he had a seatbelt sign. according to the dynamic of the trauma he underwent a ct. diagnosis: a ce-mdct revealed complete disinsertion of the oblique muscles of the left abdomen from their iliac insertion, with herniation of adipose tissue and hematoma of the soft tissues without active blushing. no other traumatic injuries were identified. therapy progressions: a conservative treatment of the hematoma of the left abdominal wall was adopted. the patients was then ischarged from hospital after days. no late complications were observed. comments: the overall incidence in all traumatic admission is . - . %. a deep knowledge of vector force involved in trauma and their influence in the specific anatomical changes of the abdominal wall muscle can lead to suspicious of this rare injuries even if no other lesion are detected. in our opinion this trauma case is useful in reminding us to look for it because the radiologist or a no well experienced trauma surgeon may miss it fondazione policlinico universitario agostino gemelli irccs, catholic university of rome, division of emergency surgery, roma, italy introduction: the best and correct management of patients with open abdomen (oa) is nowadays still unclear. our algorithm consists of using an intra abdominal negative pressure wound therapy device plus an early medial mesh mediated fascia traction (''step by step'' procedure). the aim of this study was to asses outcomes of this algorithm technique based on patient conditions and open abdomen technique performed. materials and methods: we performed a retrospective analysis of patients treated with open abdomen technique from / / to the / / . variables taken into account were: initial diagnosis, open abdomen technique used, number of surgical interventions, abdominal wall closure technique, length of stay in the icu, inhospital morbidity and mortality rates. we collected also data on the post-operative development of incisional hernias and entero-atmospheric fistula. results: / of open abdomen were done after trauma. in the remaining cases open abdomen was done for non-traumatic disease. patients have been treated following our algorithm (with negative pressure wound therapy abthera device and step by step approach with medial mesh mediated fascia traction). in this group fascial retraction was significant lower and definitive direct abdominal wall closure rate was statistically higher. conclusion: an early fascia traction mediated with a mesh lead to an earlier fascia closure with a lower need of mesh positioning for definitive closure; the rate of post incisional hernia is similar among the two groups references: case history: a year old male presented in the er with malaise, fatigue and loss of appetite. he was recently hospitalised due to a peritonsillar abscess and during investigations he was first-diagnosed with non-hodgkin lymphoma. his medical and surgical history were otherwise unremarkable. clinical findings: on admission the patient was febrile and tachycardic (hr bpm) but remained hemodynamically stable (bp: / mmhg). clinical examination revealed abdominal distention and rebound tenderness in the right abdomen. investigation/results: blood tests were significant for leukocytosis (wbc: . /ll-neut: %), acute kidney injury (urea: mg/dl, cr: . mg/dl), elevated crp ( mg/l) and ldh ( iu/l), hyponatremia (na: mmol/l) and hypoalbuminemia. chest and abdominal x-rays were non-diagnostic, while abdominal ultrasound showed increased air presence along the medial line. investigations concluded with an abdominal ct scan that revealed pneumoperitoneum, small bowel distention and multiple enlarged mesenteric lymph nodes. diagnosis: the patient was transferred to the or for an explorative laparotomy. he was diagnosed with ileo-cecal intussusception causing bowel ischemia and perforation at the ileocecal valve. enlarged lymph nodes were observed along the mesentery. therapy and progressions: the affected ileus and colon were removed and a subtotal colectomy with end ileostomy was performed. the pathology report confirmed infiltration of the dissected bowel and lymph nodes by lymphoma cells. the patient continued treatment in the icu. he was discharged on the th postoperative day. comments: intussusception is rare in adults and, contrary to children, is highly associated with malignancies. resection without reduction has been advocated-wherever possible-in order to ensure better oncological outcomes. introduction: emergency surgeries are oftenly related to contaminated/infected fields, where the implantation of non reabsorbable meshes for reconstruction of the abdominal wall may not be recomendable. we aim to evaluate the results of polyvinylidenfluoride (pvdf) meshes used for complicated ventral hernia in the acute setting material and methods: retrospective analysis of patients with vh undergoing emergency surgery on which a pvdf mesh was required, in a third level hospital (november -september ). we analyzed early and late postoperative complications and -year recurrence rates. association between grade of contamination, mesh placement and infectious complications and recurrences was investigated using binary and multiple regression. results: we collected patients with a mean age of '' years, mean bmi of '' kg/m and mean cedar index of '' . '' % of patients had a grade - ventral hernia according to rosen''s index. concomitant procedures included al least one organ resection in '' % of surgeries and previous contamined mesh explantation in '' %. a pvdf mesh was placed using an intraperitoneal onlay mesh (ipom) technique in '' % of cases and an interposition location in '' %. readmission rate was '' %, one-month recurrence '' % and recurrence after a year '' %. overall mortality rate was . %. risk of recurrence was related with patients with a rosen score over (p \ . ) and also with postoperative ssi (p = . ). higher recurrence rates were not found regarding the pdvf meshes placement. postoperative seroma and hematoma rates were '' % and '' %. enteroatmospheric fistula rate was '' %. conclusions: pvdf prosthesis seems to be an useful material for complicated ventral hernia repair, specially in the acute setting, showing similar recurrence and infectious complication (fistula, chronic mesh infection, surgical site infection) rates with regard to different prosthesis used in the literature. operative vs non-operative management in liver trauma patients in a uk major trauma centre conclusions: the airs can predict the histologic severity and the intra operative findings in patients with a high clinical suspicion of aa. airs could be useful to reduce negative appendectomy and predict the postoperative stay to evaluate the deformity progression in spine injuries (dorsal, dorsolumbar, lumbar) managed by internal fixation. introduction: there continues to be controversy surrounding the management of thoracolumbar burst fractures. numerous methods of fixation have been described for this injury, but to our knowledge, spinal fusion has always been part of the stabilising procedure, whether this involves an anterior or a posterior approach. material and methods: patients with spinal injury (dorsal, dorsolumbar, lumbar) were included. all patients had dorsal, dorsolumbar, lumbar spine injuries managed with posterior short segment pedicle screw fixation and were followed up for at least one year after surgery. preoperative, post operative and follow up lateral radiographs were examined for cobb''s angle, anterior wedge compression angle and upper and lower adjacent intervertebral disc heights anteriorly, middle and posteriorly. results: at final follow up, the mean improvement in cobb''s angle post operatively was . °. the mean loss of correction of cobb''s angle was . °with sd of . °compared to post operative. the mean improvement in anterior wedge compression angle was . °post operatively. the mean loss of reduction in anterior wedge compression angle was . °with sd of . °. the increase in cobb''s angle was statically significant (r = . , p = . ) with the loss of reduction of anterior wedge compression angle at follow up and loss in intervetebral disc height at upper intervetebral disc anteriorly only(r = . , p = . ). the mean period at which sitting and standing was initiated was . months and . months respectively and mean periods for which brace was used was . months. conclusions: pedicle screw fixation is good but related to loss in reduction of anterior wedge compression angle and decrease in upper intervertebral disc height anteriorly. references: p. l. sanderson:short segment fixation of thoracolumbar burst fractures without fusion. introduction: with the newly implemented ao upper cervical spine classification system a modern, pragmatic system has been established. to what extent the simplification is helpful or whether an adjustment of the new ao classification may be discussed, forms the question of this work. material and methods: retrospective analysis of upper cervical spine injuries with ct/mri diagnostics presented to trauma surgeons with several years' experience to do classification and suggest treatment. results: the classification according to the known systems showed a relatively good agreement in the exact classification and therapy. the classification according to the new ao upper cervical spine was simple and consistent but revealed different treatment recommendations for two subtypes (iii type a and iii type b). conclusions: the new ao upper cervical spine classification system leads to a simplification. uncertainties remain with the most frequent fractures on the upper cervical spine, the c fractures. these will be managed under iii type a. however, just these injuries require completely different treatment concepts. further adaptation is required for type iii b because there uncertainties regarding the therapy also remain. case history: a -year-old woman, on treatment with acenocoumarol due to atrial fibrillation, and interatrial communication, suffered a compression fracture of the vertebrae l to l after a lowenergy trauma. due to poor pain control, she underwent a percutaneous transpedicular kyphoplasty, with no intraoperative complications. clinical findings: during the immediate postoperative period, she developed dysarthria and claudication of barré in her right upper limb. investigation/results: an angio-ct scan was performed, showing endovascular material in the left middle cerebral artery (mca) and within the lungs, compatible with cement emboli. mri showed cortico-subcortical ischemic areas in mca territory. cement-embolism stroke after percutaneous kyphoplasty therapy and progressions: conservative treatment was chosen due to the high number of emboli and the favorable evolution of the patient, with resolution of the neurologic symptoms in h without sequelae. days later, she suffered a transient ischemic attack, with no changes in the ct-scan compared to the previous images, which also solved with no residual deficits. one month after this episode, the patient died due to a spontaneous cerebellar hemorrhage related to acenocoumarol overdose. comments: kyphoplasty is a safe technique performed to treat vertebral compression fractures in elderly patients, with good clinical results and a low complication rate. its main complications are related to the leakage of cement from the vertebral body, usually well tolerated. other complications are exceptional, such as cerebral strokes, cardiac perforation, or death. the present case, although infrequent, shows us the need to assess the risk-benefit balance when operating fragile patients, as life-threatening complications may happen in these procedures. references: . marden fa, putman cm. cement-embolic stroke associated with vertebroplasty. ajnr am j neuroradiol. nov; ( ): - . survival rate and application number of total hip arthroplasty in patients with femoral neck fracture: an analysis of clinical studies and national arthroplasty registers g. hauer , a. heri , s. klim , p. puchwein , a. leithner , p. sadoghi medical university of graz, department of orthopaedics and trauma, graz, austria introduction: total hip arthroplasty (tha) is an increasingly popular treatment option for fractured neck of femur (nof) [ , ] . the aim of this study was to systematically review all literature on primary tha after fractured nof to calculate an overall revision rate. furthermore, we wanted to compare primary tha implantations after fractured nof between different countries in terms of tha number per inhabitant. material and methods: all clinical studies on tha for femoral neck fractures between and were reviewed and evaluated with a special interest on revision rate. revision rate was calculated as ''revision per component years'' [ ] . tha registers were compared between different countries with respect to the number of primary implantations per inhabitant. results: twenty-two studies showed a mean revision rate of . % after ten years. we identified eight arthroplasty registers that revealed an annual average incidence of tha for fractured nof of . per , inhabitants (table ) . conclusions: we found similar annual numbers of thas for fractured nof per inhabitant across countries. revision rates in clinical studies are higher compared to registry data [ , , ] . the results of this analysis can be used to rank present and future national tha numbers within an international context. early clinical predictors of pneumonia in patients with acute spinal cord injury without bone injury: a retrospective study t. sakamoto , s. kanezaki , n. notani oita university, oita, japan introduction: pneumonia is still significant complication that associates with mortality and duration of hospitalization in patient with acute spinal cord injury without bone injury (sciwobi). the purpose of this retrospective study is to clarify early clinical predictors of pneumonia in patients with sciwobi. material and methods: we reviewed the medical records of patients with sciwobi who admitted between january and november . spearman's rank-correlation coefficient was used to test the relationship between each parameter. multiple logistic regression analysis was performed to determine the factors that influenced pneumonic morbidity. results: a total of patients with acute sciwobi, who were evaluated for neurological impairment within h after injury, were reviewed. pneumonia occurred in patients ( %), seven patients injured at c and four at c . according to spearman's rank method, asia motor score, beginning period of nutrition, ventilator use, neurological level of injury (nli) ] c , low prognostic nutritional index (pni) were correlated with onset of pneumonia. logistic regression found ventilator use to be most predictive of pneumonia (odds ratio [or] = . , % confidence interval [ci] . - ), followed by nli ] c (or . , % ci . - . ), beginning period of nutrition (or . , % ci . - . ), pni (or . , % ci . - . ). conclusions: in addition nli, low pni increases the risk of pneumonia. we consider that improving nutritional status, especially early initiation of enteral nutrition, decrease the incidence of pneumonia. bicycle-related cervical spine fractures e. helseth , j. ramm-pettersen , s. f. eng , i. naess , m. mejlaender-evjensvold , h. linnerud oslo university hospital, neurosurgery, oslo, norway introduction: the incidence of traumatic cervical spine fractures (cs-fx) in the norwegian population is / , /year, and % of these injuries are bicycle-related ( , ) . materials and methods: prospective cohort study of all bicyclerelated cs-fx in the south-east norwegian population ( . million) in the time period - . the data were retrieved from our quality control database for traumatic cs-fx in south-east norway. in the database all cs-fx patients (c (occipital condyle) to c /th ) are prospectively registered. results: during the four-year study period patients with bicyclerelated cs-fx were registered, ( %) were males, and mean age was years (range - ). the cs-fx was located in the upper cervical segment (c -c ) in ( %) patients, lower cervical segment (c -th ) in ( %), and at both segments in ( %). the most common fx subtype was c -fx. spinal cord injury secondary to cs-fx was registered in patients ( %). fracture stabilization was achieved with open surgery in ( %), external immobilization with a stiff collar alone in ( %,) and without treatment in ( %). conclusions: severe bicycle-related cervical spine injuries are not uncommon. the increasing political desire to move commuting from motorized vehicles to bicycles warrants a heightened focus on road safety. introduction: the need for cervical immobilization is predicted by the atls, the standard of care in trauma since , because cervical trauma is a important cause of disability. however, its discontinuation was linked to x-rays, a fact that has been changed thanks to the development of two algorithms that assess the severity of cervical trauma: the canadian c-spine rule (ccr) and the national emergency x-radigraphy utilization study (nexus). material and methods: this study aims to compare the reduction values in the number of ct scans required after the application of both algorithms in a level- trauma center and to verify the degree of adherence of residents in the use of each. cohort study with randomized application by residents of the algorithms in all patients suffering from blunt trauma with cervical collars who were admitted from august to october . the conducts had their frequencies analyzed to obtain an inference about the efficacy of each method in the abstention of x-rays and case resolution, in addition to verifying if the indicated conduct was followed by the resident, inferring on the confidence in the algorithm. results: cases were evaluated during this period, of which were by the ccr algorithm and by the nexus. the indication rate for ccr imaging was . % and nexus was . %, showing no statistical difference between them (p = , ; ci = %). in the evaluation of the effective conduct, which evaluated the reliability of the algorithm, there was no disagreement between them (p [ , ; ci = %). conclusions: neither method demonstrated superiority to the other in reducing the indication of imaging exams and its uses had equal adherence by resident physicians. panacek case history: a year old lady presented with severe neck pain following a fall and cervical hyper-extension injury. she had previously undergone anterior cervical discectomy and fusion at c / with placement of artificial interbody bone graft. postoperatively, the patient reported an excellent clinical outcome and later imaging confirmed interbody fusion. clinical findings: on examination, the patient was neurologically intact but reported severe mid-cervical neck pain with reduced range of movement. investigation/results: imaging included ct and mri of the whole spine diagnosis: imaging revealed an unstable hyper-extension injury of the cervical spine. a fracture extended through the caudal end of the fused graft-vertebral interface at c / with disruption of the posterior elements. therapy and progressions: given the severity of the injury surgery was recommended. the patient underwent uneventful c -t posterior instrumentation and fusion with excellent outcome (follow up two years). comments: this is the first report of a cervical spine fracture through the site of an anterior cervical discectomy and fusion. it is hypothesised that the fused cervical segment resulted in increased stress at the fused caudal graft-vertebral interface during hyper-extension, this combined with reduced tensile strength at the graft-vertebral interface resulted in this unusual transverse fracture pattern. the clinician should be aware that patients presenting with cervical spine trauma in the context of previous cervical spine surgery are prone to greater mechanical forces. there should be a high index of suspicion for serious injury prompting thorough assessment and investigation. pr s -screw-fixation: computer aided study prevent unguided missile r. krassnig , w. pichler , e. viertler , a. schwarz , r. wildburger , g. hohenberger auva rehabilitation clinic tobelbad, tobelbad, austria, boldin und pichler og, graz, austria, medical university graz, graz, austria, auva unfallkrankenhaus, graz, austria, medical university graz, orthopaedics and trauma, graz, austria introduction: transiliosacral screw fixation of unstable dorsal pelvic ring fractures is not much present neither in literature nor in practice. in cause of the complex anatomy and the varying narrow safe bony corridors its a demanding procedure. limited information is available on optimal placement and the geometry of safe zones for screw insertion in the pelvis. material and methods: d-reconstructions of consecutive ct scans of polytraumatic injured patients ( female, male) were the basis to insert two virtual cad bolts (representing screws) into the first two sacral segments as performing during screw fixation. results: in s the narrowest point was reached after a mean of . mm respectively . mm, depending on the selected way of measurement. for s the mean distance to the tricky constriction area amounted to . mm, respectively . mm. the average height in s measured . mm and the average width . mm. according, the average height for s was . mm and the average width . mm. the measurement results didn't show a significant difference between male and female pelvis bones for any distance of interest. conclusions: an optimal screw position is very important, because in the areas of bony narrowing are the exit points of the sacral nerves, which exit through the foramina anteriorly and posteriorly. damage to this nerve structures can cause severe long-term consequences such as numbness or paralysis. knowledge of predefined distances may aid in preoperative planning, decrease operative and radiation times and may prevent unguided missiles. clinical findings: there were absent breath sounds on the right side of the thorax, ultrasound showed an extensive pleural effusion. a chest tube was inserted and l of bloody-milky fluid was drained. investigation/results: ct scan showed fractured c -c and th -th vertebral bodies, fractured lateral osteophytes of th - and probable injury of the thoracic duct at th - level. pleural effusion analysis showed raised cholesterol and triglyceride levels. diagnosis: traumatic chylothorax; fractures th -th , th -th , c -c therapy and progressions: patient was kept on ventilatory support for days. primarily she was treated with total parenteral nutrition followed by no fat and hypolipidemic diet. the chest tube was removed after days. she was discharged in stable condition the following day. at the month check-up she was stable and eupnoic. comments: traumatic chylothorax caused by blunt chest trauma is extremely rare. there are hypotheses that injuries to the thoracic duct are caused by hyperextension of the spine or by increased thoracic/ abdominal pressure (seat-belt injuries). in our case, chylothorax probably resulted from fractured lateral osteophytes. patients are usually successfully treated with pleural drainage and total parenteral nutrition. if there is no improvement after weeks or if drainage exceeds . l/day or l/day for more than days, thoracic duct ligation should be considered. conservative treatment resulting in t-l or lumbar kyphosis can worsen the quality of life of the patient whereas traditional open surgery may be an overtreatment in some cases, considering blood loss, possible complications, hospital stay and delayed functional recovery. in this setting, a good option can be a percutaneous minimally invasive surgery. the advantages of percutaneous pedicle screw fixation are: preservation of posterior musculature, less blood loss, shorter operative time, lower infection risk, less postoperative pain, shorter hospital stay and easier implant removal after bone union. limitations such as inability to achieve direct spinal canal decompression can be dealed by combination with open techniques. the objective of this study was to report the results of ppsf on these fractures and the technical problems we had to overcome. methods: patients are included, treated with percutaneous transpedicular fixation and stabilization with minimally invasive technique from december to october . patients were males, females; average age was , years (range from to ). in all cases, system pathfinder-nxt (zimmer) was used. results: most of the patients presented an early post-operative mobilization with amelioration concerning pain and a low complication rate. limitations in mobilization were mainly due to coexistent injuries, polytrauma or non-reversible neurological deficit. conclusion: ppsf is a reliable and safe procedure which does not replace the open technique but adds to treatment options by restoring a good sagittal alignment similar to those reported for open surgery. removal of hard material is advocated after fracture healing to preserve the lumbar spinal mobility and avoid zygapophyseal joint osteoarthritis. critical surgery within the first hour of presentation: is it a feasible intervention for better trauma care outcomes in low and middle income countries? introduction: in low and middle-income countries (lmic) golden hour care concept is almost nonexistence due to resource constraints. in this study, we analyzed one novel concept of critical surgery within the first hour of admission as a possible intervention which could be applied in the existing scenario in these countries without much resource requirement. material and methods: a retrospective analysis of a prospectively maintained data registry under a project named titco (towards improved trauma care outcome) was done. registry data from a level - trauma centre in india were analyzed from october to september . all patients who admitted and underwent critical surgical interventions within the first hour of presentation were analyzed. these patients were divided into two groups depending upon primary presentation or referred from another facility. statistical analysis was done between these two groups to compare the outcome. results: sixty-one ( . %) patients were directly admitted from the site of the incident whereas forty-five ( . %) were transferred from other hospitals for surgical needs. the median time from injury to presentation for primary patients was min with interquartile range (iqr) of . in the referred patient median time gap between the injury to our center (not referring center) was min with iqr of . this difference was statistically significant. major outcome indicators in the form of median icu and total stay, as well as mortality, were not significantly different conclusions: proposed concept might be a useful hospital-based intervention in existing trauma system in lmic to improve the outcome of injured patients along with improving prehospital services. oslo university hospital, ullevål, orthopedic department, oslo, norway, extrastiftelsen, oslo, norway introduction: it is well-known that physical activity is good for us. although the skeletal muscle is the main organ which is directly affected, exercise affects the whole body. the mechanisms responsible for these beneficial effects are gradually becoming known to us through extensive research. this might make it easier for physicians to prescribe exercise as a therapy equally and even more beneficial than drugs regarding effect and risk profile. the aim of this thesis was to review the current literature on the molecular mechanisms of exercise-induced health benefits. material and methods: a search in medline and embase resulted in articles. they were sorted by title and abstract, then by reading the full text. relevant articles from the reference-lists were included. sources were found outside of the search. results: when we exercise, the skeletal muscle is subjected to several mechanical and chemical stimuli, which in turn activate a set of kinases and phosphatases. these are molecules that regulate transcription-factors and co-activators, and this leads to adaption of the muscle-cells. i addition, the muscle secretes a number of proteins called myokines, which conduct the effect of exercise to other organs and tissues. some lead to increased cerebral neuroplasticity, hypertrophy and angiogenesis (bdnf, vegf and igf- ). several interleukins have also been identified as myokines, and they mediate an anti-inflammatory effect which is favorable in the prevention and management of conditions like atherosclerosis and type diabetes. lastly, we found that exercise leads to epigenetic changes, altering the genetic expression in several types of tissues. some studies suggest that the epigenome is affected by exercise even before we are born, giving babies born to physically active mothers a favorable epigenetic expression. conclusions: we should use this knowledge to support the implementation of physical activity in treatment and preventive health care. impact on undertriage and mortality after changing from a twotiered to one-tiered trauma team activation protocol costs. prognostic factors may assist in identifying high cost groups with potentially modifiable factors for targeted preventive interventions, hence reducing costs and increase rtw rates. evaluation of long-term follow-up and consequences of gunshot and stab wounds in a french civilian population introduction: the data concerning long-temr follow-up of patients and consequences of gunshot wound (gsw) and stab wound (sw) are almost inexistent in the literature. in finland, a study showed that % of patients with trunk wounds died secondarily from alcohol-related or violent problems [ ] , highlighting the secondary importance of long-term care for these patients. the main objective of our study was to analyze the hospital and posthospital follow-up of patients with gsw or sw and to evaluate late complications and the consequences of these traumas. material and methods: from january to january , patients were hospitalized for gsw or sw management in laveran military hospital. hospital data were collected via informatic patient file and post-hospital data via a telephone questionnaire with the general physician (gp). results: median hospital follow-up was days . seventy-six patients had a follow-up visit with their gp ( %). median follow-up was mois . twenty-four patients were totally lost to follow-up ( . %). global follow-up identified patients with longterm consequences ( . %), psychiatrics and organics. seventeen cases of recurrence were found ( . %). high iss, age, gsw and gp identified in patient medical file were significantly linked to long-term consequences occurrence. conclusion: this study showed a high number of long-term consequences occurrence among patients with gsw or sw. however, the extra-hospital follow-up seems insufficient. it is therefore imperative to strengthen the compliance and adherence to the care network of these patients. awareness and involvement of medical, paramedical teams and gp role seems essential to screening and management of these consequences. introduction: focused assessment with sonography for trauma(-fast) is an effective tool for assessments of severely injured patients, especially in the settings of helicopter emergency medical service(hems) because of limited devices and time. the objective of this study is to investigate accuracy of trauma ultrasound in helicopter emergency service compared with enhanced ct scan. material and methods: we investigated the trauma patients in years which was demonstrated fast and delivered to the advanced critical care center in gifu university by hems. accuracy of the fast was determined by comparison to the presence of injury, primarily determined by computed tomography, and to required interventions. results: patients were included in this criteria. there were and patients in which we found fluids in thoracic or abdominal cavity by enhanced ct scans and ultrasounds in hems, respectively. sensitivity and specificity, positive predictive value, negative predictive value, accuracy were . , . , . , . , . . if we limited the data for abdominal fluids, each data were . , . , . , . , . . in the patients of negative fast with positive ct, no patient died due to hemorrhage in thoracic or abdominal cavity. conclusions: it has been reported that sensitivity of fast in hems was lower compared with in er. in the settings of prehospital trauma care, advantages of portable ultrasound could be limited because of peculiar environments. and also, the thoracic or abdominal fluids could increase with time by organ injuries and it causes fast negative in acute phases.in this series, we could not find cases which has possibility of death because of negative fast and might influence the treatment. repeated fast or careful assessment of patients based on the other findings could be beneficial. references: the sensitivity of fast in hems was low and demonstrating fast for several times could be effective to detect the thoracic or abdominal hemorrhage. pre-hospital trauma care in switzerland and germany: do they speak the same language? los angeles county ? usc medical center, department of surgery, divison of acute care surgery and surgical critical care, los angeles, united states introduction: field amputation can be life-saving for entrapped patients requiring surgical extrication. under these austere conditions, the procedure must be performed as rapidly as possible with limited equipment, often in a confined space, while minimizing provider risk. the aim of this study was to determine the optimal saw for a field amputation. material and methods: this was a prospective cadaver-based study. four saws (gigli, hand pruning, electric oscillating and reciprocating) were tested in human cadavers. each saw was used to transect four separate long bones (humerus, ulna/radius, femur and tibia/fibula). the time required for each saw to cut through the bone, the number of attempts, slippage, quality of proximal bone cut and extent of body fluid splatter as well as the physical space required by each device during the amputation were recorded. univariate analysis (fisher's exact and kruskal-wallis or mann-whitney u-test) was used to compare the outcomes between the different saws. results: the fastest saw was the reciprocating followed by oscillating ( . [ . - . ] sec vs . [ . - . ] sec, p = . ). the number of attempts required to amputate ( . [ . - . ] , p = . ) and the amount of slippage ( . [ . - . ], p = . ) were highest with the pruning saw. the reciprocating saw had the worst proximal bone cut quality ( % poor, p = . ) and the largest blood splatter ( . [ - ] , p = . ). the physical space required to perform an amputation ranged from cm with the oscillating to cm with the reciprocating saw. overall, the oscillating saw outperformed the others in number of attempts, slippage and quality of bone cut and physical space requirements, and was the second fastest ( table ) . conclusions: the speed, precision, safety, space required, as well as the highly adjustable blade in the oscillating saw make it ideal for a field amputation. a gigli saw is an excellent backup for when electrical tools cannot be used or fail. impact of air medical transport on the survival of major trauma patients in thailand e. surakarn , w. siriwanitchaphan bangkok hospital headquarters, bangkok trauma center, bangkok, thailand introduction: air medical transport is an alternative mode of interfacility transfer for injured patients who required a higher level of trauma care in thailand. this study assessed the impact of air medical transport on the survival of major trauma patients transferred from local hospitals to a tertiary care hospital. material and methods: trauma registry of - was reviewed. major trauma patients transferred by air ambulance were identified. injury severity score (iss), predicted mortality and actual survival to hospital discharge were studied and compared between two subgroups, the seriously injured patients (iss - ) and the severely injured patients (iss [ ) . the predicted mortality was calculated from the probability of survival (ps) of trauma and injury severity score (triss). results: there were major trauma patients (iss [ ) transferred by air ambulance in five years period. patients were severely injured (median iss = ), and patients were seriously injured (median iss = ). the range of flight time was - min. the overall survival rate was . %. the predicted mortality in the severely injured group was cases ( . %), but the actual mortality was nine ( . %), . % lower than predicted mortality. the predicted mortality in the seriously injured group was one case ( . %), while the actual number was two ( . %). the eleven deaths in this study were eight cases of severe traumatic brain injury(tbi) patients, two cases of massive bleeding with subsequent multi-organ failure and one drowning. conclusions: air medical transport significantly improved the survival of severely injured patients who need higher level of trauma care. severe tbi and the presence of multi-organ failure associated with unfavorable outcomes. however, a detailed analysis of the trends and epidemiology of rtis affecting the most vulnerable children in qatar, under years of age, has not been conducted. this study's primary objective of is to describe the epidemiology of rtis and deaths in young pediatric patients in qatar. material and methods: data, for all young pediatric [under years] victims of rti''s and rti deaths from january , , through december , , from the trauma registry of the hamad trauma center [htc], the national level trauma referral center of qatar, was analyzed. this data was correlated and compared with data from the hamad general hospital mortuary and vital statistics data from the qatar ministry of development planning and statistics, the vital statistics annual bulletin, for the years - . results: the htc attended to patients, under years, with severe rtis and in-hospital rti deaths were reported during the study period. males made up . % of the injured and % of fatalities.the average age of the injured was years and for fatalities was it was . years. the rti incidence rate per , for both sexes, under years, has been unchanged ( in and in ) . the road mortality rate, per , , has decreased significantly, from . in to . in . since , the proportion of pre-hospital deaths has been increasing, - %, and the in-hospital death rates has been reduced to %. conclusions: rapid improvements in pre and in-hospital post-crash care in qatar have resulted in marked reductions in in-hospital deaths for young children with rtis. the emergence of pre-hospital road deaths of under ''s must be made a priority for road safety in qatar. the implementation of proven prevention programsshould be fast tracked in order to directly address this issue. introduction: despite improving survival of patients in prehospital traumatic cardiac arrest (tca), initiation and/or discontinuation of resuscitation of tca patients remains a subject of debate among prehospital emergency medical service providers. the aim of this study was to identify factors that influence decision making by prehospital emergency medical service providers during resuscitation of patients with tca. methods: twenty-five semi-structured interviews were conducted with experienced ambulance nurses, hems nurses and hems physicians individually, followed by a focus group discussion. participants had to be currently active in prehospital medicine in the netherlands. interviews were encoded for analysis using atlas.ti. using qualitative analysis, different themes around decision making in tca were identified. results: the causes of bleeding were grouped into several categories.the most frequent cause with cases in a row is attributed to diverticular bleeding,other causes of bleeding were angiodysplasia,post polypectomy bleeding,gist tumor,rectal ulcers and inflammatory disease.no case presented mortal or serious complications,secondary to the procedure. only cases presented a mild complication: focal mucosal ischemia of the embolized intestinal segment that was resolved with conservative treatment.lesions in charge of bleeding in those cases in which the angiographic treatment failed,were:ulcer in cases,a case of bleeding after endoscopic polypectomy, a case of diverticular bleeding and bleeding secondary to a coagulation disorder.among these patients, the definitive treatment was the following: -a second angiographic treatment was effective in the case of bleeding due to coagulation disorder. -a case of self-limited bleeding. -surgical treatment was the definitive treatment in both cases of bleeding in the context of and patient with bleeding after polypectomy. we have not observed a significant relationship neither the type of lesion or its location with the probability of failure of the angiographic treatment. nor do we observe a significant relationship between the type of material used for embolization and the risk of treatment failure. comments: our data show that angioembolization is an effective and safe technique to treatment lgi bleeding. references: clin endosc . endoscopic therapy for acute diverticular bleeding introduction: the use of resuscitative endovascular balloon occlusion of the aorta (reboa) as adjunct for temporary hemorrhage control in patients with major torso hemorrhage is increasing. specifications and characteristics of available aortic occlusion balloons (aob) are diverse. in order to minimize the risk of failure and complications it is important to choose a device that fits the requirements per medical situation. the aim of this study is to provide guidance in the choice of an aob in a specific situation. material and methods: aob were assessed for characteristics and different properties of each are outlined. the bending stiffness was measured with a three-point bending device. results: although all aob tested are small caliber devices ranging from (er-reboa tm ) to french (codaÒ ), some need large bore access sheaths up to french (fogarty Ò and lemaitre tm ) or even insertion via surgical cut-down (equalizer tm ). the bending stiffness of the aob varied from . n/mm (± . sd) with the codaÒ to . n/mm (± . sd) with the russian prototype. guidewire-free devices are generally stiffer than over-thewire catheters. the tokai rescue balloon tm showed kinking of the shaft at low bending pressures. the er-reboa tm , fogarty Ò , lemaitre tm , reboa balloon Ò , and rescue balloon tm are the only catheters with external length marks to assist blind positioning. the only aob using a non-compliant balloon is the reboa balloon Ò . conclusions: specifications of available aob are diverse. in resource-limited settings, reboa should be performed with a rather stiff device that can be placed without wire and fluoroscopy, such as the er-reboa tm , fogarty Ò , and lemaitre tm . of these aob, the er-reboa tm is the only catheter compatible with a small french sheath. use of non-compliant balloons without real-time fluoroscopy is not advised given the potential risk of aortic rupture. when fluoroscopy is available, a guidewire can be considered. case history: year old male patient presenting with an initially uncomplicated pertrochanteric fracture, treated by an intramedullary nailing system (figs. and ) . days after the operation and mobilization without any adverse events the patient was readmitted. clinical findings: massive swelling, hematoma and pain. investigation/results: sudden fall of hb values down to , g/dl, ct scans showed the lesser trochanter located directly to the deeper femoral artery after mobilization (fig. ) . diagnosis: perforation of the deep femoral artery and several veins by the dislocated lesser trochanter therapy and progressions: blood transfusion, intraoperative cardiopulmonary resuscitation, several revision surgeries to stop the bleeding by oversewing the deep femoral artery and ligation of the veins, removal of the lesser trochanter fragment (fig. ) . admission to intensive care unit. subsequent plastic coverage. comments: according to literature, bleeding complications and injuries of the deep femoral artery can occur even several days after an initially uncomplicated pertrochanteric hip fracture. besides acute life-threatening bleeding, false aneurysm can occur ( ) ( ) ( ) . even if those late complications are very rare, the consequence for the patient can be devastating. these rare cases show the clear obligation to a thorough follow up treatment and regularly dressing changes. investigation/results: arterial colour doppler of the popliteal artery showed hypoechoeic contents and narrowed lumen. biphasic flow was seen in both popliteal and posterior tibial arteries. diagnosis: popliteal artery injury with delayed repair therapy and progressions: two incision and four compartment fasciotomy was done under regional block the next day which revealed a non contractile posterior compartment. superficial and deep muscles of the posterior compartment had doubtful viability. left distal sfa to infragenicular popliteal artery bypass graft was placed on day post injury. blood flow was established upto the ankle and foot, confirmed on check angio. however, foot drop of the patient persisted. after appearance of a healthy granulation tissue at the wound site ( days), a split thickness skin graft was placed to give coverage with % uptake of the graft. comments: blunt popliteal artery injury has been reported to result in amputation rates of nearly - %.the importance of a detailed vascular examination of a blunt trauma patient is emphasized as a limb can be salvaged if timely intervention is done. in this case even with an unfavourable mess score. case history: a healthy -year-old male, with no history of interest, suffers a high-energy trauma as a water bottle rushes over his left knee. clinical findings: go to the emergency room with pain and functional impotence in the left knee, with its anatomical deformity. knee x-ray pa and l are performed and the distal pulses that are present are taken. ankle-brachial index [ . . closed reduction is performed in emergencies under sedation and control x-ray is requested, aiming at correct reduction. it was decided to keep under observation for - h before discharge from hospital to schedule regulated ligament reconstruction surgery after studying with mri. therapy and progressions: at h of evolution after the accident and after having reduced the dislocation, the patient who has the leg with a temperature equal to the contralateral is reassessed, however, there is no palpable dorsal pedis pulse or posterior tibial palpation in the affected leg. it is decided to urgently request an angiotc and it is objective thrombosis of popliteal artery. vascular surgeon is contacted and emergency surgery is decided. a by-pass is performed with vena safena. diagnosis: traumatic knee dislocation and popliteal artery injury comments: in the st century, complementary tests in diagnosis are becoming increasingly important. however, in this case we want to management of aseptic tibial nonunion anastasios g. c. reamed interlocking intramedullary nailing for the treatment of tibial diaphyseal fractures and aseptic nonunions. can we expect an optimum result? results of a systematic approach to exchange nailing for the treatment of aseptic tibial nonunion management of tibial non-union using reamed interlocking intramedullary nailing the radiographic union scale in tibial (rust) fractures: reliability of the outcome measure at an independent centre pelvic trauma: wses classification and guidelines damage control orthopaedics in unstable pelvic ring injuries references: beuran, m. trauma scores: a review of the literature glasgow coma scale, age, and arterial pressure (mgap): a new simple prehospital triage score to predict mortality in trauma patients. critical care medicine. champion hr. a revision of the trauma score proximal femoral nail antirotation versus gamma nail for intramedullary nailing of unstable trochanteric fractures. a randomised comparative study results of the femur fractures treated with the new selfdynamisable internal fixator (sif) dhs helical blade for elderly patients with osteoporotic femoral intertrochanteric fractures the hypermetabolic response to burn injury and interventions to modify this response racial differences in propranolol enantiomer kinetics following simultaneous i.v. and oral administration propranolol dosing practices in adult burn patients the hypermetabolic response to burn injury and modulation of this response: an overview. wound heal south africa management strategies and outcome of blunt traumatic abdominal wall defects: a single centre experience blunt traumatic abdominal wall hernias: a surgeon's dilemma blunt traumatic abdominal wall hernias: associated injuries and optimal timing and method of repair traumatic abdominal wall herniation: case series review and discussion trauma patients with open abdomen: do they differ from others? a single center experience h. fagertun , a. seternes department of circulation and medical imaging, trondheim, norway introduction: treatment with open abdomen is demanding for patients, staff and hospital. a multidisciplinary approach is mandatory. the aim of this study was to compare trauma patients with open abdomen (oa) and patients treated with oa for other reasons, regarding outcome and resources spent. material and methods: retrospective study of patients treated with oa in a tertiary hospital in norway. ten were trauma patients vacuum-assisted wound closure and mesh-mediated fascial traction for open abdomen therapy-a systematic review prospective study examining clinical outcomes associated with a negative pressure wound therapy system and barker's vacuum packing technique thoracic-abdominal trauma with diaphragm lesions n. vlad , i. streanga , a. morar , i st. spiridon'' hospital iasi. we have analyzed clinical data, trauma mechanism, pathology of the lesion, time trauma-diagnostic, associated lesions, treatment, and follow-up. results: there have been patients ( men, women), mean age . location of diaphragmatic tears has been on the left hemidiaphragm ( cases), on the right hemidiaphragm ( cases), or bilateral ( cases). the trauma mechanism has been blunt ( cases) or penetrant ( cases). all patients had associated visceral lesions and had been operated right diaphragmatic injury and lacerated liver during a penetrating abdominal trauma: case report and brief literaturereview traumatic diaphrag-matic ruptures: clinical presentation, diagnosis and surgicalapproach in adults traumatic rupture of the diaphragm: experiencewith patients % ( / ) were aast grade or . in the total group, median age was years, . % were male and . % were blunt injuries. median iss in the nom group was and in the om group. median iss for those with grade or injury was . . % ( / ) underwent nom, compared to . % ( / ) of those with aast grade or . for each mmhg increase in systolic blood pressure, patients with grade or injury were % less likely to have an operation (or . , p = . ) and for each beat increase in heart rate intra-operative grade i was revealed in patients ( , %), grade ii in ( , %), grade iii in ( , %) grade iv in ( , %) and grade v in ( , %). histologic finding of catarral appendicitis was found in ( , %) patients, ( %) had phlegmonous appendicitis and ( , %) had gangrenous appendicitis. the airs difference was statistically significant with histological findings quality of publications regarding the outcome of revision rate after arthroplasty swedish hip arthroplasty register annual report joon yung lee: risk factors for failure of nonoperative treatment for unilateral cervical facet fractures in , patients were included in the trauma registry. median iss was and patients had an iss [ . of these patients / ( %) were undertriaged with a mortality of / ( %). the total mortality in was , % ( / ). i , median age was years for the patients with no tta vs years for those patients who did receive a tta (p \ . ) prognostic factors for medical and productivity costs, and return to work after trauma: a prospective cohort study l results: a total of trauma patients ( % of total study population) responded to at least one follow-up questionnaire. mean medical costs per patient (€ , ) and mean productivity costs per patient (€ , ) varied widely. prognostic factors for high medical costs were higher age, female gender, spine injury, lower extremity injury, severe head injury, high injury severity, comorbidities, and pre-injury health status. productivity costs were highest in males, and in patients with spinal cord injury, high injury severity, longer length of stay at the hospital and patients admitted to the icu. prognostic factors for rtw were high educational level, male gender, low injury severity swiss and german (pre-)hospital systems, distribution and organisation of trauma centres differ from each other [ , ]. it is unclear if outcome in trauma patients differs as well. therefore, this study aims to determine differences in characteristics, therapy and outcome of trauma patients between both german-speaking countries. material and methods: the traumaregister dguÒ (tr-dgu) was between - and - were included if they required icu care or died. trauma pattern trauma care in germany trauma systems in europe practical assessment of different saw types for field amputation: a cadaver-based test study these themes were: factual information (e.g., electrocardiography rhythm)educational programs and future guidelines. references: rosemurgy as, prehospital traumatic cardiac arrest: the cost of futility blunt vertebral vascular injury in trauma patients: atlsÒ recommendations and review of current evidence treatment-relater outcomes fron blunt cerebrovascular injuries. importance of routine follow-up arteriography provided the catheters used for this study. no other support was provided diagnosis: the probe had perforated the ivc wall. therapy and progressions: open repair was performed through a xifopubic laparotomy and the cattel-braasch maneuver to expose the ivc (fig. ). a retroperitoneal hematoma was observed anteriorly to the infrarenal ivc, without active bleeding. the ivc was dissected out sufficiently to permit proximal and distal vascular control (fig. ), the probe was then removed and the laceration on the infrarenal ivc closed with a running suture. the postoperative course was uneventful. comments: to our knowledge this is the first reported case of symptomatic ivc laceration by an ice probe during ca. references: enriquez a. use of intracardiac echocardiography in interventional cardiology complications of catheter ablation for atrial fibrilla iatrogenic percutaneous vascular injuries: clinical, presentation, imaging, and management vascular complications during catheter ablation of cardiac arrhythmias: a comparison between vascular ultrasound guided access and conventional vascular access false aneurysm of the profunda artery resulting from intertrochanteric fracture. a case report profunda femoris arterial laceration secondary to intertrochanteric hip fracture fragments. a case report with major thoraco-abdominal vascular injuries (aorta, inferior vena cava and main branches). data on demographic, clinical status and imaging was recorded. descriptive and kaplan meir survival analysis was performed. results: patients were included. median age was years (iqr - ), ( . %) were male. aorta was the most frequently damaged vessel ( , %) the median iss was (iqr - )interventional procedure. overall mortality was %, with % of deaths during the first hour, . % in the first h and . % afterwards. median survival was days (ic - ). we compared survival curves in periods abdominal vascular trauma. trauma surg acute care open history: popliteal artery injuries are frequently seen with fractures, dislocations, or penetrating injuries. a thirty one year old pathologies. references: natsuhara, k.m. et al, what is the frequency of vascular injury after knee dislocation knee dislocation and vascular injury: -year experience at a uk major trauma centre and vascular hub can vascular injury be appropriately assessed with physical examination after knee dislocation? introduction: this retrospective cohort study investigated the prevalence of and risk factors for preoperative venous thromboembolism (vte) in patients with a hip fracture and a delay of [ h from injury to surgery. material and methods: this observational study included patients with a hip fracture surgically treated at university hospital. patients underwent indirect multidetector computed tomographic (mdct) venography for preoperative vte detection after admission. overall vte risk and median time from injury to ct scan were calculated. age, sex, fracture type, time from injury to ct scan, body mass index, preinjury mobility score, previous anticoagulation treatment, previous hospitalization for vte, varicose veins, and medical comorbidities were considered potential risk factors. results: the prevalence of preoperative vte was . % ( of patients). the mean time from injury to ct scan was . days. the delay from the time of injury to ct scan averaged . days for patients who developed preoperative vte, compared with . days for patients who had not developed vte. in the adjusted models, female sex, subtrochanteric fracture, pulmonary disease, cancer, previous hospitalization for vte, and varicose veins were risk factors for vte. the final multivariate logistic regression analysis introduction: vertebral compression fractures constitute a large percentage of traumatic injuries of spine. the initial management plays an important role in the final outcomes. the present study aims to study the profile of vertebral injuries in rural & semi urban population & to analyse the role of level two hospitals in initial management of vertebral injuries. material and methods: this study was a retrospective analysis of progressively collected data of patients presenting with vertebral injuries in a level two hospital catering to semi urban & rural population in india. the initial presentation along with the age & sex profile was noted. etiological factors leading to compression fractures were noted. any neurological deficit either at the time of admission or transfer to a tertiary care neurocentre was noted as per asia scale. initial management was carried out in accordance with the atls guidelines. results: a total of out patients admitted with complaints of back pain were diagnosed to have compression fractures of the spine. the mean age was . years. male: female ratio was approx : . the lumbo sacral spine region was the most comply affected region. two patients were incidentally detected to have vertebral fractures as a result of metastatic malignancy. a due note was made regarding patients who had deteriorated during the transfer in terms of neurological deficit & evidence of spinal shock. conclusions: road traffic accidents contribute a significant portion of vertebral trauma . smaller hospitals & general surgeons have an important role to play in terms of initial stabilisation of such patients particularly the ones presenting with neurogenic shock. a good initial management has sigificant bearing on outcomes. analysis of risk factors for tracheostomy in cervical spinal cord injury without bone injury n. notani , s. kanezaki , t. sakamoto , h. tsumura oita university, orthopaedic surgery, yuhu, oita, japan introduction: there are many cases that require tracheostomy in the acute phase of cervical spinal cord injury, and various risk factors have been reported so far. however, there has been no report on cervical spinal cord injury without bone injury. the aim of this study is to evaluate risk factors for tracheostomy in patients with cervical spinal cord injury without bone injury. material and methods: we conducted a retrospective observational study. patients who were treated for cervical spinal cord injury without bone injury in our hospital were divided into groups: tracheostomy (tc) group and no tracheostomy (no tc) group. we compared variables, including age, sex, asia impairment scale (ais), neurological level of injury (nli), injury severity score (iss), vital signs, blood gas analysis, tracheal intubation, chest complication, smoking history between two groups. results: there were patients in tc group, and patients in no tc group. on univariate analysis, there were significant differences in age, ais a, tracheal intubation, nli ] c . on multivariate analysis, nli ] c was an independent predictor of tracheostomy. conclusions: in this study, we demonstrated that nli ] tc could be useful to predict tracheostomy in patients with cervical spinal cord injury without bone injury. case history: many fractures of the articular processes of the cervical spine are associated with displacement and instability, approximately % of all traumatic cervical spine injuries involve isolated fracture of the articular processes non-displaced or minimally displaced. [ ] this case demonstrates a isolated facet fracture of the cervical spine with c radiculapathy treated with minimally invasive spine surgery techniques clinical findings: a -year-old male was admitted to the neurosurgery department due to severe neck pain (vas / ). the pain radiating to the right upper extermity along dermatome c . neck and trunk rotation worsened the pain. investigation/results: furthermore, physical examination revealed hyperaesthesia in the right index finger without muscle weakness. ailments suddenly appeared weeks earlier after getting up in the morning. imaging demonstrated isolated, unilateral fracture of the right superior articular process of c diagnosis: imaging demonstrated isolated, unilateral fracture of the right superior articular process of c therapy and progressions: the patient was treated by microsurgical c decompression and fusion of c - under navigation guidance. intraoperative ct scans were performed to evaluate sufficient bone removal.after the surgery, the neck and upper extremity pain subsided. the patient had returned to his usual job and sport activities. comments: this case illustrates the value of the navigation and intraoperative ct in the evaluation of bony decompression, anatomy and location of implants. navigation minimized iatrogenic injury resulting in reducing postoperative complications like chronic pain, kyphotic deformity and muscular atrophy.introduction: resuscitative endovascular balloon occlusion of aorta (reboa) is a technique initially developed in the military for trauma patients injured in combat . recently, there has been much debate on its role in civilian trauma cases in controlling non-compressible torso haemorrhage (ncth) . this review aims to provide an update on current literature on the outcomes and concerns of this procedure. material and methods: a systematic literature search according to prisma guidelines was performed over the period of january to august across embase, medline and cochrane databases. patient characteristics, mechanism and severity of injury, survival rates and post-reboa complications between survivors and non-survivors were compared. results: a total of studies were included in this review. % and % of the reboa cases were penetrative and blunt cases respectively. the survival rates ranged from to % across the studies. systolic blood pressure (sbp) was significantly elevated post-procedure, from . to . mmhg in the survivor group (p \ . ) and . to . mmhg in the non-survivor group (p = . ). the injury severity score (iss) was lower in the survivor group ( . vs . ; p \ . ) whereas their glasgow coma scale (gcs) was higher ( . vs . ; p = . ). the survivors also had a shorter duration of aortic occlusion ( . vs . min; p = . ). common complications noted following the procedure include renal injury, lower limb ischaemia and thrombosis. conclusions: pre-reboa sbp, iss, gcs and duration of aortic occlusion were found to be associated with survival. complications directly due to the procedure were difficult to ascertain. a prospective study in a multiple trauma centre is needed for further evaluation of the indications, feasibility and complications involved in reboa. references: introduction: traumatic vertebral artery injury (vai) is a wellknown complication of cervical spine injury and often causes posterior circulation stroke. we report preventive effect of acute phase endovascular intervention for traumatic vai. material and methods: all patients with cervical spine injury were surveyed with post-contrast computed tomography for vai. when vai was diagnosed, the affected vertebral artery (va) was occluded with endovascular intervention before spine reduction and fixation. brain ischemic lesion was evaluated before and after the treatment. results: forty-one patients with vai associated with cervical spine injury underwent endovascular intervention. the affected va was occluded with endovascular coils before cervical spine reduction and fixation in patients, and after treatment in one patient. va stenting was done for another two. six presented new brain infarctions after spine surgery. of these, two had endovascular intervention after spine reduction. out of patients who had endovascular embolization before spine reduction, four had newly developed infarctions after spine surgery, of which two were symptomatic. there were no complications related to the endovascular procedure. conclusions: in conclusion, endovascular embolization for traumatic vai before spine reduction and fixation was found to be effective to prevent symptomatic brain infarction. introduction: the use of drug coating balloons (dcb) in primary or secondary angioplasty for peripheral vascular disease is a new tendency. the use of paclitaxel decelerates the growth and hyperplasia of neo-intima tissue which can cause re-stenosis and total occlusion in the spot of pta is a very promising technique in long lasting results of balloon ptas. purpose: to demonstrate our experience and results of the technique of dcb pta with the use of drag coating balloons. material and method: in the period between march and september , patients with sfa lessions were treated with pta with dcb for acute limb ischemia. were males and females. mean age was , y.o (± . ). patients were examined pro operationally and immediate post operationally in abi difference and their post operational follow up included measurement of abi and u/s triplex scan on the st, rd, th and th month(where chronically available) after pta. results: the mean immediate post operative increase of abi was , (± , ). were chronically available the increase of abi remained to , in the months follow up, , in the months and , in the th month follow up while patency of the artery treated remained in all patients. of the patients suffered from acute complications during or short after the pta ( with peripheral embolization and with retroperitoneal hematoma) which were treated immediately and left no consequences. conclusions: the use of dcb for pta in acute ischemia is a quite new, promising technique for maintaining patency of treated arteries for long time post operative period. the medium time results from its use in our clinic seem to be satisfactory. jichi medical university hospital, tochigi, japancase history: a -year-old male hit his neck hard against the fence. thereafter, he experienced difficulty in breathing and severe neck pain. he was brought to the emergency center by ambulance. clinical findings: his vital signs on arrival were gcs: e v m , hr: , bp: / , rr: , spo : ( lo ). significant neck edema and tracheal deviation were noted. inspiratory stridor was not heard with no signs of retracted breathing or subcutaneous emphysema. investigation/results: an enhanced ct scan of the neck revealed tracheal deviation and compression with ruptures of the left thyroid lobe. a large hematoma and arterial extravasation from a branch of the inferior thyroid artery were noted. diagnosis: rupture of the left thyroid lobe and injury around the distal portion of the left inferior thyroid artery. therapy and progressions: after securing the airway by intubation, angiography of the neck was performed; extravasation from a branch of the left inferior thyroid artery was suspected. angioembolization was continued for hemostasis using gelatin sponge. neck edema improved in the intensive care unit. following extubation on the hospital day , the patient was discharged on the th day with no complication. comments: thyroid injury due to blunt neck trauma is rare and surgical intervention such as hemithyroidectomy is generally prescribed. the patient''s condition, in this case, improved by angioembolization without any invasive surgical procedures. catheter procedure may, thus, be effective for hemostasis on thyroid injury after the confirmation of airway placement. introduction: the indication for resuscitative endovascular balloon occlusion of the aorta (reboa) is hemodynamically unstable patients in life-threatening hemorrhage below diaphragm. it was unclear that the difference of indications for reboa affects mortality in trauma.material and methods: this study used data from the japan trauma data bank (jtdb) ( - ), a nationwide trauma registry, to describe the epidemiology of reboa. adult trauma patients used reboa were included. patients were excluded if they had cardiac arrest at the scene or dead on arrival, or had an unsurvivable injury of any region of the body as defined by the abbreviated injury scale. patients were classified by whether patients had indications for reboa. the indications for reboa were defined by indications for hemostasis to intraabdominal, retroperitoneal, pelvic or extremity hemorrhage. the indications were decided by the delphi method with the cooperation of experts in trauma for this study. the contraindications were defined by brain injury needed intervention and hemorrhage above diaphragm. the logistic regression was used to assess the mortality after adjustment for injury severity score. as a sensitivity analysis, a generalized linear mixed model with random effects of a facility was performed. results: of , patients registered in the jtdb, patients underwent reboa. had indications for reboa and underwent reboa without indications. the physiological variables were similar, but the consciousness was worse in the no-indications group. injury severity of brain and chest were higher in the no-indications group. the indications group had . % and the no-indications group had . % contraindications for reboa. the mortality was similar ( . % versus . %, or . , %ci . - . ). a sensitivity analysis showed the same result as the primary analysis (or . , %ci . - . ). introduction: most incident first responders have a primary nonmedical role, but are frequently the only professionals initially at the scene. early hemorrhage control via advanced techniques such as resuscitative endovascular balloon occlusion of the aorta (reboa) can save lives. training first responders these techniques has therefore the potential to improve outcomes. this study evaluates the ability to train quick response team fire fighters (qrt-ff) to gain percutaneous femoral artery access and place a reboa catheter, using a comprehensive theoretical and practical training program. material and methods: six qrt-ff participated in the training. sof medics from a previous training served as control group. a formalized training curriculum included basic anatomy and endovascular materials for percutaneous access and reboa catheter placement. key skills were: ( ) preparation of an endovascular toolkit, ( ) achieving vascular access in the model and ( ) placement and positioning of the reboa catheter. results: qrt-ff had significantly better baseline knowledge of surgical anatomy (p = . ) compared to medics. they also scored significantly better on using endovascular materials (p = . ), performing the procedure without unnecessary attempts (p = . ) and overall technical skills (p = . ). the median time from start to reboa inflation was : min for qrt-ff and : min for medics. procedure times improved in all qrt-ff and of the medics in a second attempt of gaining vascular access and reboa placement. conclusions: our comprehensive theoretical and practical training program proves suitable for percutaneous femoral access and reboa placement training of qrt-ff without prior ultrasound or endovascular experience. repetition reduces procedure times. training in the use of advanced hemorrhage control techniques such as reboa, as a secondary occupational task, has the potential to improve outcomes for severely bleeding casualties in out-of-hospital settings. prytime medical tm devices, inc. provided the reboa access task trainer (ratt) and the catheters used for this study. no other support was provided.the authors declare that there are no conflicts of interest that could inappropriately influence (bias) their work. introduction: angioembolization (ae) has become an important component in the management of bleeding from severe pelvic fractures. timely availablity of ae is required for both, level and trauma centers. the aim of this study was to assess the utilization of this procedure in level and trauma centers and effect on oucomes. material and methods: retrospective, -year ( - ) study using the the american college of surgeons tqip database, including adult patients with isolated severe pelvic facture (ais [ ] [ ] [ ] . patients who underwent laparotomy or preperitoneal packing within h from admission were excluded, operative management for bleeding control between and h was considered as failure. univariate analysis was used to compare patients in level vs centers, multivariate regression analysis was performed to determine factors predictive for mortality and overall complications.results: patients ( in level ; in level centers) met the criteria for inclusion. overall, ( . %) underwent ae, with a trend toward higher ae rate in level centers ( . % vs . %, p = . ). no significant differences were observed in timing and failure rate of ae between the levels. particulary in the ae subgroup there was a significantly lower blood product utilization in the first h in level i centers (prbc . vs . units, p = . ; plasma . vs . units, p = . ). mortality and overall complication rates were similar. table the level of trauma center was not a predictive factor for mortality (or . , p = . ) and overall complications (or . , p = . ). conclusions: in isolated severe pelvic fractures, there was a trend toward higher ae rate and significantly lower utilization of blood products in level centers. there were no significant differences in mortality or complications. the ae subgroup in level centers had a higher blood products use without outcome benefit, suggesting more restrictive transfusion policy may be considered. portal vein thrombosis after distal splenopancreatectomy: successful recanalization using fogarty balloon catheter case history: intraoperative lesion of smv during distal splenopancreatectomy is repaired using peritoneal patch harvested from anterior abdominal wall clinical findings: postoperative increase in serum lactate and d-dimer without signs of peritonitis prompts bedside doppler us showing no blood flood within portal vein (pv) investigation/results: ct angiography is performed suspecting acute mesenteric ischemia, but no abnormal bowel enhancement/ thickness is seen despite complete pv thrombosis. anticoagulation with unfractioned heparin is started, but clinical conditions deteriorate diagnosis: at reintervention, bowel is viable, so the surgeon performs fogarty balloon catheter thrombectomy successfully reestablishing blood flow within pv. no intestinal resection is required therapy and progressions: pv patency is regularly evaluated with us. anticoagulation with low molecular weight heparin is prosecuted for months and then suspended since no recurrence is recorded meanwhile comments: pv thrombosis is uncommon but can follow injury to portal venous axis during surgery. anticoagulation with heparin should be started as soon as the diagnosis is made and maintained for at least - months postoperatively to prevent recurrence. patients with persisting/worsening symptoms - h after initiation of anticoagulation, or those with peritonitis who are poor surgical candidates may be considered for interventional radiological treatment. otherwise, surgical intervention is required and may encompass resection of necrotic bowel. thrombectomy and/or balloon dilation/vascular stent placement may be helpful in recently developed pv thrombosis since risk of recurrence seems to be decreased references: acute mesenteric ischemia: guidelines of the world society of emergency surgery (world j emerg surg ); mesenteric venous thrombosis (j clin exp hepatol ); contemporary management of acute mesenteric ischemia in the endovascular era (vasc endovascular surg ) key: cord- -vgjz ts authors: nan title: th international congress of the european association for endoscopic surgery (eaes) sevilla, spain, – june date: - - journal: surg endosc doi: . /s - - -x sha: doc_id: cord_uid: vgjz ts nan laparoscopic cholecystectomy is one of the most commonly performed operations worldwide. bile duct injury (bdi) is a rare but very serious complication of the procedure, with a significant impact on quality of life and overall survival. the high frequency of bdi with laparoscopic cholecystectomy was first considered to be a consequence of the initial learning curve of the surgeon, but it later became clear that the primary cause of bdi is misinterpretation of biliary anatomy. intraoperative cholangiography (ioc) has been advised by many authors as the technique reduces the risk of bdi. however, the procedure has inherent limitations and is therefore reserved for select cases. fluorescent cholangiography using indocyanine green(icg) is a novel approach, which offers real-time intraoperative imaging of the biliary anatomy. a comparative study was contacted by administering icg intravenously or intrabiliary during the operation. forty patients scheduled to undergo an elective lap. cholecystectomy were randomly divided in two groups: in group a icg was administered in a dose . mg in ml solution intravenously hour before surgery. in group b icg was injected intrabiliary in a . mg/ml solution mixed with the patient's bile. also, we observed and analysed the following parameters, liver function, b.m.i, asa score and possible complications, before and after operation. results: group a. intravenous icg was administered in patients. there was no any reaction and the extrahepatic biliary anatomy was identified well. there was no bdi or any complication related to the procedure. group b. icg was injected intrabiliary in patients during the laparoscopic procedure. in all but one patient the extrahepatic biliary tree was delineated very well. in one patient part of icg solution was injected into the gallbladder wall and this resulted in a partially confusing image. there was no bdi and no postoperative complication conclusions: fluorescence cholangiography can be used during laparoscopic cholecystectomy to obtain fluorescence images of the bile ducts following intrabiliary injection during the operation orintravenous injection h before the procedure. the later technique is more easy to perform and does not require catheterization of the biliary tree. endoscopia digestiva chirurgica, policlinico universitario ''a. gemelli'', rome, italy; ihu, strasbourg, france; camma group, icube, university of strasbourg, cnrs, ihu strasbourg, strasbourg, france; ircad, strasbourg, france; digestive and endocrine surgery, nouvel hopital civil, university of strasbourg, strasbourg, france; digestive and endocrine surgery, ihu-strasbourg, strasbourg, france aim: surgical societies are united in promoting the critical view of safety(cvs) during laparoscopic cholecystectomy(lc). nonetheless, reports have shown a discrepancy between the operative reports and the correct application of cvs, which may explain the stability of bile duct injury rates. therefore, surgeons and computer scientists at our institution are developing a machine-learning algorithm to automatize cvs assessment. however, the lack of a consistent cvs video assessment framework limits the ability to generate data to train the artificial intelligence. here we describe and test a method for cvs evaluation in videos. method: between march and july , consecutive videos of lc performed at nouvel hospital civil(strasbourg, france) were recorded. two independent reviewers assessed the achievement of cvs in the s video sequences preceding clipping of cystic duct and artery. in addition to the 'doublet view' method, a 'binary' video evaluation method was tested: each of the criteria composing the cvs( structures entering the gallbladder, clearance of the hepatocystic triangle and lower part of the cystic plate) was classified as achieved or not. if the criteria were met, then the cvs was considered achieved. inter-rater agreement for cvs and for each of the criteria was evaluated. results: twenty-two videos( fundus first and partial lc, and broken videos) were excluded from the cvs analysis. cvs elements were assessable in all but one s videos sequences( . %). after mediation, cvs was achieved in / ( . %) of lc. the cystic plate was identified in only . % of videos. inter-rater agreement using the doublet view vs. the binary method was as follows: . %(? = . ) vs. . %(? = . ) for cvs achievement, . %(? = . ) vs. . %(? = . ) for the structures, . %(? = . ) vs. . %(? = . ) for the hepatocystic triangle and . %(? = . ) vs. . %(? = . ) for the cystic plate ( fig. ) . conclusions: reliable cvs assessment is crucial to generate consistent data for machine-learning algorithms aiming at decreasing bile duct injury after cholecystectomy. our binary cvs video assessment method showed higher inter-rater reliability than the doublet view, originally described for assessment of photos. further studies are on going to validate the cvs assessment in videos and support our initial results. surg endosc ( ) the vital role of surgeries in healthcare requires a constant attention for improvement. surgical process modeling is an innovative and rather recently introduced approach for tackling the issues in nowadays complex surgeries, involving complex logistics, much technology, and large teams. surgical process modeling allows for evaluating the introduction of new technologies and tools prior to the actual development and is beneficial in optimization of the treatment planning and treatment performance in operating room. in this study, we first discuss the concepts associated with surgical process modeling, aiming to clarify them and to promote their use in future studies. next, we apply these concepts to analyze the procedure of challenging interventions, minimally invasive liver treatment (milt) methods, with the ultimate goal of improving and optimizing the treatment procedure. the procedure model of current treatment activities and planning of various milt methods and the associated techniques, are analyzed and combined into a generic procedure model of milt, which provides a firm foundation for qualitative and quantitative analysis of different milt procedures. the generic procedure model is validated by data from erasmus medical center (rotterdam, the netherlands) and oslo university hospital (oslo, norway) . the proposed procedure model is designed to be a basis for improvement of the procedure and to determine how and where the new technologies can be best, effectively and efficiently, employed in the clinical practices prior to and/or during actual development of the new technologies for milt. as a conclusion, the current work illuminates the importance of surgical process modeling for improving different aspects of treatment procedures and provides an overview of various modeling strategies that can be used to establish surgical process models. the generic procedure model of various milt methods, including laparoscopic liver resection, laparoscopic liver ablation and percutaneous ablation, is introduced and validated which is a basis for introduction of the optimized procedure model of milt objective: to determine the most appropriate time to start total laparoscopic living donor right hepatectomy (tldrh) based on the experience with laparoscopic liver resection (llr). summary background data accumulation of experience in llr is essential before starting tldrh to ensure donor safety. methods: we retrospectively reviewed data of and consecutive patients who underwent llr and donor hepatectomy, respectively, between and . operative outcomes of laparoscopic major hepatectomy (lmh) were compared between two periods based on tldrh introduction (phase i - vs phase ii . learning curve of llr was evaluated using the cumulative sum (cusum) method to determine the optimal time of tldrh introduction. conclusion: accumulating an experience of at least lmh cases is needed in low-volume lt centers before starting tldrh to ensure donor safety. introduction: the number of surgical adverse events is still too high. an important number of these adverse events occur within the operating room (or) and are in fact preventable. in order to reduce adverse events in the or, we simply need to know what went well and what can be done better. the aim of this study was to analyze and debrief a predefined selection of surgical procedures, with the use of an operating room 'black box', to identify commonly observed safety threats and resilience support events. methods: in the period - , predefined gastro-intestinal laparoscopic cases were recorded by the or black box'. the postoperative surgical team assessment record (star) questionnaire was used. the recordings were analyzed by specifically trained raters, using the systems engineering initiative for patient safety (seips) model of work system and patient safety to identify relevant safety threat and resilience support events. qualitative data analysis was used to identify the most commonly discussed events during the team debriefings. results: in only . % (n = ) of times or team members, when asked direct following surgery, indicated that they had noticed aberrations (n = ) during the case. a mean number of . (sd . ) relevant positive and negative events (e.i. aberrations) per surgical procedure were identified using the black box performance report. on average, . (sd . ) of events identified by the black box were rated as safety threats. most events discussed during the team debriefings were related to communication. conclusion: these results once again highlighting the importance of clear and closed-loop communication in the operating room. theatre staff underestimated the number of aberrations occurring in the or, when asked to retrieve from memory. postoperative structured team debriefing may be important for resolving incorrect assumptions between operating team members to avoid future unnecessary miscommunication. background: the eaes has recently published an intraoperative adverse event classification to assist the direct measurement and routine reporting of minimal access surgery interventions. we aimed to explore the clinically validity and reliability of the classification. methods: a prospective evaluation utilising case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision surgery randomised controlled trial was performed (isrctn ). enacted adverse events identified with the observational clinical human reliability analysis technique were graded with the eaes classification by two blinded, independent assessors. test-retest reliability was explored using grades previously applied during the development of the classification with intraclass correlation co-efficients calculated. clinical validity was assessed using -day morbidity events, the clavien-dindo classification and the highest eaes grade per case. results: laparoscopic cases ( h of surgery) contained error events which were all successfully categorised. excellent inter-rater and test-retest reliability was seen (icc . , % ci . - . , p \ . and icc . , % ci . - . , p \ . respectively. % of patients experienced post-operative morbidity (median event, range [ ] [ ] [ ] [ ] [ ] [ ] . labelling analysed cases by their highest eaes classification grade gave % grade , % grade and % grade procedures. % of grade cases developed a morbidity event, but this significantly increased in grade and operations ( % and %, p = . ). the number of complications and highest recorded clavien-dindo grade increased with each additional grade ( . ± . vs. . ± . vs. . ± . , p = . and median vs. vs. , p = . respectively). anastomotic leak and re-operation were correctly captured by the allocated eaes grade ( . % vs. . % vs. %, p \ . and % vs. % vs. %, p \ . respectively). there was a significant rise in length of stay observed with increasing eaes grade (median vs. vs. days, p \ . ). conclusion: in the context of major laparoscopic surgery, the eaes intraoperative adverse classification is seen to be a clinically valid and reliable assessment method. psychological medicine, nuhs, singapore, singapore aims: neurobiological feedback in surgical training could translate to better educational outcomes such as measures of learning curve. the variation in brain activation of medical students when performing laparoscopic tasks before and after a training workshop is not properly studied before and we planned to do this using functional near infrared spectroscopy (fnirs) which is a non-invasive optical brain imaging tool that measures cortical oxygenation change which is used as a marker of pre-frontal cortex activity (pfca). methods: this randomised controlled trial examined the pfc activity differences in two groups of novice medical students during the acquisition of basic laparoscopic tasks. 'trained-group' had standerdised oneto-one training on the tasks, while the 'untrained-group' had no prior trainining and was just shown a video of the tasks. the pfca was measured pre and post intervention using a portable fnirs device. primary outcome was the difference in the pfca pre and post intervention. secondary outcomes were the differences in pfca between the tasks and between the sexes. results: trained and untrained medical students with an equal sex distribution and a comparable age distribution were invovlved in the study. all students were right handed. trained group had a significantly attenuated pfca in the 'precision-cutting' (p = . ) and 'suture-insertion' (p = . ) tasks compared to the untrained group. subgroup analysis based on sex revealed significant attenuation in pfca in trained females compared to untrained females across of the laparoscopic tasks: 'pegstransfer' (p = . ), 'precision-cutting' (p = . ), 'suture-insertion' (p = . ). no significant pfca attenuation was found in male students who underwent training compared to untrained males. conclusion: a standardised laparoscopic training workshop promoted greater pfca attenuation in female medical students compared to males. this suggests that female and male students respond differently to the same instructional approach. these results may have implications for surgical training and education such as a greater focus on one to one surgical training for female students and use of pfca attenuation as a form of neurobiological feedback as a measure of learning curve in surgical training. robot assisted versus laparoscopic advanced suturing learning curve e. leijte , i. de blaauw , c. rosman , s.m.b.i. botden surgery, radboudumc, nijmegen, the netherlands; pediatric surgery, radboudumc, nijmegen, the netherlands aims: compared to conventional laparoscopy, robot assisted surgery is expected to have most potential in difficult areas and demanding technical skills as minimally invasive suturing. this study was performed to identify the differences in the learning curves of laparoscopic versus robot assisted advanced suturing method: novice participants, with the knowledge of basic surgical procedures, were recruited and performed three suturing tasks on the eosim laparoscopic augmented reality simulator or the robotix robot assisted virtual reality simulator. each participant performed an intracorporeal suturing, tilted plane needle transfer and anastomosis needle transfer task. to complete the learning curve, all tasks were repeated for maximal twenty repetitions or until a plateau was reached three consecutive times. clinical relevant and comparable parameters regarding time (seconds), movements and safety were recorded. intracorporeal suturing was used to visualize and compare the learning curves between the groups. results: forty-six participants completed the learning curve, of which laparoscopically and robot assisted. when comparing the suture time, the plateau was reached much faster in the robot assisted group ( - repetitions) than the laparoscopic group ( ) ( ) ( ) repetitions) as shown in figure . there was a significant difference in 'time per suture', during the whole learning curve with median values of versus (first knot), versus (fifth) and and (eighteenth), all with a p \ . . however, the parameter 'adequate surgical knot' was reached earlier in the laparoscopic group than in the robot assisted group. first: % versus %, fifth: % versus %, and eighteenth: % versus %. when assessing the 'needle out of view' parameter, the robot assisted group scored a median of . and . s during the first, respectively eighteenth knot, and the laparoscopic participants had their instruments out of view for and s during the first respectively eighteenth knot. conclusion: the learning curve of minimally invasive suturing can be reduced with the use of robot assisted surgery, with a specific reduction in operation time. the rate of adequate knots seemed to remain lower in robot assisted surgery, although this could be due to the virtual reality aspect of the simulator. introduction: endoscopic sleeve gastroplasty (esg) is a novel promising bariatric endoscopy treatment. gastric volume reduction and delayed gastric emptying are the mechanisms driving weight loss. however, little is known about the factors influencing the effectiveness of weight loss overtime. the present study aims at evaluating the correlation between endoscopic suture appearance and excess weight loss (ewl%) at and months follow up. patients and methods: all patients who underwent follow-up endoscopy at and months after esg were included. esgs were classified in groups according to endoscopic appearance of the gastric sutures: optimal (group ) when all stitches were in place and tights; suboptimal (group ) when one or more stiches were displaced; loose (group ) when all the sutures were completely disrupted. bmi at enrollment and ewl% at and months were recorded and compared to the endoscopic appearance. results: a total of patients were included in the analysis. at months, ( . %) patients had an optimal esg, ( . %) had a suboptimal sleeve and ( . %) had complete sutures failure. bmi at enrollment and ewl% were respectively . ± . and . ± . % for group , . ± . and . ± . % for group and . ± . and . % ± . % for group . twenty five patients had months egds: ( %) presented an intact esg and were classified in group , ( %) in group and ( %) in group . twelve months ewl% was respectively . ± . %, . ± . and ± . %. initial bmi significantly correlated with suture status at both (rho - . ; p \ . ) and months (rho - . ; p = . ) follow-up. furthermore, the sutures' appearance itself correlated with ewl% at both time points (rho ? . ; p = . and rho . ; p = . respectively). conclusion: our preliminary results show that the aspect of the endoscopic suture has a significantly impact on ewl% at and months after esg. furthermore, bmi at enrollment seems to predict endoscopic suture duration overtime. larger studies and longer follow-up are needed to further validate our preliminary findings. background and aim: endoscopic sleeve gastroplasty(esg) is a relatively novel endoscopic procedure that reduces the gastric lumen with proven less complications and less months weight loss compared to laparoscopic sleeve gastroplasty (lsg) . at present there are no studies investigating the role of multidisciplinary approach in esg. the aims of the present study were to evaluate the role of multidisciplinary assessment(ma) prior esg, weight loss outcomes, quality of live improvements and adverse events. material and methods: from may to may all patients that underwent esg were retrospectively evaluated from a prospective database. until september before esg only psychiatric evaluation was requested, while after this date we adopted the guidelines of the italian society for obesity surgery and all patients were evaluated on a multidisciplinary fashion prior esg. the multidisciplinary team was composed by:gastroenterologist, surgeon, psychiatrist, endocrinologist and dietitian. patients were divided in two groups:group were patients with esg before ma and group were patients with esg after ma. we compared this two groups in terms of weight loss outcomes, quality of live improvements and adverse events. quality of live was measured with the bariatric analysis and reporting outcome system(baros).all procedures were done with the apollo overstitch suturing system(apollo endosurgery) and a double channel gastroscope olympus tgif- (olympus japan).all procedures were done in general anesthesia and with insufflation of co . all patients had ambulatory visit t , and months after esg and weight loss outcomes were measured in terms of excess weight loss (%ewl),the total body weight loss (%tbwl) and baros scale were assessed. statistical analysis was done with chi-square test and \ . value was considered significant. results: patients were identified ( female; mean age . , range - ) . mean bmi at inclusion was . (range . - . ). mean %ewl and %tbwl at months was . and . respectively (table ) .non procedure related complications were observed. comparing the two groups there was significant(p \ . ) difference in terms of %ewl and %tbwl (table ) ,with better results in group . there was also a significant improvement in the baros scale in the patients in group . conclusions: ma before esg has a fundamental role in terms of better procedure outcomes for both weight loss and quality of live in obese patients. gastroenterology, hadassah medical center, jerusalem, israel, israel aims: the over-the-scope clip (ovesco) is a novel endoscopic tool that enables non-surgical management of gastrointestinal defects. the aim of this study was to report our experience with ovesco for patients with staple line leaks following laparoscopic sleeve gastrectomy (lsg). methods: a prospectively maintained irb-approved institutional database was queried for all patients treated with ovesco for staple line leaks following lsg from to . primary outcome was complete resolution of leak following ovesco as defined by return to complete oral nutrition and no evidence of leak on imaging. secondary outcome was the number of additional endoscopic or surgical procedures needed following ovesco. results: twenty-five patients ( males, females) were treated with ovesco for staple line leaks following lsg. the median age was years (range - ), and mean body mass index was kg/m . nine patients ( %) were referred from an outside hospital. the median time from index operation to leak diagnosis and from leak diagnosis to ovesco was days (range - ), and days (range - ), respectively. all patients had upper staple-line leaks near the gastroesophageal junction. initial treatment included antibiotics- patients; computed tomography guided drainage and antibiotics- patients; and laparoscopic drainage- patients. ovesco led to final resolution of leak in patients ( %) within days of clip deployment (range - ). leaks which persisted following ovesco were eventually resolved with a combination of ovesco and stent- patients ( %), total gastrectomy and esophago-jejunostomy- patients ( %), and endoscopic suturing- patient ( %). one mortality was noted in a patient who suffered multiorgan failure. the number of additional endoscopic sessions ranged from to (median ). no procedure related complications were noted. all patients were treated with total parenteral nutrition and the total length of stay was days (range - ). conclusions: despite its low success rate, ovesco should be part of the bariatric surgeon's non-surgical armamentarium in treating staple line leaks following lsg. r. bademci , r. vilallonga , p. alberti , r. renato , c. yuhamy , s.s. cordero , l. posadas general surgery, istanbul medipol üniversitesi, istanbul, turkey; bariatric surgery, vall d'hebron, barcelona, spain background: in cases of morbid obesity, treatment is generally applied as either a surgical or endoscopic approach. the number of primary obesity surgery endolumenal (pose) procedures is increasing but the reliability and effectiveness is unclear as yet. the aim of this study was to present a series of cases that required revision surgery due to pose failure and to reveal possible alternative surgeries. materials and methods: a retrospective comparison was made of the data of obese patients with pose failure and conversion to surgical procedures between and in respect of operation, medical illness and bmi results. results: the patients comprised % females, % males with a mean age of . ± . years and mean follow-up period of . ± . months. on average, patients lost . ± . kg, with an average excess weight loss of . %. conclusion: no firm conclusions can be drawn from such a small group. although sg seems to be a safe procedure and should be considered as the first technique to be applied following pose failure, it is possible to perform gastric bypass on patients with this endoscopic precursor. introduction: the population of post bariatric surgery patients is rapidly increasing worldwide. due to the altered anatomy post roux-en-y gastric bypass (rygb), conventional endoscopic management for choledocholithiasis is challenging. these patients are now commonly managed by means of a laparoscopic assisted ercp. although effective, this requires significant resource utilization and potential morbidity related to the need for surgical intervention. we present our preliminary experience with a purely percutaneous management of choledocholithiasis in bariatric patients post-rygb. methods: a retrospective single center review identified five patients with choledocholithiasis after bariatric rygb who underwent percutaneous cbd access and treatment by interventional radiology. four patients underwent percutaneous transhepatic cbd access while one patient underwent percutaneous trans-cholecystic cbd access. in three of the five patients conscious sedation alone was sufficient to perform the procedure. results: all patients had radiologically confirmed choledocholithiasis and were clinically symptomatic prior to intervention. the biliary tree was successfully accessed percutaneously and cleared in all five patients. in the four patients where a percutaneous transhepatic access was utilized, three patients required only fluoroscopic balloon sphincterplasty and sweep of the cbd to clear the ductal stones, while the fourth required percutaneous cholangioscopy assisted lithotripsy for clearance. in the fifth patient with non-dilated intrahepatic bile ducts a trans-cholecystistic approach into the cbd was utilized with percutaneous cholangioscopic assistance to clear the ductal stones. all procedures were completed successfully with no post procedure complications. conclusion: percutaneous clearance of cbd stones in bariatric patients presents a minimally invasive alternative to current surgical practice. the use of conscious sedation and the purely percutaneous approach may potentially reduce morbidity and resource utilization for this increasingly common clinical scenario. laparoscopic narbona-arnau procedure to control the gerd after lsg- years results of a prospective study i.c. hutopila, c. copaescu background: after the laparoscopic sleeve gastrectomy (lsg) alone or associated with calibration of the esophageal hiatus, for some patients the reflux symptoms worsen postoperatively due to development of a hiatal hernia (hh) or due to the recurrence of the hh previously repaired. for these situations, when the conservative treatment fails, are proposed some surgical solutions, one of them cardiopexy with teres ligament-narbona arnau. objective: is to establish a standardized laparoscopic technique for cardiopexy using the teres ligament (narbona arnau technique) and to analyze the procedure's outcomes. methods: the study was performed in a bariatric and metabolic center of excellence-ponderas academic hospital. all the patients undergoing narbona arnau procedure to control gerd after lsg since were included and prospectively analyzed. the selection criteria included lsg patients, presenting hh and symptomatic gerd. preoperative investigations were upper gastrointestinal endoscopy, radiological contrast study, ph-metry, computed tomography with oral contrast. results: patients were included into the study. gerd and hh were preoperatively documented in all the cases. one patient was excluded after years of follow up after being converted to a laparoscopic roux-en-y gastric bypass, for intense relux symptoms. no incidents during surgery. for cases laparoscopic narbona arnau technique was performed concurrent with re-sleeve gastrectomy and gastric curvature plication. without postoperative complications. postoperative follow-up at months, , and years, the percentage of patients without gerd symptoms and free of treatment with ppis was , %, , %, , %, respectively . %. at years postoperatively the upper gi endoscopy showed remission/ improvement of the degree of esophagitis for patients. for the same period of follow-up, the ph-metry highlighted a normal value of demeester score for . % o patients (all the patients had preoperatively high de meester scores). no objective signs of hiatal hernia recurrence at imagistic investigations and upper gastrointestinal endoscopy were encountered. conclusions: complete preoperative evaluation is mandatory for choosing the optimal intervention. laparoscopic narbona arnau technique after lsg is proved to be a good option for the treatment of symptomic gerd, but further studies with high-volume patients are necessary. introduction: the aim of this study was to investigate the influence of baseline glycated hemoglobin level (hba c) level in bariatric patients on postoperative outcomes. we found scarce of clinical data regarding influence of baseline hba c on bariatric surgeries postoperative morbidity and readmission what was inspiration to conduct this multicenter retrospective study. methods and procedures: retrospective cohort study analyzed patients who underwent laparoscopic: sleeve gastrectomy (sg), roux-en-y gastric bypass (rygb) or mini-gastric bypass (mgb) for morbid obesity in seven referral bariatric centers. patients were divided into groups depending on preoperative hba c : hba c \ . %; . - . % and c . %. primary endpoints: influence of hba c level on perioperative ( -days) and postoperative ( -months) morbidity rates, operation time, length of hospital stay (los) and readmission rate. results: study group included , % females and % males. median age was ( - ) years. median hba c was . ( . - . ). hba c \ . % was present in % patients, hba c . - . % in %, and hba c c . % in %. percentage of male patients increased in groups from % in hba c \ . % to % in hba c c . % significantly. same tendency through groups we observed in case of bmi and age. uncontrolled diabetes (hba c c . %) was present in . % patients, while . % patients were not on antidiabetic medications despite having hba c c . %. median operative time in patients was significantly longer than in hba c \ . % and hba c . - . %. -days morbidity rate was . % and did not differ groups significantly, as -months morbidity rate (excl. -days) of . % . los did not differ groups significantly. patients having hba c in range of . - . % and with hba c c . % did not have significantly increased odds for perioperative morbidity, -months postoperative morbidity as compared with those with hba c \ . %. patients with hba c c . % had increased or for prolonged los as compared to those with hba c \ . % (or . ; % ci . - . ). hba c did not influence or for readmissions. patients with baseline hba c c % had significantly increased chances for hospital readmission (or . , % ci . - . ). conclusion: baseline level of hba c did not influence chance for perioperative morbidity, -months postoperative morbidity and prolonged los. patients with hba c c % have increased chance for hospital readmissions. surg endosc ( ) :s -s introduction: surgical resection is crucial for curative treatment of rectal cancer. through improvements in treatment and minimally invasive techniques, -year survival improved to over % of patients. the most recently introduced surgical technique is robotic-assisted surgery (ras). ras and conventional laparoscopy (cl) seem equally effective in terms oncological control. however, ras possibly provides further advantages e.g. d vision or the endowrist function, which have the potential to maximize the precision of surgery and thus has benefits for functional outcomes such as sexual function as well continence. therefore, the aim of this systematic review and meta-analysis was to compare functional outcomes of cl and ras for rectal cancer. materials and methods: this review was done according to the prisma and amstar guidelines andregistered with prosper-o(crd ). the search was planned with the pico criteria and conducted on medline (via pubmed), web of science and central. two independent reviewers first screened titles and abstracts and then eligible full-texts. inclusion criteria were original studies, comparative studies for cl vs. ras for rectal cancer as well as reporting of functional outcomes. quality assessment was done with the newcastle-ottawa-scale for non-randomized studies and the cochrane tool to assess risk of bias for randomized trials. results: the search retrieved hits, of which studies with patients met inclusion criteria. preliminary results yielded a lower rate of urinary retention for ras (odds ratio (or)[ %-confidence interval (ci)] . [ . , . ] ) while there were no differences for ileus (or[ci]: . [ . , . ] ). erectile function (iief) showed no differences after (mean difference (md)[ci] . [- . , . ] , (md[ci] . [- . , . ] ) and months (md[ci] . [- . , . ] ). in terms of urinary problems (ipss) there were no differences postoperative (md[ci] - . [- . , . ]) and month postoperative md[ci] - . [- . , . ] ), but advantages for the cl group after months md[ci] - . [- . , - . ] ). discussion: ras and cl seem to provide similar functional outcomes after rectal cancer surgery. however, the results need to be interpreted carefully as none of the studies had any functional outcome defined as primary endpoint. future studies should evaluate both surgical approaches in terms of functional outcomes and should be appropriately powered. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm .intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ) all patients required a reoperation. intraoperative findings at shows in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to non-infectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. conclusion: our results shows that the use of knotless barbed suture for enterotomy closure after laparoscopic intracorporeal right colectomy is safe, reproducible and associated with shorter operative time. aims: the accurate measurement and staging of rectal cancer, in particular the distal margin of low rectal tumours, is of paramount importance to optimise oncological surgical resection whilst preserving function. it is well recognised that the lower the tumour, the greater the technical challenges, operative time and the possibility of a temporary or permanent stoma. accurate localisation of the tumour is also essential to assist the multi-disciplinary team when considering neo-adjuvant chemoradiotherapy (crtx). the objective was to compare tumour height as reported on magnetic resonance imaging (mri) with endoscopic measurement. methods: a retrospective analysis of rectal tumour heights on pre-operative endoscopy and mri in patients undergoing radical colorectal surgery with curative intent over years from january . rectal tumours were identified as within cm of the anal verge (av). all mri measurements were reported by one of two specialist gastrointestinal radiologists. measurements were taken from the lowermost point of the tumour to the av. endoscopic measurements were as recorded by endoscopists including rectal surgeons, general surgeons, gastroenterologists and a clinical nurse specialist endoscopist. results: records of eighty one patients with histologically confirmed rectal adenocarcinoma were reviewed. median age was years ( to ). twenty three patients had or more endoscopies. on mri the median tumour height from the av was . cm ( . - cm) . on endoscopy the median tumour height was cm ( - cm) . on comparing endoscopy with mri, the median difference was cm ( - cm) . for over a third of patients ( %) tumours were lower on mri than endoscopy, median difference . cm ( . - cm) . only rectal surgeons documented tumour height in relation to the rectal folds. the majority of the repeat endoscopies were performed by surgeons to locate tumours more accurately pre-surgery. on no occasion was it documented whether the tumour had been measured during insertion or withdrawal of the endoscope. conclusions: precise localisation of rectal tumours is imperative to plan complex surgery and give informed counsel to patients. this study demonstrates the urgent need for a standardised protocol for all endoscopists to use while recording the distal extent of rectal tumours. objectives: the aim of the present rct was to compare the incidence of genitourinary (gu) dysfunction after elective laparoscopic low anterior rectal resection and total mesorectal excision (lar ? tme) with high or low ligation (ll) of the inferior mesenteric artery (ima). secondary aims included the incidence of anastomotic leakage and oncological outcomes. background: the criterion standard surgical approach for rectal cancer is lar ? tme. the level of artery ligation remains an issue related to functional outcome, anastomotic leak rate, and oncological adequacy. retrospective studies failed to provide strong evidence in favor of one particular vascular approach and the specific impact on gu function is poorly understood. methods: between june and december , patients who underwent elective laparoscopic lar ? tme in italian nonacademic hospitals were randomized to high ligation (hl) or ll of ima after meeting the inclusion criteria. gu function was evaluated using a standardized survey and uroflowmetric examination. the trial was registered under the clinicaltrials.gov identifier nct . results: a total of patients were randomized to hl (n ) or ll (n ). gu function was impaired in both groups after surgery. ll group reported better continence and less obstructive urinary symptoms and improved quality of life at months postoperative. sexual function was better in the ll group compared to hl group at months. urinated volume, maximum urinary flow, and flow time were significantly (p \ . ) in favour of the ll group at and months from surgery. ultrasound measured post void residual volume and average urinary flow were significantly (p \ . ) better in the ll group at months postoperatively. time of flow worsened in both groups at months compared to baseline. there was no difference in anastomotic leak rate ( . % hl vs . % ll). there were no differences in terms of blood loss, surgica l times, postoperative complications, and initial oncological outcomes between groups. conclusions: ll of the ima in lar ? tme results in better gu function preservation without affecting initial oncological outcomes. hl does not seem to increase the anastomotic leak rate. introduction: robotic single-site cholecystectomy (rssc) has been known to have some advantages such as reducing stress of the surgeon compared to single incision laparoscopic cholecystectomy (silc). however, there are few studies comparing the perioperative outcomes of these two operative methods. patient and methods: between march and february , rssc and silc were performed for benign gallbladder disease in our center. propensity score matching was performed to control variables including sex, age, body mass indes (bmi), diagnosis, american society of anesthesiologist (asa) score and cohorts were selected among the silc group through : matching. the perioperative data of these patients were analyzed retrospectively. the diagnosis was classified into acute cholecystitis, chronic cholecystitis, and gallbladder polyp. results: patient demographics between the two groups were evenly matched. total operation time including docking time was slightly longer in rssc group ( . min vs. . min, p \ . ), but real working time except the docking or set-up was shorter in rssc group ( . min vs. . min, p \ . ). conversion to additional robotic arm or additional port was frequent in silc group ( vs. cases, p = . ). intraoperative bile spillage rate ( . % vs. . %, p = . ) and postoperative hospital stay ( . days vs. . days, p = . ) were comparable in both group. conclusion: both surgical procedures performed safely. but the rssc demonstrated the better performance of the operation with shorter working time and the advantage of overcoming unexpected difficulties during the surgery with low conversion rate compared to silc. even though laparoscopic cholecystectomy(lc) is the gold standard procedure for cholelithiasis, patients are still suffering from various causes of pain. one of main causes is high pressure by pneumoperitoneum which makes peritoneal stretching and diaphragmatic irritation. however, there are few well-designed studies for evaluating pneumoperitoneum. therefore, we conducted a study to compare the postoperative pain after lc at serial different pressure methods. a prospective randomised double blind study was done in patients with benign gallbladder disease. they were divided into groups. each patients underwent lc with different pneumoperitoneum method; group a: far-low ( - mmhg), goup b: low ( - mmhg) and group c: standard pressure ( - mmhg). three groups were compared for pain intensity, duration, analgesic requirement and complications. post-operative pain score was significantly least in far-low pressure group as compared to low or standard pressure group during late periods ( , h). but, there were no pain score difference between far-low and low groups during early period ( , , , h) even though scores of standard group were significant higher than those of low group. number of patients requiring rescue analgesic doses and intraoperative complications were not significantly different among groups. this study demonstrates reducing the pressure of pneumoperitoneum results in reduction in intensity of post-operative pain. this study also shows that low pressure technique is safe with comparable rate of intraoperative complications. however, in immediate postoperative period, there is limitaton of pain relief after low pressure surgery. therefore, there may need new alternatives for pain. background and aim: anatomical hepatectomy with the glissonian approach is widely accepted as an important technique to ensure surgical safety and curability of the carcinoma. however, the histomorphological structure of the hepatic connective tissue is not sufficiently understood by surgeons. this study aimed to clarify the hepatic connective tissue structure using modern tissue imaging and analytical techniques. materials and methods: in total stained thin slices were loaded onto the computer and were reconstructed as dimages and analyzed. results: when the liver capsule enters the liver at the hepatic hilum, it becomes a sheath which envelops the portal pedicle. the hepatocytes in a row that constitute the periportal limiting plate at the edge of the hepatic lobule are firmly supported by the framework of the reticular fiber. the hepatic lobule and the portal area are in contact via the periportal space of mall. the framework of the limiting plate plays a role of a capsule of hepatic lobule (proper hepatic capsule) on the side in contact with the portal area. the binding site between the hepatic capsule and proper hepatic capsule (ppbs) is loose binding and is a layer that is easy to apply to surgical procedures. in order to enter between the liver capsule which became the sheath of the portal pedicle and the proper hepatic capsule at the hepatic hilum, the liver capsule must be dissected to reach the surface of the proper hepatic capsule. then, on the one hand, the portal pedicle is firmly gripped and pulled, on the other hand, the hepatic parenchyma covered by the proper hepatic capsule is pushed to expand between the portal pedicle and the liver parenchyma. at this time, the portal area (glisson's sheath) branched from the sheath of the portal pedicle into the gap of the hepatic lobule breaks like a string. with this dissecting plane, dissecting layer can reach to the next branch of the portal pedicle without entering into the portal pedicle or liver parenchyma. conclusion: understanding the connective tissue constituting the liver and conducting surgery turns the laparoscopic systematic hepatectomy into a standardized procedure. background: postoperative pancreatic fistula (popf) is the primary contributor to morbidity after distal pancreatectomy (dp). to date, no techniques used for the transection and closure of the pancreatic stump showed a clear superiority over the others. this study aimed to compare the rate of popf after pancreatic transection conducted with the reinforced stapler (rs) and ultrasonic dissector (ud) following dp. method: consecutive patients underwent dp from to were retrospectively reviewed. we included dps where pancreatic transection was performed by rs or ud and excluded dps extended to the pancreas head. to overcome the absence of randomization, we conducted a propensity matching analysis according to risk factors for popf. results: overall, patients met the inclusion criteria. the rs was employed in patients and ud in cases. after the one-to-one propensity matching, patients were selected from each group. the matched rs and ud cohort have no differences in baselines characteristics except for the mini-invasive approach, that was more common in the ud group ( % vs. %, p = . ). overall, patients ( %) developed a popf, a grade b ( %) and ( %) a grade c. in the rs group the rate of popf was % (n = ) and the ud group % (n = ) with a p \ . . conclusion: the results of this study suggest that the use of rs for pancreatic transection, reduces the risk of postoperative pancreatic fistula. a randomized trial is needed to confirm these preliminary data. aim: this study compares clinical and cost outcomes of robot-assisted single port and open longitudinal pancreaticojejunostomy (rlpj and olpj) for chronic pancreatitis. single incision mis needs more manual skills than conventional multiport operation. the advantage of better operation course is d vision and dedicate instrument. this paper aims to evaluate the feasibility and safety of the robot-assisted single incision with single port platform for chronic pancreatitis. materials and methods: clinical and cost data were retrospectively compared between open and ralpj. we collected patients since july, to september, . the patient was supinely placed in reverse trendelenburg position. the assistant surgeon was located between patient's legs. under general anesthesia a trans-umbilical . cm skin incision was made. a single incision advanced access platform with lagis port, glove portÒ (nelis, s. korea) and gelpoint combined with the da vinci si and xi surgical system (intuitive surgical, sunnyvale, ca, usa) pure or plus one was performed. the three arms, no. , no. , and da vinci scope, were in dwelled through the glove portÒ. pneumoperitoneum of mmhg was established through the port. a rigid -degree up scope was used during operation. results: twenty-one patients underwent lpj: open and ralpj. no robot-assisted cases converted to open were noted. patients undergoing ralpj had less intraoperative blood loss, a shorter surgical length of stay, less postoperative pain and lower medication costs. operation supply cost was higher in the ralpj group. no obvious difference in hospitalization cost was found. conclusions: versus the open approach, ralpj performed for chronic pancreatitis shortens hospitalization, less postoperative pain and reduces medication costs; hospitalization costs are equivalent. a higher operative cost for ralpj is mitigated by a shorter hospitalization and less pain control. robotassisted puestow procedure using single port platform is feasible and safe method. the total procedures by da vinci robotic system are safe and easily performed in highly selected patients. general surgery, hospital universitario infanta sofia, madrid, spain; general surgery, hospìtal quirón la luz, madrid, spain aims: the concomitant presence of abdominal wall midline hernias and diastasis recti is frequent. diastasis recti might be a risk factor not only for having but for recurrence of midline hernias. most open surgical procedures not consider the treatment of both pathologies, nor laparoscopic most spread out approaches. the author presents a novel endoscopic, extraperitoneal and retromuscular hernioplasty technique and its preliminary results. methods: a serie of patients is presented. a ct abdominal wall study is performed preoperatively. they all presented abdominal wall midline hernias in presence of a [ . cm concomitant diastasis recti. there were females and males. a totally endoscopic, extraperitoneal and retromuscular repair was performed, that included a midline anatomic restoration, tension-free hernia gap closure, omphaloplasty and skin treatment, if needed in every case. the tension-free massive-meshed hernioplasty included a bilateral totally endoscopic posterior components separation when needed. no drainages were used. all procedures included a bladder catheterization. results: all patient were dispatched within a period under h. no reoperations were needed in postoperative period. postoperative pain was measured by an eva scale. % of the patients have no pain medication after - h dispatching from hospital. % of the patients have a skin suffusion or hematoma. a male patient presented a temporary abdominal asymmetry due to a unilateral posterior component added to his technique. the mean following-up is to months ( - months) . no recurrence was observed. conclusions: preliminary results demonstrate this new approach to be a safe, feasible and a reproductible procedure. the 'terra' novel technique could provide of a new minimally invasive approach to abdominal wall midline hernias repair in the presence of a diastasis recti. only time and new results can predict the spreading out of this 'third way'. results: this study comprised males and females. mean age was years (range - years) and mean body mass index was . gh and mh were found intraoperatively in and cases respectively. mean operative time for all hernias (gh/mh) was min (range - min); min for gh (range - min); and min for mh (range - min). in . % of cases, hernia operative measurement was larger than preoperative size, especially in cases of incisional hernias ( . %). in . % of cases, laparoscopy found additional abdominal wall defects previously undetected by physical examination and by us-and/or ct-scan. a composite mesh and a non-composite mesh (up to cm in size) were used in . % and . % of cases respectively. the ethicon securestrap?? absorbable fixation device straps for sm fixation were employed in . % of cases. mean length of hospital stay was . days. mean follow-up time was months (range - months). in our study, there was one early (\ days) postoperative seroma ( . %), plus one late, small ( cm) symptomless recurrence, but neither needed reoperation. conclusion: the sutureless sm technique facilitates intra-abdominal introduction, as well as the handling and fixation of large/very large meshes. this new approach is safe and fast, even in cases of gh/mh repair. aims: any ventral hernia (vh) combined with rectus muscle separation (rms) must be repaired along with repairing the rms, otherwise there is a high risk for hernia recurrence. open rms repair is vast and traumatic surgery and laparoscopy is not effective. at a new era of repairing abdominal wall hernia by assisted endoscopy started with wolfgang reinhold's milos procedure. these procedures are somewhat complexed and real reconstruction of the linea alba (la) was limited, which done better by ferdinand koeckerling's elar technique. we perfected the elar technique to be fully endoscopic with wide mesh fusing to the muscles immediately by fibrin glue: extended endoscopic hernia & linea alba reconstruction glue (eehlarglue), achieving a low traumatic mis for vh and rms with excellent surgical and cosmetic results. methods: our eehlarglue is a totally endoscopic based technique used since . penetrating with optiview trocar and co pressure to the anterior rectus sheet (ars) level is followed by an extensive endoscopic dissection of the sub-cutaneous fat tissue from the ars. three trocars are inserted at the supra-pubic line enabling the dissection up to the xiphoid and costal margins laterally. any hernia sac is dissected, and the content reduced back to the abdominal cavity. relaxing incisions of the ars are performed longitudinally in the lateral aspect. the la is reconstructed by running two layers of non-absorbable sutures from xiphoid to pubis. a light mesh x cm is applied over the repair and the mesh is fused immediately to the muscles by fibrin glue. results: patients underwent the eehlarglue with follow up of months. all had significant rms of - - cm combined with primary or recurrent vh. recovery was smooth with - days of simple analgesics and return to regular activity within - days. no one had recurrent vh, but two males had limited rms and two early cases seroma formation. conclusions: our eehlarglue enables endoscopic vh repair and la reconstruction with extrastrength received by immediate mesh fusion to muscles with fibrin glue. thus, achieving low traumatic mis, easy recovering and very effective results-a perfect solution for patients with vh combined with rms. results: twelve blinded prospective rcts were used. when compared to tep repair, tapp repair has comparable seroma formation rates (chi = . ; (p = . ); ci - . , . ; i = %) and post-op pain at h (chi = . ; (p = . ); ci - . , . ; i = %). however, tep repair is associated with a significantly shorter operative time (chi = . ; (p = . );ci . , . ; i = %), post-op pain at hour (chi = . ; (p = . ); . , . ; i = %) and shorter hospital stay (chi = . ; (p = . ); ci . , . ; i = %). conclusion: tep is significantly better than tapp repair with regards to operative time, post-op pain at h and hospital stay. there is no significant difference with regards to post-op pain at h and seroma formation. background: primary hyperhidrosis (ph) is a neurological condition characterized by excessive sweating most often of the face, palms or axillae . palmar hyperhidrosis is treated through sympathetic chain clipping or transection .we aiming to compare the efficacy and results obtained with both techniques. patients and methods: sixty four patients underwent of sympathetic procedures from march to february . the patients were categorized into two groups: right sided transection sympathectomy and left sided clipping . patients were evaluated to compare the rates of success, satisfaction, compensatory sweating and recurrence either with transection or clipping of the t andt ganglion .mean follow up was ? _ months. results: sixty four patients males and females undergoing electro-coagulation sympathectomy on the right side and clipping on the left side. with mean age was years (range to years). all patients had balanced demographic data . no statistical difference between the two groups according rate of success. compensatory sweating was observed in patients ( . %) overall with cases of severe unsatisfied compensatory sweating. recurrence was reported in one case with transection and cases in clipping. satisfaction was occurred in cases in transection group and cases in clipping group .pnumothorax was occurred in cases in transection group compared to one case in clipping. no gustatory sweating and over dryness were reported in both groups. conclusion: both thoracoscopic sympathetic transection and clipping of t t ganglion are safe and effective procedure in palmar hyperhidosis treatment. with no differences regarding recurrence rate,satisfaction and incidence compensatory sweating. keywords: thoracoscopic sympathectomy,palmar hyperhidrosis, clipping, compensatory hyperhidrosis. introduction: primary ventral hernias and ventral incisional hernias pose a challenge for surgeons throughout the ages. even though minimally invasive surgery and hernia repair have evolved rapidly, there is no standardized method that adequately decreases postoperative complications. hybrid hernia repair is a surgical repair, which has not been adopted widely. it combines both a laparoscopic and open component allowing sac excision, primary defect repair as well as laparoscopic mesh insertion. aims: to evaluate the short-term and long-term outcomes of hernia recurrence for patients undergoing hybrid ventral repair (hvr) for the treatment of primary and incisional ventral hernias. methods: between october- and june- , hybrid vhr was performed in -patients at st mary's hospital, imperial college london. the medical records of these patients were reviewed retrospectively for demographics, comorbidities, prior surgeries, body mass index (bmi), hernial defects, hybrid technique used; mesh selection, operative time, complications and recurrences over a -year follow-up. results: twenty-four patients who underwent hybrid vhr were included with surgery performed by two surgeons. the mean age is -years with a mean bmi of . kg/m . % had incisional hernias and % had primary hernias. the number of hernia defects ranged from to , with the average mesh size used was x cm. extensive adhesionolysis was performed in % of patients. -day postoperative complications; patients developed post-operative seroma, paralytic ileus in , pain control in and urinary retention in patient. there were no conversions to open procedures. the mean length of hospital stay was -days. none of the patients developed chronic pain and only one recurrence over the -year follow-up period. conclusions: the hybrid technique for vhr is safe and feasible, and has important benefits over an open or purely laparoscopic approach, including a low rate of seroma formation, chronic pain and fiveyear hernia recurrence. future investigation may include randomized controlled trials, to fully evaluate the benefits of hybrid vhr, with careful assessment of patient-centred end-points including quality of life and postoperative pain. surgery, medical faculty-university of tetove, tetove, macedonia; general medicine, medical faculty-university of tetove, tetove, macedonia; anestesiology, medical faculty-university of tetove, tetove, macedonia; surgery, clinical hospital-tetove, tetove, macedonia laparoscopic cholecystectomy is widely used operative technique and it's characterized with less postoperative hospitalization and side effects. duration of the hospitalization after laparoscopic surgery depends on several factors of which pain and physical weakness are the most important. dexamethasone is well known; not only for its anti inflammatory effects but at the same time for analgesic and antiemetic effects, although the mechanism of this effects are not clarified yet. objectives: the aim of our study is the evaluation of analgesic effect of dexamethasone on reducing postoperative pain after laparoscopic surgery. patients and methods: in this study, patients aged - years old undergoing laparoscopic surgery, were classified into two groups, patients in each group. the first group were treated with a intravenous injection of mg dexamethasone preoperatively and another dose the next day after operation. the second group received a intravenous injection of normal saline. we evaluated the dose of consumed analgesics and antiemetic's drug during the first h in both groups. results: according to our experience results the total dose of tramadol in a postoperative period in dexamethasone receiving group was smaller than in normal saline group. measure of postoperative pain was assessed using the paper-based vas scale. our result shows that the intensity of post operative pain in a period during first h, after surgery in a group of patients treated with dexamethasone was lower compared with the group of patients treated with normal saline. nausea and vomiting during the first h was significantly lower in the dexamethasone group than in the normal saline group. surgery, hospital quiron sagrado corazon, sevilla, spain; surgery, hospital virgen macarena, sevilla, spain; surgery, hospital virgen del rocio sevilla, sevilla, spain aims: closing the defect (cd) during laparoscopic ventral hernia repair (lvhr) could be related to a reduction of seroma formation or bulging (hernia mesh) compared to conventional lvhr. but tension of the midline may contribute for some authors to a higher incidence of pain, recurrence in medium size defects and suggest to perform a component separation (cs) for restoring the midline in medium-large defects.we have developed a new technique for restoring the midline in medium ventral hernias (lira technique) and weanalyzed our results in terms of pain and recurrence compared to our conventional cd series (ccd). methods: we conducted a prospective controlled study of lvhr with ccd from january to december and a prospective controlled study performing lira technique from january to january . we analyzed and compared both techniques in medium size defects ( - cms) in terms of postoperative pain ( , days, , months and year) using a visual analogue scale (vas), bulging (return to prior distance among rectus muscles with the mesh in the sac in ct that didn't need surgical treatment)and recurrence (by physical examination and tomography). results: ccd was performed in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ) and lira technique in patients (mean age was . ± . years old and mean bmi was . ± . kg/m ). the mean average follow-up in both series was year. mean average vas in ccd was . ± . ( day), . ± . ( days) . ± . ( month) . ± . ( months ) and at year. in lira series vas was . ± ( h) . ± . ( days), . ± . ( month), ( months) and ( year) . there are cases of bulging in ccd series and recurrence. bulging and recurrence were absent in lira series. conclusions: lira technique might be a safe procedure in medium size defects for restoring the midline in lvhr, and could be related to a lower pain rate compared to ccd with no recurrence or bulging. surg endosc ( ) :s -s background: the desire of pediatric surgeon to reduce incision related morbidity and pain while achieving good cosmetic results has recently led to the introduction of single incision pediatric endo-surgery [sipes] and needlescopic surgery. intracorporeal suturing and knot tying during sipes remains challenging. the aim of this study is to introduce a novel and simple technique for intracorporeal suturing of the pediatric inguinal hernia after needlescopic disconnection of hernia sac using just needles rather than laparoscopic instruments. it is an imitation of the principles of sewing machine. methods: the first author discussed the idea of the technique with the co-authors and a demonstration was done on a silicon pad before application of the technique on children with congenital inguinal hernia [cih] for peritoneum closure after needlescopic disconnection of the hernia sac. the main outcome measurements were; feasibility of the technique, knot quality, suture placement accuracy, performance and suturing time and recurrence rate. results: the sutures were snugly applied to the ridges of silicon pad with good approximation and the knot was firmly tightened in all experiments. after applying and mastering the technique on a silicon pad, we shifted to use it on children with hernia defect. all operations were completed by the needlescopic technique without the need for insertion of any laparoscopic instruments. the time required for suturing of the peritoneum around internal inguinal ring [iir] and knot tying, decreased considerably from min s in the first operation to less than min after the fifth operation and stabilized at approximately minute s. no major intraoperative complication and no recurrence. the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of patients were enrolled ( of them underwent tapp and olr). drop out occurred in cases ( of tapp and of olr group). patient characteristics were statistically similar between the groups. tapp procedure had less early post-operative pain (p = . ), a shorter length of stay (p = . ) and less postoperative complications (p = . ) when compared with the olr approach. a slightly higher recurrence rate in the tapp group was found. additionally, there is a trend towards a higher postoperative quality of life and less chronic pain in the tapp group. conclusions: tapp procedure for bilateral inguinal hernia effectively reduces early postoperative pain, hospital stay and postoperative complications. cannizzaro hospital, catania, italy aim: the purpose of this study was to evaluate the long-term results in terms of safety and efficacy of a new technique to repair incisional ventral hernias with a self-gripping mesh, after a mean follow-up period of months. methods: a retrospective, single-centre study was performed from june to june . all patients undergoing elective incisional ventral hernia repair were included. hernias were diagnosed based on clinical examination at the outpatient clinic. in case of doubtful diagnosis, ct-scan was used to confirm the diagnosis. the component separation technique and, when needed, tar were performed. the self-gripping mesh was placed in sublay position (overlap cm) with the self-gripping surface face down. in all cases drainage tubes were placed in retromuscular and supraaponeurotic position. the following characteristics were collected: age, sex, body mass index (bmi), smoking, comorbidities, number of previous surgical operations, defect size (ehs classification), mesh size, postoperative complications, duration of follow-up. all patients were interviewed by telephone every six months. when patients complained recurrence or other symptoms, visits were organized and when there was the doubt of recurrence a ct-scan was performed. results: a total of patients were included in this study, males, mean age was years. % of patients had bmi [ , smokers and diabetics were respectively % and %. the mean defect size was cm . component separation technique was associated with tar in patients. in cases the size of mesh was cm, while in patients the size of mesh was cm and in cases this was cm. in the other patients the mesh sizes were tailored to defect dimensions. subcutaneous seromas occurred in patients, they were treated conservatively in cases and with percutaneous punction in cases. long-term follow-up demonstrated recurrences in one case, while in another one ct-scan revealed a bulging. no cases of mesh infection, pain or sensation of mesh. conclusions: this study with a mean follow-up period of months demonstrated that the use of self-gripping mesh in sublay position is safe and effective to treat incisional ventral hernias. aim: morgagni hernias present technical challenges. the laparoscopic approach was described at first in , however, as they are uncommon in adult life and, little data exist on the optimal method of surgical management. this study purpose was to analyse a method for laparoscopic repair of morgagni giant hernias using laparoscopic primary closure. methods: this case series describes a method of laparoscopic morgagni hernia repair using primary closure. in all patients a laparoscopic transabdominal approach was used. the content of the hernia was reduced into the abdomen and the diaphragmatic defect was closed with a running laparoscopic suture using a self-fixating suture. clips were placed at the edges of the suture to secure the pledged sutures to both the anterior and posterior fascia. demographic data as age, gender and bmi were collected. operative data (operative time, rate of conversion, blood loss) and post-operative data (short and long term complications, length of hospital stay, need of readmission and reoperation) were recorded. results: retrospectively collected data about patients were analysed. there were ( . %) male and ( . %) females. the median bmi was . ± . kg/m . median operative time was ± min. there were no intraoperative complications nor conversion to open surgery. patients began a fluid diet on the first post-operative day and were discharged after a median hospital stay of ± . days. in a median follow up of months we did not observe any recurrences. conclusions: transabdominal laparoscopic approach with primary closure of the diaphragmatic defect is a viable approach for repair of morgagni hernia. in our experience, the use of laparoscopic transabdominal suture fixed to the fascia allowed the closure of the defect laparoscopically with minimal tension on the repairs. can we predict the success of the laparoscopic approach in the adhesive small bowel obstruction? c. tellez marques, e. sebastian valverde, e. membrilla fernandez, l. grande posa, i. poves prim general surgery, parc de salut mar-hospital del mar, barcelona, spain aims: the laparoscopic approach in the acute adhesive small bowel obstruction and internal hernias (asbo) has shown superior to laparotomy in terms of morbidity and hospital stay. especially, in patients who present simple adhesions or internal hernias. according to this, the aim of the study is to determine those preoperative factors associated with simple adhesions and internal hernias, and consequently, improve the success of the laparoscopic approach in asbo methods: a retrospective study of patients who underwent urgent surgery for asbo was conducted from january to may . we compare preoperative variables between single adhesions and internal hernias vs complex adhesions. a p value \ . was considered statistically significant. results: we analysed patients who underwent surgery for asbo, ( %) by laparoscopy and ( %) by laparotomy. conversion rate in laparoscopy was . %. . % of patients presented a single adhesion or internal hernia; and . % were considered complex adhesions. sex and age did not correlate with the type of adhesions. previous surgery (p \ . ), number of previous surgeries (p \ . ), asa (p \ . ) and previous abdominal wall mesh (p = . ) were significantly associated with complex adhesions. laparoscopy as the only surgical history was significantly associated with simple adhesions (p = . ). only appendectomy (p = . ) or supramesocolic (p = . ) previous surgeries tended to present single adhesions but it did not reach statistical significance. the need for intestinal resection was not related to the type of adhesions (p = . ). there was a significant correlation between the findings in the ct (computed tomography) and the type of adhesion found (p = . ). signs of ischaemia on ct were related to the need for intestinal resection (p \ . ). in the multivariate analysis, the number of previous surgeries, asa and ct scan findings were identified as independent factors related to the type of adhesion. conclusions: according to our study, a lower number of previous surgeries, asa i-ii and internal hernia in the ct scan are associated with single adhesions and internal hernias. patient selection is a key factor for the success of laparoscopic approach in asbo. aims: there aims of this study were: (i) to compare england with the united states in the utilisation of minimal access surgery (mas) and in-hospital mortality from four common abdominal surgical emergencies (appendicitis, incarcerated or strangulated abdominal hernia, small or large bowel perforation and peptic ulcer perforation). (ii) within england to evaluate the influence of mas upon in-hospital and long-term mortality. methods: between and , the rate of mas and in-hospital mortality for four abdominal surgical emergencies were compared between the united states and england. univariate and multivariate analyses were performed to adjust for underlying differences in baseline patient demographics. results: , admissions in england for four abdominal surgical emergencies were compared to an estimated , , admissions in the united states. after adjustment for patient demographics, mas was used less commonly england for three conditions; appendicitis (odds ratio (or) . , % ci . - . ), abdominal hernia (or . , ) and small or large bowel perforation (or . , ). in-hospital mortality in multivariate analysis, was increased in england compared to the united states for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , ) and peptic ulcer perforation (or . , % ci . - . ). in england, after adjustment for patient demographics, open surgery was associated with increased in-hospital mortality for three conditions; abdominal hernia (or . , % ci . - . ), small or large bowel perforation (or . , % ci . - . ) and peptic ulcer perforation (or . , . similarly open surgery was associated with increased long-term mortality for three conditions; abdominal hernia (hr . , % ci . - . ) , small or large bowel perforation (hr . , % ci . - . ) and peptic ulcer perforation (hr . , % ci . - . ). conclusions: minimal access surgery was used less commonly and inhospital mortality was increased in england compared to the united states for common abdominal surgical conditions. given the benefits of mas shown in this large study, strategies to enhance adoption of mas in emergency conditions in england need to be optimised and include appropriate patient selection and improved surgeon mas training and experience. surg endosc ( ) :s -s background: in the treatment of inguinal hernias, there is little hard evidence concerning the economic reimbursement in the diagnosis-related-group (drg) era. factors that affect whether a hospital may earn or lose financially depending on open or laparoscopic approach is still underexplored. the aim of this study is to provide a reliable analysis of in-hospital costs and reimbursements in inguinal hernia surgery. methods: this retrospective study analysed the -year experience in inguinal hernia repair in patients undergoing open lichtenstein (ol), laparoscopic totally extraperitoneal unilateral (utep) or bilateral (btep) hernia repair. demographics, results, costs and drg-based reimbursements were recorded and analysed. results: during the study period, patients underwent ol, patients utep and patients btep. the average total cost amounted to eur in ol, eur in utep and eur in btep groups (p \ . *). the hospital reimbursement amounted to eur, eur and eur in the ol, utep and btep groups respectively (p \ . *). finally, the mean hospital earnings were eur, eur and - eur for each patient in ol, utep and btep respectively (p \ . *). conclusions: in-hospital costs were higher in utep and btep as compared to ol. the drg-based reimbursement provided adequate compensation for patients with unilateral inguinal hernia, whereas hospital earnings were profitable in ol group only, and led an overall financial loss in the btep group. surgeons should be conscious that clinical advantages of the laparoscopic approach are not adequately compensated for, from an economic point of view. aims: umbilical hernias are common anatomical defects in swine which become a suitable model for surgical training and research in the field of surgical meshes. the aim of this study was to develop a surgical protocol for a successful laparoscopic implantation of stem cell-coated surgical meshes. methods: large white pigs, weighing - kg and with congenital abdominal hernia were anesthetized for the surgical procedures. non absorbable polypropylene surgical meshes were coated with fibrin glue (fg) (control group) or with fg admixed with porcine bone marrowderived mesenchymal stem cells (fg/bm-mscs). approximation of hernia's borders was performed by intracorporeal suture. the meshes were carefully rolled inside the trocar for laparoscopic implantation. the surgical implantation was performed by laparoscopy using helicoidal staples. laparoscopic inspections and biopsies of the tissue surrounding the mesh were performed at , and days post-implantation. at day , the animals were euthanized and macroscopically evaluated. ultrasonography was used at day , , and to evaluate the size of the hernia. the biopsies were then processed for the histological analysis. results: ultrasonography demonstrated that the mean size of umbilical hernias before mesh implantation was . ± . cm. a decrease in hernia mean size was observed at day and . the laparoscopic procedures allowed a successful mesh implantation in all animals. in most of cases, the implantation site did not show excessive inflammation or tissue adhesions. but one animal showed hernia maintenance. one animal had peritoneal and implant-site infection. foreign body reaction was noted in the histological analysis, although no significant difference was found between the control, and bm-msc group. conclusions: the anatomical similarities between humans and pigs in umbilical hernias make this animal model useful to: i) improve minimally invasive surgical procedures for hernia treatment; ii) evaluate new surgical meshes, and iii) introducing stem cell therapy to hernia surgical repair. the laparoscopic approach is efficient and safe for the implantation of stem cellcoated meshes. gene and protein expression analysis are required to evaluate the molecular changes between the conventional and the stem cell surgical approach. aims: fluorescence angiography with indocyanine green (icg) is used as a marker in the assessment of tissue perfusion, being more frequently used in colorectal procedures. this technology has shown to be a good technique to reduce complications related to vascular supply to the anastomosis. in esophagogastric procedures blood supply to the gastric pouch, jejunum and esophagus could be evaluated by icg fluorescence imaging. it could be also used in bariatric surgery to evaluated the anastomoses, during gastric bypass, and blood supply to the gastroesophageal junction and the angle of his during sleeve gastrectomy. methods: we have collected data during gastric resection due to adenocarcinoma and bariatric procedures that were performed by the same surgeon, using icg fluorescence to evaluate blood supply. the icg was infused before performing the anastomosis in order to evaluate the need to change the transaction line (tl). we analyzed those cases in which the tl was changed and the number of leaks in those cases that we changed this line. results: all the cases were performed by laparoscopic approach: subtotal gastrectomy (sg), total gastrectomy (tg), gastric sleeve (gs) and gastric bypass. there were no changes regarding the tl before performing the anastomosis in any of the four types of procedures (sg, tg, gs, gb). in the analyzed data there is anastomotic leak in one sg procedure ( . %). conclusions: icg fluorescence angiography could be helpful in assessing blood supply during gastrointestinal anastomosis, although we have not find an influence in the results during bariatric and gastric procedures. however, we do not have the sufficient evidence to determine the value of this technology in this entities, being needed more volume and data to improve the significance of the results. aims: hyperspectral imaging (hsi) combines a spectrometer with a camera to analyze the tissues' optical properties in a broad wavelength range, without the need for a contrast agent. it provides extensive real-time information about tissue physiology, including oxygen saturation (sto ). fluorescence-based enhanced reality (fler) is a software solution providing a dynamic, quantitative analysis of the signal evolution of a systemically administered fluorophore, during fluorescence angiography (fa) . the aim of this study was to compare the performance of hsi and fler to assess bowel perfusion, in a porcine, non-survival model of bowel ischemia. methods: in pigs, an ischemic small bowel segment was created and imaged after hour of ischemia. the imaging modalities were applied sequentially to the same area.hsi was performed first, to acquire the sto spectra, by means of the tivita tm system (diaspective vision, pepelow, germany), which provides a spectral range of - nm and a nm resolution. subsequently, fa was performed using a nir-capable laparoscopic camera (d-light p, karl storz, germany), after intravenous injection of . mg/kg of indocyanine green (icg; infracyanine, serb, paris, france). the fluorescence flow was recorded during s, then the slope of the fluorescence flow was analyzed using a proprietary software to obtain a virtual perfusion cartography. the virtual cartography was overlaid onto real-time images to obtain the enhanced reality effect. ten adjacent regions of interest (rois) were selected from hsi datasets and were superimposed to fler-generated cartographies using a custom plug-in software function, allowing for a quantitative comparison of both imaging modalities. hsi was repeated after icg injection. results: the r correlation coefficient between hsi-sto and the fler slope was . . at control hsi after icg injection, the correlation coefficient dropped significantly (r . ). the interference of icg on hsi imaging was clearly identified in the spectral curves. conclusion: sto given by hsi provided results comparable to those obtained with fler in our bowel ischemia model, without the need to inject a contrast agent. icg interferes with hsi datasets, disrupting sto values. surgical treatment is one of the most effective options for treatment of giant hiatal hernia. laparoscopic approach became is a 'gold standard' over the time demonstrating all advantages of minimally invasive techniques over the open procedures. however the utility of robotic operations still remains controversial. aim of the study: evaluate the initial experience of robotic fundoplication in compare to laparoscopic procedures. materials and methods: since the january till the december of thirty operations were operated on. mean age was . ( - ), among them ( %) were female and ( %) were males. mean bmi was . ( . - . ) . laparoscopic procedures were performed in patients ( st group), robotic procedures with davinci system were performed in patients of the second group. nissen fundoplication modified was performed in patients, toupet fundoplication was used for patients. results: the median operative time in laparoscopic group was min, in robotic group- , min. there were no statistical differences between two groups (p = . ). blood loss was minimal in both groups. mean postoperative hospital stay was . days ( - days) in the st group and , days ( - days) in the second. there were no statistical differences between two groups (p = . ). postoperative course was uneventful in all patients of both groups. surgical stress response is associated with systemic inflammatory syndrome, sepsis, multiorgan dysfunction syndrome. robotic assisted surgery has been introduced to overcome the limitations of conventional laparoscopy. this technique has potential advantages over laparoscopy, such as increased dexterity, three-dimensional view, and a magnified view of the operative field. these advantages could result in limited intra-abdominal trauma and hence in attenuated surgical stress response over conventional laparoscopy. aims: this study aimed to synthesize data on the effect of robot assisted surgery on surgical stress response. methods: electronic databases were searched with the search terms 'surgical stress', 'stress response', 'oxidative stress', 'robotic assisted surgery', 'c-reactive protein', 'interleukin ', 'interleukin ','cortisol',;'oxidative stress markers', 'antioxidants', 'antioxidant status', 'mda', 'glutathione', 'cortisol', 'acute phase response' up to and including march . results: one hundred forty studies were identified and their title and abstract were reviewed. one randomized controlled trial, six non randomized comparative studies, one experimental study and one case report met inclusion criteria. data were discordant. one prospective trial concluded that cortisol and il- were lower in laparoscopic assisted distal gastrectomy compared with robot assisted distal gastrectomy in another study comparing robotic assisted laparoscopic radical prostatectomy with open radical prostatectomy based on plasma measurements of il- , il- a and c-reactive protein, it was demonstrated that robotic assisted laparoscopic radical prostatectomy induces lower tissue trauma than open radical prostatectomy. in another study, it was reported reduced expression of genes associated with surgical stress response in patients treated with robotically assisted radical prostatectomy compared with patients treated with open prostatectomy. the case report concerned a case of polymyalgia rheumatic after robotic assisted laparoscopic prostatectomy. the experimental trial demonstrated that cortisol and substance p were significantly higher with open thoracic approach versus robot assisted thoracoscopic oesophageal surgery. conclusion: further research is needed to elucidate the effect of robotic surgery on surgical stress, based on a well standardized protocol for the measurement of surgical stress response. purpose: tissue compression is essential to prepare the tissue for proper staple formation. this study evaluates the risk factors of compression injury on the circular stapling line in vitro. methods: to reproduce the artificial bowel wall, a collagen plate was prepared by mixing collagen extracted from porcine with glycerin. artificial collagen plates with mm and mm in the thickness were made for dry and healthy condition and immersed plates in the tap water for min to make wet and edematous condition. circular stapler (cdh a, ethicon, usa) was applied in the collagen plates (dry and wet condition) and optimal compressions. compression line was evaluated for compression injury score. risk factors for excessive compressions and unacceptable injury were analyzed. results: in the dry condition, optimal compression didn't cause unacceptable injury. in the wet condition, excessive compressions were occurred in . % with optimal approximation. unacceptable injury was significantly different in proper and excessive compression cases as . % and . %, respectively. on the univariate analysis, thickness ( mm), wet condition, proximal side, maximal compression, and excessive compression were associated with unacceptable injury. on the multivariate analysis using logistic regression model, excessive compression was significant independent factor to cause tissue injury (p \ . ) and this significance was also proved in the optimal compression group (p = . ). background: minimal invasive appendectomy gained much popularity due to its better cosmoses, early recovery and less wound site infections. single incision laparoscopic appendectomy (sila) has many disadvantages such as, long operative time, bad ergonomics, surgical site infections, high conversion rate and port site hernia. needlescopic appendectomy (na) using mediflexÒ facial closure needle expected to be more superior over sila. here in we compare our results of needlescopic appendectomy with single-incision one. material and methods: one hundred and twenty patients with acute non complicated appendicitis were randomly assigned to na and sila children for each group during the period between january to october . the main outcome measurements included, demographics, operative time, intraoperative complication, conversion rate, post-operative hospital stay, surgical site infection, port site hernia and cosmetic results. results: a total of children underwent appendectomy. there were children who underwent na and children who underwent sila. there were no difference in age ( . vs . years, p = . ), weight ( . vs . kg, p = . ) and hospital stay ( . vs . days, p = . ) between the two groups. there were no intraoperative complication during the two surgical approaches. operative time for na group is significantly shorter than single incision group ( . vs . min, p = . ). no single case of conversion in na group and cases needed conversion in sila group. seven cases of sila showed surgical site infection. cases of sila group presented with port site hernia. the na group was superior as regard ergonomics. the two groups showed equal excellent cosmetic results. conclusion: needles scopic appendectomy and sila are comparable as regard cosmetic results and hospital stay. na proved to be safe, applicable, repetitive and superior over sila as regard better ergonomics, less operative time, absence of surgical site infection and port site hernia. aims: to objectively analyze the surgical performance and surgeon's ergonomics in the use of a novel flexible laparoscopic instrument during intracorporeal suture, and compare it with the use of a conventional laparoscopic needle holder. methods: three experienced laparoscopic surgeons performed five laparoscopic sutures on an organic tissue using the novel flexible instrument (flexdexÒ) and five sutures using a conventional needle holder with axial handle. the new device is based on a mechanical design with no electrical components, which transfers the surgeon's hand, wrist, and arm movements to the instrument tip in an intuitive manner. the use of the instruments was organized in a random fashion. prior to the study, participants conducted a -minute training session with the new flexible instrument. execution time and quality of the suture were assessed for each repetition. besides, flexion and radioulnar deviation of the wrist were recorded using an electrogoniometer (biopac systems, inc.) attached to the surgeon's hand and forearm. the intensity of the forearm's muscle activation was also analyzed by means of a myo armband (thalmic labs). results: surgeons required more time to perform the intracorporeal suture using the novel laparoscopic instrument ( . ± . s vs. . ± . s; p \ . ), but the quality of the suture was similar with both instruments. the wrist flexion ( . ± . °vs . ± . °; p \ . ) and wrist ulnar deviation ( . ± . °vs . ± . °; p \ . ) were significantly lower when using the flexible instrument. during the suturing tasks, the use of flexdexÒ instrument led to a higher muscular activation of the flexor ( . ± . vs . ± . rms; p \ . ) and extensor ( . ± . vs . ± . rms; p \ . ) muscle groups of the forearm. conclusions: the presented novel instrument allows surgeons to perform robotic-like laparoscopic suturing. we believe that with a longer training period surgeons could potentially reduce surgical times with this device. the preliminary results of this study suggest that the use of this new instrument provides a quality of the suture similar to that obtained with a conventional laparoscopic needle holder and an ergonomically more adequate wrist posture. aims: the intraoperative real-time evaluation of tissue perfusion is one key element for successful visceral surgery. traditionally, tissue evaluation is performed visually by surgeons. newer devices for objective quantification have in majority been based on the application of the fluorescent dye indocyanin green (icg). a novel method derived from geographic research is hyperspectral imaging (hsi). the aim of this study was the evaluation of hsi as a promising method for the evaluation of tissue perfusion and its implementation in the evaluation of the gastric conduit during esophagectomy in a porcine model. methods: the hsi camera records a dimensional data cube from a dimensional surgical situs obtaining wavelengths between and nm. the absorption at different wavelengths is tissue-specific and influenced by the amount of oxygenated haemoglobin and other pigments. a software calculates different indices in real-time including oxygen saturation. a porcine model (n = ) is used for esophagectomy with gastric conduit formation. ischemia is induced artificially by magnets simulating staplers. different shapes of the gastric conduit and anastomosis formation are evaluated for perfusion metrics in order to obtain recommendations for the optimal formation of esophagogastrostomy. conclusion: hsi is a promising method for intraoperative evaluation of tissue perfusion that does not require application or injection of any agents. the preliminary results in this study showed that the gastric conduit receives its main blood supply from the gastroepiploic arteries and not via the mucosa. further results from the current evaluations enable formation of an optimized gastric tube and esophagogastrostomy in esophagectomy. surg endosc ( ) pediatric surgery, al azhar university, giza, egypt; pediatric surgery, beni suef university, beni suef, egypt background: varicocele is one of the most common causes of infertility. many surgical interventions are used for varicocele ligation including open and conventional laparoscopic multiport or single incision techniques. the aim of the study is to present a new needlescopic lymphatic sparing varicocele ligation using mediflexÒ facial closure needle and gauge vascular access cannula. material and methods: twenty-two male children with bilateral varicocele of grade ii-iii. all children were counseled by clinical examination, doppler ultrasonography, abdominal ultrasonography, and routine laboratory investigations. testicular lymphatics were delineated by subcutaneous injection of / cm methylene blue in anterior wall of the scrotum min prior to surgery. the testicular vessels (both vein and artery) were ligated one cm above the deep inguinal ring using two mediflex needles with preservation of lympatics. the main outcome measurements included; operative time, hospitalization, testicular atrophy, hydrocele formation, recurrence of varicocele and intra or postoperative complication. results: a total of twenty-two male children with grade ii-iii varicocele subjected to needlescopic lymphatic sparing technique. twenty one were bilateral. background and aims: even if the clinical outcomes of robotic rectal resections are under investigation, the related robotic costs have not yet been well addressed, and the differences between the robotic rectal resection costs and the laparoscopic approach are still not well known. we have therefore performed a prospective comparative study of robotic rectal resections (rrr) and laparoscopic rectal resections (lrr) performed at our centre with the aim to evaluate the cost-effective outcomes of robotic versus laparoscopic surgery. study design: this is an observational, comparative prospective non-randomized study which includes patients that underwent laparoscopic and robotic rectal resection reaching a minimum of months of follow up from february to march , at the sanchinarro university hospital, madrid. an independent company performed the financial analysis and fixed costs were excluded. outcome parameters included surgical and post-operative costs, quality adjusted life years (qaly), and incremental cost per qaly gained or the incremental cost effectiveness ratio (icer). the primary end-point was to compare clinical outcome as well as cost effectiveness study between both groups. results: a total of rrr and lrr were included. the mean operative time was significantly lower in the lrr approach ( versus min; p = . ). the main pre-operative data, overall morbidity, hospital stay and oncological outcomes were similar in both groups, except for the readmission rate (rrr: . %, lrr: . %;p = . ).the mean operative costs were higher for rrr ( . versus . €; p = . ); however, the mean overall costs were similar ( . € for rrr and . € for the llr; p = . ). mean qalys at year for rrr group ( . ) was higher than that associated with lrr ( . ) (p = . ). at a willingness-to-pay threshold of , € and , €, there was a . % and . % probability that rrr group was cost-effective relative to lrr approach. conclusion: this study provides data of cost-effectiveness differences between rrr and lrr approach showing a benefit for the rrr aim: the efforts were aimed to the introduction of novel surgical technologies to overcome the intrinsic anatomical and technical constraints of rectal surgery. this was the case of the introduction into the clinical practice of laparoscopy and later on of robotic surgery for rectal surgery. however, whether robotic surgery is actually superior to laparoscopy is still debated. the aim of this study was to compare d laparoscopy and robotic surgery for rectal cancer on technical and oncological outcomes. methods: this was a single-center, prospective, randomized controlled trial. all patients more than years of age undergoing elective surgery for rectal cancer situated from to cm from the anal verge were included. patients undergoing abdominal perineal amputation and/or with t and/or m tumours were excluded. patients were randomized before surgery into two arms: arms a ( d laparoscopy) and arm b (robotic), and gave their consensus to the study. demographic data, data regarding the tumour, operative and post-operative data were collected. patients with a follow up shorter than months were excluded as well. results: twenty patients were enrolled in arm a and in arm b in the period time of one year. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. robot-assisted rectal resection results in comparable operative time ( . vs min; p = . ). the conversion rate was significantly lower for arm b ( vs p = . ). postoperative morbidity was comparable between groups. hospital stay was comparable but time required to resolve post-operative ileus was shorter in arm b ( . vs . days, p = . ). overall survival and disease-free survival were comparable between arms ( . % vs . %, p = . , and . % vs . %, p = . , respectively) conclusions: d laparoscopy and robotic surgery are two viable options for rectal surgery. robotic surgery can add some in terms of post-operative outcomes and ergonomics. aim: currently, robotic surgery for rectal cancer is a surgical operation that is being performed worldwide. we also introduced robotic surgery in . however, after robotic surgery, we observed a rise in creatinine kinase (ck), which is unlikely to happen in other surgeries. we studied the postoperative complications of rectal cancer patients who underwent either robotic surgery or laparoscopy during the same period of time. methods: from january to november , patients underwent surgery using robotassisted rectal resection (da vinci si cases and xi cases) and patients underwent laparoscopic rectal resection. in this study, abdominoperineal resection, intersphincteric resection, and lateral lymph node dissection were excluded. result: the operation time for the robotic surgery group was significantly longer than that for the laparoscopic group ( min vs. min; p \ . ). the ck value of the robotic surgery group on pod was significantly higher than that of the laparoscopic group ( iu/l vs. iu/l; p \ . ). in addition, one case of compartment syndrome was observed in the laparoscopic group. there were no significant differences in age, body mass index, intraoperative bleeding, tumor invasion depth, urination disorder, or postoperative hospital stay. in robotic surgery, it is considered that the increase in ck value is caused by the extended operation time, contact of the patient's cart with the left thigh of the patient, and the extra force applied to the abdominal wall caused by the displacement of the remote center. conclusion: in robotic surgery, it is suggested that the measurement of postoperative ck value is important. therefore, an attempt to shorten the operation time and paying attention to the surgical field are necessary to improve the outcomes. aims: anastomotic leak remains as one of the most important and life threatening post-operative complications in colorectal surgery. this complication has important consequences, both acute and long term, longer hospital stay, re-intervention, and increased morbidity and mortality. among all different circumstances that have been related to this entity, blood supply is an important factor that might have influence. fluorescence with indocyanine-green (icg) is used as a marker in the assessment of tissue perfusion in colorectal surgery which might reduce the numbers of leaks. methods: a multicenter analysis of the experience of centers in spain is collected in order to assess the value of icg in colorectal anastomosis. colorectal procedures were performed using icg to evaluate vascular supply in the anastomosis. icg was infused before performing the anastomosis analyzing the number of cases in which the transection line (tl) was changed. we also analyzed the number of leaks in those cases that we changed this line. results: out of the cases performed, cases were performed by open surgery, by laparoscopy, by single-port and with transanal total mesorectal excision(tatme). the following procedures were performed: right colonic resection(rc), splenic flexure partial resection(sf), left colonic resection(lc), subtotal colectomy(sc), total colectomy(tc), hartman reversal surgery(hr), low anterior resection with partial mesorectal-escision(lar) and ultra low anterior resection with total mesorectal-escision(ular). leak rate(lr) was . % ( . %rc, . %lc, . %sc, . %lar, . %ular). overall lr was . % in colonic surgery and . % in rectal surgery. the tl was changed due to icg in . % of the cases ( . %rc, . %sf, . %lc, % tc, . % lar, . % ular), being . % in colonic resection and . % in rectal resection. the relation between leaks and the cases in which the tl was changed, were % ( . %rc, %lc, . %ular). conclusion: icg fluorescence may play a role in anastomotic tissue perfusion assessment. the lr after colorectal surgery might decrease using icg to detect the proper tl before to perform the anastomosis. however, we do not have the sufficient evidence to determine that the changing transaction line can lead to avoid leaks. surg endosc ( ) aims: to analyse the value of postoperative day crp as an early predictor of safe discharge in robotic rectal cancer surgery. methods: a retrospective analysis was performed, including patients who had undergone robotic total mesorectal excision (tme) in a single centre over a -year period (may -september . patients who had a permanent stoma (abdominperineal resections or hartmann's procedure) were exluded from the study, leaving patients for further analysis. as the los is currently used as a performance tool in assessing outcomes in colorectal surgery (with a cut-off established at days), we compared the crp values in these groups. results: fourty one percent of patients were discharged home within days. they had an earlier peak of crp on postoperative day (pod) (median . , ) . the group of patients that were discharge home after days ( %) had a crp peak on pod (median , ). on pod , the group of patients that went home within days had a lower crp ( - -vs. - -) compared to the group of patients that were discharge after days, p = . ). conclusions: a crp peak on pod in robotic tme can predict an early and safe discharge (los within days). background: purposelateral pelvic lymph node dissection (lpnd) is suggested to treat suspected lymph node metastasis in pelvic side-wall in patients with rectal cancer who underwent preoperative chemoradiotherapy (crt). however, technical difficulties make it possible that lateral pelvic lymph nodes (lpns) are not dissected completely and, thus, remained in the narrow pelvis. near-infrared fluorescence imaging (fi)-guided surgery is expected to help visualization and complete excision of nonvisible lymph nodes during cancer surgery. this study aimed to evaluate the efficacy of fi using indocyamine green (icg) to identify lpns during robotic lpnd. methods: rectal cancer patients who were suspected lpn metastasis and had received preoperative crt were prospectively enrolled. icg in a dose of . mg was injected around tumor preoperatively. all procedures were performed with a totally robotic approach. after completing lpnd, fi was checked again for identifying remained lpns and resecting them completely. results: the lpns were successfully detected in ( . %) of the patients. however, after accounting for eight cases, having finished adjusting icg injection, the lpns were successfully detected in ( . %) of patients. the fi-guided lpnd group (n = ) showed similar mean operative time for unilateral pelvic dissection and complication rate, compared to patients who underwent conventional robotic lpnd (n = ). however, the mean number of unilateral harvested lpns was . in the fi-guided lpnd group, which was greater than the mean of . in the conventional group. lpn metastasis was identified in % of the fi-guided lpnd group, which was higher than that of the conventional group, . %. conclusion: fi-guided lpnd identifies lymph nodes in pelvic side-wall with great reliability. this contributes to increased number of lpns yield compared to conventional robotic lpnd. this technique should be considered to dissect them completely by preventing subsequent missing of nonvisible lpns. aims: to compare the medium-term oncological outcomes of laparoscopic total mesorectal excision (l-tme) vs. robotic total mesorectal excision (r-tme) for rectal cancer. methods: a retrospective analysis was performed including patients who underwent l-tme or r-tme resection between - . patients with disease stage iv at diagnosis or r resection were excluded. patients were initially included, and cases of r-tme were matched based on age, gender, stage and time of follow-up with an equal number of patientswho underwent l-tme. we compared -year disease free survival (dfs) and overall survival (os). in adittion, a multivariate analysis was performed in order to idenfity independent prognostic factors for -year dfs and os. results: pathological outcomes were similar between groups. however, major complications were lower in the robotic group ( . % vs. . %, p = . ), highlighting the anastomotic leakage rate, which was . % in the r-tme vs. . % in the l-tme group (p = . ). overall, the -year dfs rate was % in the laparoscopic group and % in the robotic group (p = . ). the -year os rate was % in the l-tme groups and % in the r-tme group (p = . ). for disease stage iii, -year dfs was significantly higher in the r-tme group. os was also significantly superior in the robotic group for every stage, reaching % in the stage iii. in the multivariate analysis, r-tme was a significant positive prognostic factor for distant metastasis (or . %ci . , . , p = . ) and os (or . %ci . , . , p = . ). conclusions: r-tme for rectal cancer can achieve better oncological outcomes compared to l-tme, especially in stage iii rectal cancers. the robotic approach has demonstrated to be a significant positive prognostic factor for local recurrence and overall survival, due to the better postoperative outcomes. however, a longer follow-up period is needed to confirm the oncologic findings. university hospital for visceral surgery, university of oldenburg, oldenburg, germany; bremen spatial cognition center, university of bremen, bremen, germany aims: in clinical settings, realistic assessment of one's own abilities can enhance performance and promote patient safety, especially in surgical residents, who inevitably have to acquire skills during real surgery. this study thus implemented the global assessment of laparoscopic skills (goals) questionnaire with the aim to explore divergences between resident self-evaluation and specialist's evaluation on laparoscopic performance, as a first step to implement the goals questionnaire as a tool for constructive and objective feedback. methods: between july and october , seven residents from the university hospital for visceral surgery at the pius-hospital oldenburg participated in this study. at the end of every laparoscopic operation where the resident acted as the primary surgeon, the resident and the supervising surgeon independently evaluated the resident's operative performance using the goals questionnaire. the five dimensions evaluated were depth perception, bimanual dexterity, efficiency, tissue handling and autonomy. a cumulative goals-score (with being the highest possible score) was calculated for n = laparoscopic operations. resident's year of training, the level of case difficulty and the type of laparoscopic procedure performed was also analysed. results: residents overestimated their laparoscopic abilities in . % of the operations (goals-scores: residents: median = , mean = . ; specialists: median = , mean = . ; p \ . ). residents in the first three years of surgical training were more likely to overestimate their performance (residents: median = . , mean = . ; specialists: median = , mean = . ; p \ . ) than those with more than three years of surgical experience (residents: median = , mean = . ; specialists: median = , mean = . ; p = . ). goals score differences did not depend on case difficulty and laparoscopic procedure. conclusions: surgical residents tend to overestimate their intraoperative laparoscopic performance when compared to specialist evaluation. overestimation was found to depend on one's own laparoscopic experience and seem to disappear with gained expertise. these results signify the importance of individually adapted training and the greater need for objective feedback for surgical residents. this approach could in return increase the skill acquisition rate of the resident and in return contribute towards enhancing patient safety. introduction: the delivery of safe surgical care is dependent of various, complex and interrelated factors. substantial data exist regarding the impact of training in human factor skills on surgical outcomes. however, except for the standardized time-out process, the best way to go about improving these skills remains unclear. the aim of this study was to gain more insights in the theatre staff's perception of human factors and their importance on surgical outcome in the operating theatre. methods: the surgical team assessment record (star) questionnaire was used to study the role of human factors, such as communication, situational awareness and organization, contributing to surgical team performance. the self-assessment questionnaire was filled out by the theatre staff, directly after the surgical procedure. conditional logistic regression was used to identify the impact of the role in the operating theatre on the yes versus no answers. results: some questionnaires were completed. the theatre staff rated their team's performance with a median of (iqr . , -point likert scale). the surgical fellows (n = ) rated their personal factors significantly lower compared to the rest of the operating team (median versus , p-value \ . ). the staff surgeon (n = ) indicated significantly more often that there were many distractions ( . %, yes n = ) and noticed aberrations ( . %, yes n = ) during the surgical procedure (pvalue \ . ) when compared to the rest of the operating team. most aberrations reported by the surgeons were related to technical performance. conclusions: human factors play an important role in the surgical environment. situational awareness may be less developed in members of operating teams, compared to the surgeon of that team. further work is needed to elucidate the impact of human factor skills on team performance. a team-based approach to safety interventions is recommended. future studies should determine what type of aberrations and distractions are most relevant and valuable to embark on with team training. dept. of digestive surgery, school of medicine, tokushima university, tokushima, japan; dept. of digestive surgery, tokushima university, tokushima, japan background: the qualitative evaluation for laparoscopic training of medical students was performed using rubric evaluation, and weak points in conjunction with the lack of anatomical knowledge were derived. to conquer these weak points, virtual reality (vr) ? augmented reality (ar) training for understanding of regional anatomy was investigated. materials and methods: one hundred and six students in th grade of tokushima univ. participated basic laparoscopic task training (gummy band ligation, beads transfer, delivery of beads, gauze excision) with training box and sham laparoscopic cholecystectomy with virtual simulator. rubric evaluation, as qualitative evaluation, which includes the evaluation standards for each maneuver were performed before and after basic task training and sham operation. the group which self-evaluation was higher in a rubric evaluation was investigated. the d image of vessels and bile duct obtained from mdct of real patient was projected in reality space with microsoft hololens. training of ar image using hololens was performed for understanding of regional anatomy. after training of regional anatomy with hololens, sham laparoscopic cholecystectomy was performed again, and quality of procedure was evaluated by rubric. anatomical questions were. results: rubric evaluation in basic task training showed no difference between self-evaluation and evaluation by tutor before and after practice. in sham laparoscopic cholecystectomy, several students showed higher score than tutor, especially in part of extension of operation field by elevation of the gall bladder, exposure of triangle of calot, and exposure of cystic duct. after ar training, all students showed high score in questions related regional anatomy during operation. especially, rubric evaluation of students who showed high self-evaluation in sham operation showed same score with tutor. conclusions: as rubric evaluation showed weak points of detailed parts of maneuver, and vr ? ar was useful for understanding details of regional anatomy for laparoscopic training. background: the eaes has recently published an intraoperative adverse event classification to aid reporting of minimally access surgery events. this includes capture of non-consequential errors. we aimed to investigate the clinical impact of these apparent 'near miss' events. methods: case videos and clinical data from a completed multi-centre laparoscopic total mesorectal excision randomised controlled trial was utilised (isrctn ). the eaes classification was applied by two blinded assessors to all enacted adverse events identified on video analysis using the observational clinical human reliability analysis technique. the total number of grade (non-consequential) errors were compared with the number and nature of day morbidity events (graded with the clavien-dindo system) and length of stay. results: cases ( h of surgery) contained error events of which ( . %) were classified as eaes grade (median per case, interquartile range - , range - ). there were significantly more inconsequential errors recorded in patients that developed any early morbidity event than those who had an uneventful post-operative recovery (median . (iqr - ) vs. ( - ), p = . ). a stepwise increase in the sum of eaes grade errors is seen for each additional day morbidity event reported ( . vs. vs. vs. , p = . ) and the highest clavien-dindo grade experienced ( vs. vs. vs. . p = . ). positive correlation is observed between the sum of eaes grade a errors and length of post-operative stay (r s = . , p = . ). conclusion: in the context of major laparoscopic surgery, near misses are commonplace and correlate with surgical outcomes. this may represent a novel surrogate assessment method for intraoperative performance. aims: diagnostic laparoscopy (dl) is an under-utilised procedure that can replace non-therapeutic exploratory laparotomies in many contexts. to date, no validated education programme for dl exists. this study seeks to evaluate the feasibility, acceptability and face, content, construct validity of the laplat curriculum (laparoscopic learning for abdominal trauma; a simulationbased curriculum for trauma dl). this is in addition to the development of a novel d-printed bench-top model for abdominal inspection. methods: this prospective and observational pilot study involved novice medical students and junior doctors. surgeons from the uk and international (n = ) were involved in a two stage delphi-process to determine the components of the training course which were used to formulate a final curriculum. in the absence of an adequate model for abdominal inspection, a novel dprinted abdominal inspection model was designed and produced. after an introductory familiarisation session as well as pre-course cognitive lectures, the novices performed tasks on a virtual reality and bench-top simulator with repetitions of each in a half-day session. outcome measures for construct validity were total time to complete task, accuracy, percentage of horizon maintained and economy of movement. face and content validity as well as acceptability was evaluated by a qualitative and quantitative survey. results: face, content and construct validity as well as acceptability was established. face validity was demonstrated in all components of the course (including pre-course cognitive content and technical tasks) in addition to content validity. all also met an acceptability threshold of / on a -point likert scale. one-way anova tests demonstrated construct validity in all tasks (p \ . ) with learning curves in reducing time observed. using a performance improvement metric, one-way anova tests showed similar rates of improvement per participant between most tasks (p [ . ). the course was rated overall mean . / (± . ). conclusion: this pilot study has demonstrated the feasibility, acceptability and face, content and construct validity of the laplat curriculum as well of the novel d-printed abdominal inspection model. randomised controlled trials are needed to establish higher-quality evidence, as part of a wider curriculum with transfer needed to the clinical environment. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology-surgical propedeutics, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania; urology, training and research center, prof. dr. sergiu duca, cluj-napoca, romania; general surgery, training and research center, prof. dr. sergiu duca, cluj-napoca, romania aims: to evaluate the benefits of systematical use of ex vivoliver model and ct imaging in the planning process for swine laparoscopic liver resections done by residents during training programs. methods: twenty four general surgery residents were equally divided into two groups: first one which performed laparoscopic liver resections without planning stage and the second one which systematically used anatomical data from a swine liver model and interactive ct scans d reconstructions. the planning stage included an interactive tutorial for establishing the strategy for the next resection followed by performing open liver dissection and the same resection on an ex vivoswine model. a total of twelve models were used during this step. afterwards, laparoscopic procedures were performed on sixteen anesthetized domestic pigs, two swine for every team, composed of three residents. both groups were part of a dedicated and continuous training program and used the same 'step by step' protocol for resections. results: the average time for imagistic planning was . min and for open dissection and resection was . min. all teams successfully completed the interventions and followed the standardized protocol without trainers' interventions and with no conversions. the second group obtained better results regarding the time needed for completion and blood loss. also, when the planning stage was applied the resection was more accurate and less functional parenchyma was removed. the 'warming up' by adding the imagistic and anatomical data to the core protocol offer more clarity before laparoscopic liver resections. this also makes an upgrade for our 'step by step' protocol and provides sufficient data to admit this planning stage as mandatory for laparoscopic liver resection on swine during a training program. introduction: submucosal tunnel endoscopic resections (ster) had been increasingly performed for treatment of gastric subepithelial tumors. one of the limitations for ster is the risk of incomplete tumor resection due to close dissection and bridging of tumor capsule. endoscopic full thickness resection (eftr) allowed complete resection of the tumor with margins to prevent recurrence. this study aimed to review the techniques and outcomes of eftr for treatment of gastric subepithelial tumors. method: patients who received endoscopic resection for gastric subepithelial tumors were recruited. the gastric subepithelial tumors were considered eligible for endoscopic resection with size \ mm. all patients received preoperative assessments including eus and ct scan to define the extend of tumors and the proportion of extra and intralumenal components. all the procedures were performed under general anesthesia with co insufflation. eftr started after injection with mucosal incision up to % of tumor circumference, followed by submucosal dissection to identify tumor margin. further dissection was performed using esd devices. after adequate exposure of lateral margins, incision into muscularis propria was performed to achieve full thickness resection. luminal defects were closed by either clips, clip-loop crown method or overstitch suturing. results: from to , patients received eftr for gastric subepithelial tumors. the mean age was . years, and were male. the gist were located at greater curvature ( ), cardia ( ) , lesser curve ( ) and antrum ( ) . the mean size was . mm ( - mm) . most of the eftr were performed in operation theatre while two were done at endoscopy. the mean hospital stay was . days, and mean operative time was min ( - mins). there was no conversion to laparoscopy. closure of luminal defect were performed mostly with clips ( ), followed by overstitch ( ) and clip and loop crown closure ( ) . most patients resumed full diet on day , and all the pathologies confirmed gist tumors with clear resection margins. conclusion: endoscopic full thickness resection is technically feasible and safe procedure for treatment of gastric gist. future research should focus on refining the techniques of eftr and closure of the defect. next generation endoscopic intervention (project engine), osaka university, suita, japan; gastroenterological surgery, osaka university, suita, japan; research & development, -d matrix, ltd., chiyoda-ku, tokyo, japan; research & development, fuso pharmaceutical industries, ltd., cyuou-ku, osaka, japan background: hemostatic peptides have received increased attention. self-assembling peptides (tdms) comprise synthetic amphipathic peptides that immediately react to changes in ph and/or inorganic salts to transform into a gelatinous state. since tdms do not carry a risk of infection, their clinical application as new hemostatic agent is expected to increase. the first generation of these peptides (tdm- ) is currently used as a hemostatic agent in europe. however, tdm- exhibits slow gel-formation and low retention capabilities on tissue surfaces. the second generation (tdm- ) was therefore developed to encourage faster gel-formation and better tissuesealing capabilities, and we subsequently verified its usefulness and increased performance relative to tdm- in preclinical open surgery. aim: the aim of this study was to verify the efficacy of tdm- in terms of its hemostatic effect in endoscopic surgery. materials and methods: evaluation of the hemostatic effect in endoscopic surgery (animal study) was performed using eight female ( kg) pigs in spine position. following systemic heparinization, we established a bleeding model by utilizing flexible endoscopic grasping forceps on the anterior wall of the stomach and duodenum. in the hemostasis method, an endoscope with a distal hood was brought into contact with the bleeding point, and ml tdm- was applied to the wound. after tdm- gelation, the endoscope was removed, and the acute hemostatic effect (after min) was confirmed. histologic evaluation was subsequently performed on resected specimens. results: in the endoscopic bleeding model, of the cases ( . %) showed complete hemostatic effects on the anterior wall of the stomach, whereas on the anterior wall of the duodenum, of cases ( %) showed complete hemostatic effects. moreover, none of the gels were displaced from the anterior walls of the stomach and duodenum, and histologic evaluation confirmed no infiltration of inflammatory cells. the new self-assembling peptide (tdm- ) displayed improved hemostatic effects relative to the previous generation (tdm- ) in endoscopic surgery. tdm- had potential usefulness for upper gastrointestinal bleeding. our future work will assess its usefulness for laparoscopic surgery. objective: indocyanine green (icg) is a dye used in medicine since the mid- s for different applications in ophthalmology, cardiology and hepatobiliary surgery; thanks to its selective hepatic uptake and biliary excretion, it can be used to evaluate hepatic function in patients scheduled for hepatic resection surgery. the aim of this study is to evaluate the efficacy and the feasibility of icg guided surgery in the intra-operative localization of liver tumors, comparing the pre-operative radiological aspect, the intra-operative visualization and the post-operative histopathological features of the tumors. materials and methods: icg was intravenously injected for a routine liver function test (limonÒ) in patients who underwent hepatic resection surgery for primitive and secondary liver tumors in the period between november and september . for each patient was performed an intraoperative visualization of the stain both in vivo and ex vivo, using a nearinfrared imaging system. all the images were recorded. results: a correct differentiation between liver parenchyma and tumor area was obtained in . % of cases. five patients were not evaluable due to widespread uptake or complete absence of uptake; it was probably the first cases enrolled in the study for which we were not able to set doses and timing of administration of icg. in patients in which the method had been feasible, we observed a prevalence of nodular pattern in patients with hepatocellular carcinoma ( %) and a predominance of rim pattern in both cholangiocarcinoma ( %) and metastasis ( %). furthermore, in patients with hccs well-intermediate differentiated (g -g ) was found predominantly a nodular pattern ( . %), whereas in poorly differentiated ones was prevalent a rim appearance ( %). regarding radiological correlations, the only one patient who presented an atypical radiological feature in pre-operative evaluation, showed a lesion with no icg captation in intra-operative visualization. conclusions: icg fluorescence imaging is a safe, minimally invasive and quite inexpensive method, that can be easily administered for routine evaluation of pre-operative liver function. it can be a useful support tool in the intra-operative detection of liver tumors, especially in laparoscopic surgery where it is not possible to directly touch the tissue. surgery, bundang cha medical center, seongnam-si, korea; surgery, severance hospital, seoul, korea; surgery, nhimc ilsan hospital, ilsan, korea; surgery, seoul national university bundang hospital, seongnam, korea; surgery, asan medical center, seoul, korea backgrounds & aims: robotic surgical system had been widely accepted in various surgical field with the expectations of overcoming the limitation of laparoscopic surgery. however, robotic liver resection had not generalized, so far. thus, this study aimed to evaluate the feasibility and safety of robotic major liver resection by prospective multicenter study. methods: from july to december , five surgeons who were novice in robotic liver resection but experienced a lot in open and laparoscopic liver resection in five tertiary hospitals performed cases of robotic major anatomical liver resection. perioperative patient's clinical data and surgical data were prospectively collected. results: cases of left hemihepatectomy, case of extended left hemihepatectomy, cases of right hemihepatectomy, cases of right anterior sectionectomy, cases of right posterior sectionectomy, and one cases of central bisectionectomy were performed. the most common indications were hepatocellular carcinoma for cases following intrahepatic cholangiocellular carcinomas for cases, liver metastases for cases, sarcoma for case, intraductal papillary neoplasms for cases, mucinous cystic neoplasm for case, hemangioma for case, and intrahepatic duct stones for cases. surgical resection margins for all tumor cases were negative. total average operation time was . ± . min and estimated intraoperative blood loss was . ± . ml (minimal to ml). in terms of severe surgical complication, there were cases of postoperative fluid collection treated with drainage and one case of bile leakage treated with percutaneous trans-hepatic biliary drainage. only one case out of cases was converted to the conventional open left hemihepatectomy because of bleeding. conclusions: in this study, robotic anatomic major liver resection might be safely performed even by robotic beginners but advanced open and laparoscopic liver surgeons. surgical technique: with the patient at °on right lateral decubitus, access is gained through the path of the percutaneous drainage catheter after opening of the aponeuroses of the oblique and transverse muscles of the abdomen. a mm laparoscopic trocar is inserted and a cavity is created with pneumoretroperitoneum at mmhg. it is accessed with an optic of °and mm, and the work space is extended with aspiration and hydrodissection. with mm grippers, the necrotic material is removed, washed and drained. a two light silicone probe is left, one light for drainage and another one for washing. results: the mean age was . background: minimally invasive surgery has achieved worldwide acceptance in various fields, however, pancreatic surgery remains one of the most challenging abdominal procedures. in fact, the indication for robotic surgery in pancreatic disease has been controversial. the present study aimed to assess the safety and feasibility of robotic pancreatic resection. methods: we retrospectively reviewed our experience of robotic pancreatic resection done in sanchinarro university hospital. clinicopathologic characteristics, and perioperative and postoperative outcomes were recorded and analyzed. aim: this work aims to study the contact pressure between the moving capsule and a synthetic small intestine in order to provide design guidance for prototyping the self-propelled capsule robot for small-bowel endoscopy. method: since small-bowel peristalsis consists of peristaltic contraction and wave distension, the contacts between the capsule and the small intestine are multimodal. we consider three contact cases for the capsule robot. case : the capsule moves on a flat small intestinal surface; case : the capsule moves in a collapsed intestine with a flat surface support; and case : the capsule moves in a surrounded small intestine. by considering these three contact cases, experimental testing and finite element analysis (fea) were conducted by measuring the contact pressure between the small intestine and the capsule. introduction: traditional laparoscopic instruments have limited degrees of freedom and are not ergonomic. this results in severe limitations in performing complex, and even simple tasks in surgery, limiting many surgeons from performing a variety of minimally invasive procedures. handx tm is a hand-held, electromechanical smart instrument with robot-like features. the instrument is composed of a sophisticated user interface that enables unrestricted hand movement, and a novel, motor driven articulating tool that is controlled by the interface. the instrument is . mm in diameter, lightweight, and can be easily moved between laparoscopic trocars and perform complex motions in the surgical field. after the regulatory process was completed we have tested the device clinically through a structured, approved, clinical trial. materials and methods: after irb approval patients were recruited to the trial. we have included a variety of procedures that require suturing and complex tissue manipulation. two experiences surgeons performed all procedures. after completing each procedure the surgeons completed a detailed standard usability (sus) questionnaire. results: procedures were completed successfully without complications or device malfunction. there were female and male patients with an average bmi of . procedures performed were right hemicolectomis with intra-corporeal anastomosis, paraesophageal hernia repairs and fundoplication, diagnostic laparoscopies, tapp procedures, ventral hernias with fascial suturing, and laparoscopic cholecystectomies. the average performance score was . / . the results suggest that the handx device is safe and easy to use and may offer a simple solution for enhancing minimal invasive surgery capabilities and possibly reduce conversion rates while maintaining current standard surgery flow.the handx could potentially extend the surgeon's abilities to access hard to reach anatomy and perform complex maneuvers and present a cost-effective alternative to large console-based robotic systems. objective: endoscopic submucosal dissection (esd) has become widely accepted treatment for rectum neuroendocrine neoplasm. the aim of this study is to evaluate the safety and efficacy of esd with dental floss-assisted suspension traction for rectal neuroendocrine neoplasm. methods: we retrospectively reviewed the medical records of the patients, who underwent esd for rectum neuroendocrine neoplasm at endoscopy center of zhongshan hospital, fudan university. the data of operation time, r resection and adverse events were collected analyzed.in dfs-esd group: after the mucosa was partly incised along the marker dots, the next step was to construct traction device, similar to others in esd, with dental floss and hemoclip. the dental floss was tied to any arm of the metallic clip. the hemoclip was attached onto the incised mucosa, another hemoclip was attached onto normal mucosa opposite to the lesion in the same way. the submucosa was clearly exposed with the traction of dental floss and the resection could proceed. results: patients were enrolled in the study. there were patients treated by esd with dental floss-assisted suspension traction and patients treated by conventional esd. the average tumor size was ( . ± . )cm in both group. the operation time was . ± . min in conventional esd group and ( . ± . ) min in dfs-esd group (t = . , p = . ). according to pathological grading about rectal neuroendocrine neoplasm, there were grade (g ) and grade (g ) in conventional esd group while grade (g ) and grade (g ) in dfs-esd group (? = . , p = . ). among cases in this study, all the basal resection margins were negative, the en blot resection rate was % and the curative resection rate was %. however, pathological results showed tumor tissue close to the burning margin in cases of conventional esd group and in cases of dfs-esd group (? = . , p = . ). conclusions: esd with dental floss-assisted suspension traction for rectum neuroendocrine neoplasm can assist exposing tumor borders, provide good vision during the procedure and offer clearer anatomic structure, so as to simplify operation, reduce operation time and ensure the negative basal margin. it is especially suitable to be promoted in primary hospitals. surg endosc ( ) aims: force feedback and assessment provides detailed insight into tissue manipulation skills. the aim of this study is to evaluate learning curves for basic laparoscopic skills based of force and motion learning curve patterns. morevover, we aimed to detect the favourable time span for this curriculum for each individual trainee. methods: in this prospective cohort study, first year surgical residents participated in a three week at home training course. a mobile box training was equipped with forcesense system for objective force, motion and time based assessment. the system provides seventeen unique metrics. the training goal was set by the mean score of proficient laparoscopic surgeons. each repetition was captured and made available for analyses. continuous force feedback was provided during training. curve fitting was used to estimate the learning curve plateau and the number of repetitions needed to approach the plateau phase and to reach proficiency level. finally, a comparisson between novices and experts was executed. results: a total of attempts, executed by residents were captured and analyzed. significant improvement of motion analysis parameters (e.g. path length and time) was observed for all training tasks, except for the fifth tasks. tissue manipulation skills (i.e. maximum and mean applied force) significantly improved by training tasks , and . learning curve analysis revealed various shapes and lengths of the individual learning curves. a large range in learning curve plateaus was found between trainees and between tasks. each trainee managed to accomplish the preset goals within three weeks. conclusion: force-and motion based assessment provides insight into both tissue manipulation and instrument handling skills. when combined in learning curve analysis, these parameters effectively show progression towards proficiency for each individual trainee over time. we emphasize the variation in learning curves between trainees. therefore, we recommend individually tailored courses provided with objective force-and motion-based learning curve tracking. aims: the posterior retroperitoneoscopic adrenal access represents a challenge in orientation and working space creation.the aim of this experimental acute study was to evaluate the impact of computer-assisted quantitative fluorescence imaging on adrenal gland identification and perfusion assessment in the posterior retroperitoneoscopic approach. methods: six pigs underwent synchronous (n = ) or sequential (n = ) bilateral posterior retroperitoneoscopic adrenalectomy (pra, n = ). fluorescence imaging was obtained via intravenous administration of ml of indocyanine green (icg) using two near-infrared camera systems. fluorescence-based visualization of adrenal glands before vascular division (n = ), after main vascular pedicle ligation (negative control, n = ) or after adrenal division (n = ) was followed by completion adrenalectomy. one of the animals had undergone icg injection h previously, during another study. the dynamic evolution of fluorescence signal intensity over time was recorded and analyzed using a proprietary software. the computed color-coded perfusion cartography was superimposed onto real-time images obtained by corresponding left (l) and right (r) camera systems. the slope of fluorescence signal intensity evolution over time in the regions of interest (roi) served to assess adrenal perfusion by means of quantitative fluorescence signal analysis. results: in the retroperitoneum, the adrenal glands were promptly highlighted after primary intravenous icg administration or showed an increase in fluorescence signal intensity upon reinjection (both glands in a recovery pig and one gland in the sequential approach). after left adrenal main vascular pedicle ligation, the gland displayed low perfusion (blue; rois a -a in figure ), while a weak fluorescence signal after completion adrenalectomy suggests perfusion via collateral vessels. with intact vascular supply, the caudal segment of the right adrenal (a ) gland showed a significantly higher perfusion rate (red) than the ischemic cranial segment (a ). quantitative analysis of logarithmic fluorescence intensity showed a statistically significant difference between perfused and ischemic zones (p = . ) allowing to assess gland vascularity. kidneys (k) and adrenal glands showed distinct perfusion curves ( figure ). conclusions: prior to dissection, fluorescence imaging allows to easily discriminate the adrenal gland from surrounding retroperitoneal structures. during adrenal gland surgery, icg injection complemented by a computer-assisted quantitative analysis helps to distinguish between wellperfused and low-perfused segments. giant adrenal tumors:technical considerations and surgical outcome a. giordano, g. alemanno, c. bergamini, p. prosperi, v. iacopini, a. dibella, a. valeri sod chirurgia d'urgenza, aou careggi, firenze, italy objectives: giant adrenal tumors are tumors with size more than cm. these are rare cancer associated with malignancy in % of cases. the size of these tumors is an important topic in literature because of their higher probability of malignancy and possible technical limitations of laparoscopic approach. we report our center's experience on laparoscopic adrenalectomy. materials and methods: in the last ten years we performed about adrenalectomies for benign and malignant adrenal tumors. of these were giant tumors. the medium size was . cm ( - cm). tumors were on the left adrenal gland and on the right. there were women and men, the average age was ( - years). of these cancers were laparoscopically removed and with open approach. cases of open conversion. results: betweenn the tumors laparoscopically removed we recorded cases of carcinoma, endothelial cysts, adenomas ( with aldosterone and with cortisol hypersecretion), myelolipomas, pheochromocytomas and metastases from lung carcinoma. the surgical outcomes in these patients were optimal in terms of good pain control and hospital stay (median days). the average time of the intervention was min with very low blood less ( ml). no postoperative complications were recorded. the removal of the adrenal gland necessitated or trocars. in the dissection and resection phases we always used radiofrequency scalpel. the follow up after and months didn't show local recurrences. conclusions: laparoscopic adrenalectomy offers significant advantages over the open approach. the size of these tumors is still at the center of debate for the choice of the technique. the tumor size is only a predictive parameter of possible malignancy. the laparoscopic approach is a safe and feasible method in terms of surgical and oncological outcomes also for the giant adrenal tumors, only if performed by expert surgeons and in high-volume centers. vascular or adjacent organs infiltration is a contraindication to the laparoscopic approach. aims: adrenal gland size greater than cm is considered a contraindication to laparoscopic adrenalectomy (la). aim of the present case-control study is to compare the surgical outcomes in patients undergoing la for adrenal gland measuring = cm versus = . cm in diameter. methods: from january to august , las were performed in the two authors' centers which follow an identical treatment protocol. eighty-one patients with an adrenal gland size = cm (intervention group) were included in the study. based on body mass index (bmi) class [ kg/m ) , lesion side (right or left), surgical technique (anterior transperitoneal for right and left-sided lesions, anterior transperitoneal submesocolic for left-sided lesions) and lesion type (conn-cushing, pheocromocytoma, primary adrenal cancer or metastases, other type of lesion), patients with an adrenal gland lesion measuring = . cm in diameter were included (control group) and paired to the intervention group. results: comparing the intervention and control groups, statistically significant differences were observed in mean lesion size ( conclusions: the only significant difference between the two groups was the operative time which was longer in the intervention group. conversion and complication rates were also higher in the intervention group but the difference was not statistically significant. based on the present data, adrenal gland size measuring more than cm in diameter is not a contraindication to a laparoscopic approach. ; and orthopaedics and urologists for the remaining . %.the costs from these claims, differed from to % of the total damage burden per year. the review of medical charts of claims related to laparoscopic gynaecologic surgery showed that % of claims were filed for visceral and/or vascular injuries ( % bowel injuries, % ureter). % of the injuries were entry-related. a delay in diagnosing injuries was the primary reason for financial compensation. conclusion: evaluating and learning from complications and claims will improve medical health care. in contrast to overall trends and developments considering medical claims, claims concerning laparoscopic surgery decreased, possible due to a rising learning curve. considering laparoscopic surgery, extra caution is required at moment of entry and the early recognising complications and at pre-operative counselling from patients. the aim of the study was to determine indications and contraindications for laparoscopic splenectomy in abdominal trauma patients and to analyze results of the operations. patients and methods: the study involved patients with spleen injury grade iii who were admitted in our institute in the years of - . the patients were divided on two groups. laparoscopic splenectomy was performed in patients (group i) and 'traditional' splenectomy was carried out in patients (group ii). there was no difference in the demographic data and trauma severity between the two groups.non-invasive investigations, such as laboratory investigations, serial abdominal ultrasound examinations (us), x-ray in multiple views and computed tomography (ct) had been performed before the decision about necessity of an operation was made. results: patients after laparoscopic operations had better recovering conditions compare to patients with the same injury after 'traditional' splenectomy. neither surgery related complications no mortalities were registered in both groups. laparoscopic splenectomy was more timeconsuming operation than 'traditional' splenectomy. we suggest that as experience of laparoscopic splenectomy is gained the operation time will be reduced. conclusion: laparoscopic splenectomy is a safe feasible operation in patients with spleen injury. the operation is indicated in patients with spleen laceration more than cm of parenchymal depth with moderate continuing bleeding or expanding hematoma and contraindicated in patients with hemodynamic instability and high bleeding rate (more than ml/h on serial us examinations). the isolated hydatid disease of the spleen is a quite rare condition, liver and lungs being the most common locations. the treatment requires usually splenectomy, open or laparoscopic. there are few reports in the literature describing a spleen-preserving type of surgery. we present a case of a female patient, y.o., with a large cystic lesion of the spleen, cm in diameter. lab tests and ct scan confirmed that is a hydatid cyst. after albendazole treatment and vaccination the patient was referred to us for surgical treatment. the procedure was performed under general anesthesia and laparoscopic approach was performed with the intention to preserve the spleen. after the cyst was identified and adhesiolysis was done, the area was isolated from the rest of the abdominal cavity with sponges with a betadine solution in order to prevent contamination. a needle aspiration of the cyst allowed the evacuation of ml of purulent content, an indicator of a dead cyst. betadine solution was injected into the lesion. laparoscopic excision of the cyst was performed using advanced electrocoagulation devices and the spleen removal was not deemed necessary. two drainage tubes were placed in the remnant cavity. an abdominal ultrasound was performed in the third postoperative day and no collections were identified. the postoperative outcome was uneventful; the patient was discharged in the th postoperative day. the conclusion is that in selected cases, with the cyst located in the anterior part of the spleen, with proper equipment and experienced laparoscopic teams, the cyst can be successfully treated without splenectomy. deep neuromuscular block was induced with rocuronium . mg/kg. in group , forty patients were enrolled for reversal of profound neuromuscular block during thyroid surgery (sugammadex mg/kg, after identification of vagus nerve). in group , thirty-five patients were enrolled profound neuromuscular block during thyroid surgery(without reversal of nmbd). tof-watch acceleromyograph was recorded in response to adductor pollicis muscle for ulnar nerve stimulation in patients with both groups; recovery was defined as a train-of-four (tof) ratio = . .to prevent laryngeal nerve injury during the surgical procedures, all patients were neurophysiologically detected using ionm. results: the total duration of surgery was higher in group than group ( . ± . , . ± . ;p \ . ). the mean time to recovery of the tof ratio to . was higher in group than group ( . ± . , . ± . ; p \ . ). the mean duration of vagus reverse (v : , milisecond) was higher in group than in group ( . ± . , . ± . ; p \ . ). no significant difference was found between left and right v -v and r -r values in group following nerve monitoring, whereas in group , a significant difference was found between left v -v , left r -r and right v -v values ( introduction: oeosphagogastric oncology trials have often lacked robust methods of monitoring and surgical quality assurance (sqa), leading to difficulty in interpretation of trial results. this study aims to assess expert opinion regarding challenges to sqa in oncology trials and potential mitigating strategies. method: a purposive international cohort of expert stakeholders with experience in oncology trials were recruited including: surgeons; oncologists; trial methodologists, and; trial managers. semi-structured interviews were thematically analysed using grounded theory. spss was utilised to assess differences between trial stakeholders' opinions. results: emergent themes were identified and consensus themes emerged on qualitative analysis of stakeholder responses. key consensus challenges to implementation of sqa in oncology trials included: insufficient resources; limitations of surgical volume in centre selection; differing oncological beliefs and resistance to change adoption; overly prescriptive protocols and standardisation contributing to difficulty in surgeon recruitment; and cultural factors leading to difficulties in providing and receiving feedback. seminal consensus mitigating strategies to overcome challenges to sqa in oncology trials included: trial centre selection according to case volume (n = , %); requirement for specific centre attributes for inclusion in trials including specialist centre designation and participation in national audit (n = , %); consideration for surgeons learning curve in surgeon selection (n = , %); flexible standardisation of trial operating (n = , %); operation manual utilisation to aid standardisation of surgical interventions (n = , %); case monitoring using video (n = , %) or photographs (n = , %); direct intraoperative observation by an expert (n = , %), and; histopathological assessment of resected specimens (n = , %). other methods of monitoring surgical quality advocated included: recording post-operative outcomes; lymph node yield; case report forms; and real time data monitoring (n = , %). oncologists were significantly more likely to state the importance of standardisation of surgery in oncology trials (p \ . ), and trial methodologists significantly more likely to advocate consideration of surgeons' learning curve in surgeon selection (p \ . ). conclusion: surveying international expert stakeholder opinion revealed a wide variety of perceived challenges across all domains of surgical quality assurance. proposed mitigating solutions require consensus opinion to formulate a framework to aid design of sqa measures within future oncology trials. research group did not register a single case of ega leakage while patients in control group (? \ , ). had the leakage which was stopped by means of 'endovac' system. there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). months after surgery, the number of post-operative strictures increased in both groups, but was lower in the research group and amounted to cases in the research group and cases in the control group (? \ , ). there were cases of esophagus postoperative strictures which developed months after the surgery in the research group which was less than in the control group which saw cases of strictures of ega (? \ , ). neither of the groups had any cases of post-operative mortality. purpose: to investigate the prognostic effects and risk factors of the omission and delay of postoperative chemotherapy of ii/iii gastric cancer (gc), with the goal of providing a reference for interventions of related departments. methods: the clinicopathological data of patients undergoing radical gastrectomy for ii/iii gc were collected and retrospectively analyzed. we defined the chemotherapy delayed until more than days after radical gastrectomy and the complete omission of chemotherapy as unacceptable chemotherapy initiation (uac group), while the chemotherapy conducted within days of radical gastrectomy was defined as acceptable chemotherapy initiation (ac group). the survival between the two groups was compared, and the trends and risk factors of uac were analyzed. results: the total number of patients who underwent totally laparoscopic distal gastrectomy with uncut roux-en-y and delta shaped billroth-i anastomosis was and , respectively. the mean reconstruction time was longer in uncut roux-en-y than in delta shaped billroth-i, ( . ± . vs. . ± . min, p \ . ). the uncut roux-en-y was used more cartridge than delta shaped billroth-i anastomosis ( . ± . vs. . ± . , p \ . ). however there was no significant differences in operation time, estimate blood loss, number of retrieved lymph node and postoperative course between reconstruction methods. postoperative complications more than clavien-dindo grade iiia occurred in cases ( . %) of postoperative early complications and cases ( . %) of late complications. the endoscopic findings showed excellent short and long-term outcomes in terms of very low incidence of bile reflux and reflux-induced remnant gastritis in uncut roux-en-y compared with delta shaped billroth-i anastomosis. conclusions: uncut roux-en-y gastrojejunostomy was a useful reconstruction method with totally laparoscopic distal gastrectomy for cancer, especially for diverting enteral contents from the remnant stomach and preventing remnant gastritis. therefore, it is recommended for young patients with early stage disease who have a long time to live after distal gastrectomy for cancer. operative technique: the seromuscular layer above the tumor is dissected, while the mucosa is kept unbroken. when seromuscular layer is dissected all around the tumor, the full layer is lifted, and the mucosa is stretched. the mucosa is then transected with a stapling device to execute fullthickness resection of the specimen. finally, the seromuscular defect is repaired by hand-sewn suture. results: since december , clean-net has been performed in patients with gastric smts. all tumors were resected en-blocwithout rupture. the average operation time ranged from to min with an average of . min. the postoperative course was uneventful. microscopically the surgical margin was tumor-negative (r resection) in all cases. the margin width was small with an average of . mm ± . . conclusions: clean-net is a useful option in the laparoscopic surgical treatment of gastric smt, when excessive sacrifice of the healthy gastric wall surrounding the endophytic tumor should be avoided. background: the type of fundoplication-complete or partial is still controversial for the surgical treatment of gerd. laparoscopic toupet ( wrap) fundoplication has less post op dysphagia and gas bloating compared to nissen fundoplication ( wrap) and is advised to be the procedure of choice when esophageal manometry findings are abnormal, however it is considered by some less effective and more difficult to perform. the aim of this research was to determine in the functionality and efficacy of the different types of fundoplication. methods: explanted pigs stomachs weighing - kg were studied. two different studies of the les were performed: distensibility and failure point (occurrence of reflux according to volume added to the stomach). for both studies we first disrupted the lower esophageal sphincter using a rigiflex tm dilating balloon. we then performed three different fundoplications-nissen, toupet, dor and measured the distensibility of the egj after each fundoplication. the failure point was determined following each fundoplication type. results: we used pig stomachs for the distensibility study and pig stomachs for the failure point study. there was no statistically significant difference between the nissen and toupet fundoplications when distensibility was measured, however the egj was more distensible following dor fundoplication (p = . for nissen, . for toupet). when the failure point was measured, nissen fundoplication was significantly more effective than toupet, and toupet was significantly more effective than dor (p = . ,p = . respectively) conclusions: we studied the differences between the mechanical effects on the egj following three different fundoplications, encompassing , , and of the esophagus. we demonstrated that there is a significant difference between dor fundoplication and nissen/toupet when distensibility was measured. there was no difference in the distensibility of the egj following a or wrap. there was, however a significant difference of effectiveness between all three fundoplications. these findings suggest that the and fundoplications have similar functionality while the wrap mechanically prevents possible reflux and support proponents of toupet fundoplication rather than nissen due to the similar functional results while decreasing the post op dysphagia and gas bloating complications. surg endosc ( ) aim: to describe patients undergoing surgical treatment of incident gastro-oesophageal reflux disease and the use of anti-reflux treatment in a danish population-based cohort. methods: all adult danes - undergoing upper endoscopy and receiving a diagnosis of gerd within days were identified. patients with previously diagnosed gerd, peptic ulcer-disease, barrett's oesophagus or cancer of the gastrointestinal tract were excluded. in this study, only patients undergoing anti-reflux surgery within two years of gerd-diagnosis were subsequently included. age, sex, charlson comorbidity index (cci), anti-reflux surgery (primary and re-operative) and endoscopic dilatation were identified using the danish national patient registry. mortality was identified using the national civil registry. pharmacological treatment of gerd (proton pump inhibitors, h \ su \/su-blockers and other prescription anti-reflux drugs) as well as use of nonsteroid anti-inflammatory drugs (nsaid) and anti-thrombotic treatment were identified using thethe danish national prescription registry. all data was linked on an individual level using the unique identification number that all danish citizen are assigned to at birth or first immigration. results: a total of first-time fundoplications were performed, hereof . % performed laparoscopically (n = ) and . % performed using open technique (n = ). at one-year followup, . % (n = ) had undergone endoscopic dilatation and . % (n = ) had undergone reoperation. the -day mortality was \ . %. patients had a median age of years ( - years) and were predominately male ( . %-n = ). a total of . % had cci (n = ). diagnoses were gerd with esophagitis ( . %-n = ), gerd without esophagitis ( . %, n = ) and gerd without specification ( . %, n = ). before initial endoscopy, , % (n = ) used at least one type of anti-reflux drug, dropping to . % (n = ) in the year after anti-reflux surgery. however, even when censoring patients with barrett's esophagus or peptic ulcer disease after initial endoscopy and patients undergoing concomitant treatment with nsaids or antithrombotic drugs, . % still used at least one type of anti-reflux drug after surgery. conclusion: in this population-based study, anti-reflux surgery was safe and lowered the use of pharmacological treatment. however, even when adjusting for competing reasons for use of antireflux drugs, . % used at least one type of anti-reflux drug one year after surgery. the new approach to perform nissen fundiplication m. paranyak, v. grubnyk surgery, odessa national medical university, odessa, ukraine nearly % of patients who undergo laparoscopic anti-reflux surgery at long-term follow-up need for surgical reintervention mostly because of hiatal hernia (hh) recurrence, wrap migration or disruption. purpose: the aim of our prospective study was to evaluate and compare several technics of wrap fixation and determine whether modified nissen fundoplication(mnf) reduce failure rate in the long term follow up. materials and methods: this was a prospective, randomized, controlled trial. from november to october one hundred and thirty-eight gerd patients who underwent anti-reflux surgery were divided into two groups. excluded criteria for our study ware diagnosed hiatal hernia (hh) type iii. in the i group which include patients we performed the following manipulations: nf was supplemented with suturing wrap to the diaphragmatic crura ( patients) on each side using two non-absorbable stitches. such technique permit us to create more symmetrical wrap. in case of weak conditions of crura or short esophagus ( patients) fundoplication wrap was sutured to the body of stomach using two non-absorbable stitches on each side. control group ( patients) underwent classic nissen fundoplication (nf) without wrap fixation. all patients were assessed before and after surgery using validated symptoms and quality of life (gerd-hrql) questionnaires, -h impedance-ph monitoring and barium-swallow. results: baseline characteristics were similar between groups. there were no conversion to open procedure or mortality. mean hospitalization was . days ± . days. at , months (range -- ) of followup, the overall rate of complications after mnf was , % ( hh reccurence) and nf , % ( hh reccurence, slipped wrap). patient in mnf group show significant improvement in gerd-hrql score, from . ± . (preoperatively) to . ± . (postoperatively) (p? \ ? . ). complete ppi independence was achieved in %. in the ii group of patients mean gerd-hrql score decline from . ± . (preoperatively) to . ± . (postoperatively), postoperative ppi treatment was necessary in %. conclusions: according to our study mnf minimized risk of slipped wrap and intrathoracic migration of the wrap and can make positive impact on reducing the failure rate of laparoscopic anti-reflux surgery. aims: comparative evidence across laparoscopic antireflux procedures does not exist. aim of this project was to identify direct comparative evidence between laparoscopic antireflux procedures and synthesize evidence using network meta-analytical methods. methods: the databases of medline, amed, central, opengrey were interrogated. pairwise meta-analyses for each pair of interventions using a random-effects model and network metaanalysis in stata was performed using the mvmetacommand and self-programmed stata routines. differences between direct and indirect evidence were explored by comparing direct and indirect estimates though computing the inconsistency factor within each closed loop of evidence. the ranking probabilities for all treatments of being at each possible rank for each intervention were computed using the mvmetacommand in stata. a hierarchy of the competing interventions was obtained using rankograms. quality of evidence was assessed using grade-nma and the cinema application. results: forty-three publications reporting on randomized trials and some patients were identified. the network of treatments formed a closed loop between °, °and anterior °; and star network between °and other treatments; and between anterior °and other treatments. laparoscopic °, °, anterior °and anterior °were equally effective in the control of heartburn and this was supported by low quality of evidence according to grade-nma. the odds for dysphagia were lower for anterior °(high quality evidence), anterior °( moderate quality evidence), °(moderate quality evidence) and proton-pump inhibitors (moderate quality evidence) compared to °. the odds for gas-bloat were lower for °and anterior °compared to °(low quality evidence). the odds for regurgitation, morbidity and reoperation were similar across treatments, albeit these were associated with very low quality evidence. anterior °had a % probability of being the best treatment in terms of dysphagia. conclusion: under consideration of treatment effect estimates, evidence quality as assessed with grade-nma and other parameters, anterior °, anterior °and °should be preferred over °. further research needs to focus on the comparison between °and °/ °. aims: we have recently demonstrated that the tension of crural closure can be reliably measured intraoperatively (alsgbi conference december ). the aims of this study were to further characterise tension at the diaphragmatic hiatus from our prospective pilot study of patients. methods: a prospective analysis was performed of patients undergoing laparoscopic hiatal hernia repair between april and december . patients underwent crural tension measurement intra-operatively. patients had a pre-operative ct scan of the abdomen within one-year of surgery. hiatal surface area (hsa) was measured intraoperatively and a sauter-fh universal digital force gauge was used to measure the tension of crural closure during cruroplasty. outcome measures included the mean tension of the crural closure and the presence of muscle splitting during the cruroplasty. results: for all patients, the mean crural tension measurement was . n and the mean hsa was mm . pre-operative ct was positively correlated with post-dissection intra-operative hsa (r = . , p = . ), however, strength of association was weak (r = . ) and ct consistently overestimated the size of hiatal defect intra-operatively (mean of differences mm , p = . ). crural tension was positively correlated with age (r = . , p = . ), hiatal height (r = . , p \ . ), hiatal width (r = . , p \ . ) and hsa (r = . , p \ . ). crural tension was correlated to the hiatal width to height ratio to assess the shape of defect and there was positive correlation (r = . , p = . ). tension was calculated for the posterior and anterior halves of the suture cruroplasty. anterior tension was significantly higher when compared to posterior tension ( . n vs . n, p \ . ) . patients had evidence of muscle splitting during the cruroplasty. the group with muscle splitting were significantly older ( vs , p = . ), had larger hsa ( mm vs mm , p \ . ) and higher crural tension ( . n vs . n, p \ . ). the lowest observed mean crural closure tension causing muscle splitting was . n. conclusion: there is now a possibility to optimise this operation with objective measures years after it was first described. initial findings suggest that crural closure up to * n could be the permissible tension threshold for suture cruroplasty and higher tension may benefit from the use of mesh reinforcement. background: endoscopic submucosal dissection (esd) and endoscopic full thickness resection (eftr) are advanced endoscopic techniques which can be time consuming using traditional endoscopic instruments. a new endosurgery platform, designed by fortimedix surgical, was developed featuring flexible articulating instruments to use in combination with a standard flexible endoscope. the platform is intended to perform endoscopic cutting, dissecting, and hemostasis. aim: evaluate feasibility of the platform in the upper gi-tract. project description: the platform was tested in a dry esophageal model as well as a second series with a porcine esophagus and stomach. the system has an external docking station affixed to the operative table to stabilize both flexible instruments for the right and left hand of the surgeon. at the tip of the endoscope, a cap containing instrument lumens is attached to allow advancing and removing the flexible instruments. the endoscope with the cap and instrument lumens attached is advanced via an overtube with outer diameter . mm. in the first series, flexibility and range of motion of the endeffectors was assessed. additionally, the ability to advance the instruments to the intraluminal target area from the docking station and along the scope was evaluated. in the second series, the functional capabilities of the system and instruments were evaluated in a porcine model. preliminary results: : in the dry model, the platform was adequately deployed to the target then range of motion was tested as well as cutting and grasping gastric wall with instrumetn triangulation achieved. the grasping forceps provided enough force to pull the mucosal wall and expose the dissection plane. in the pig model, the distal esophagus and stomach could successfully be accessed and platform deployed. esd was performed using newly designed flexible articulating scissors, dissection-hook, and graspers with good triangulation and sufficient grasping force with traction/counter-traction. the new fortimedix surgical endo-surgery platform applied to a standard flexible endoscope is feasible to perform esd. future studies are planned to determine learning curve and compare it to traditional endoscopic instruments. background: in laparoscopic surgery, we usually observe the organs in the same direction to avoid a mirror-image situation. therefore, we are unable to recognize how far the dissection has proceeded on the other side of the target organs or lesions, especially when the plane of dissection is under the mesentery or organs. this becomes a problem not to understand how far the dissection has progressed and how much more dissection is needed. aim: to solve this problem, we developed a laparoscopic device with tip illumination. project description: the device is configured by the long and narrow part made of polycarbonate resin and a battery-powered light-emitting diode to illuminate the tip by shining light through the polycarbonate resin. during the surgery, the tip of the device is inserted into the deepest part of the dissection area, and the transmitted light indicates how far the dissection has progressed. the tip of the device has a prism structure and light is emitted in a direction perpendicular to its axis. tip position can thus be more clearly identified even with insertion in the same direction as the laparoscopic view. to verify the utility of this instrument, laparoscopic surgeries were performed in a porcine model and cadavers. preliminary results: we performed some laparoscopic surgery such as the medial-to-lateral approach to the white line of the left side of the descending colon for sigmoidectomy, dissection of the posterior surface of the pancreas to the upper edge of the pancreatic body or splenic artery for distal pancreatectomy, and the separation of the anterior surface of the inferior vena cava from the liver to the area between the right and middle hepatic vein for right hepatectomy. we quickly and easily identified the deepest part of the dissection area even if identification had been difficult using other techniques such as placing gauze in the deepest position, inserting forceps into the dissection area or simply depending on the experience of the operator. background: recent advancements within surgery have seen artificial intelligence transform traditional approaches. robotic assistive devices have demonstrated particular success, as safe and cost effective, and are widely supported via industry and local government as a step closer to the future standard of practice. an example of seamless and touchless robotic assistive technology is based on touchless and interactive eye tracker glasses worn by the surgical team thereby enabling the team to perform wider surgical tasks, more efficiently and reduced human error. we introduce a perceptually-enabled, smart operating room (smart-or) based on a novel real-time framework for theatre-wide d gaze localisation in a mobile fashion. this framework enables dynamic gaze based user interaction with a robotic scrub nurse to facilitate meaningful practical integration of human and technology intra-opertively. aims: we tested participant acceptability of a novel robotic scrub nurse during simulated surgery. project description: surgeons performed segmental resection of pig colon and handsewn end-to-end anastomosis while wearing eye-tracking glasses to select surgical instruments on a screen. the robotic scrub nurse(rn) picked up and transferred the instrument to the surgeon. the study compared human nurse(hn) vs rn. gaze-screen interaction was based on a d gaze framework we developed with synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for real-time d reconstruction of the environment. nasa-tlx and van der laan's technology acceptance questionnaires were collected and analysed using anova. preliminary results: overall, teams of surgeons(st) and scrub nurses(sn) participated. nasa tlx feedback for st and sn revealed no significant difference between in mental, physical or temporal demand. importantly, st and sn reported no significant difference in task overall performance. st reported more significant frustration with rn vs hn. van der laan's scores showed positive usefulness and satisfaction scores in using the rn platform. overall, all outcomes were more positive by sn vs rn. conclusions: this is the first platform of its kind. overall, quantitative and qualitative feedback was positive. the source of frustration has been understood and we believe it can be improved by appropriately modifying robot behaviour. importantly,there was no difference on perception of performance. background: endoscopic tumor resections in the gi tract may be facilitated by more advanced instruments for dissecting and suturing. we have focused on developing an endoscopic suturing technique using a standard flexible pediatric endoscope with new, flexible instruments allowing for complex end-effector movements. aim: perform flexible endoscopic suturing using a standard flexible scope in the gi tract project description: a standard flexible pediatric endoscope and a standard gastroscope were used for testing the new technique. via an overtube, the endoscope and newly designed fortimedix surgical flexible instruments (needle holder; grasper) with a diameter of mm were inserted into the esophagus. suture training was performed in an experimental setting in a box in the dry lab and porcine model . the flexible needle holder was advanced into the esophagus next to the scope, and a suture of the esophageal wall was performed, followed by extracorporeal knot-tying with knots. the test series consisted of training with both resident trainees and surgeons to evaluate the learning curve. each participant performed sutures on the box model and in the pig-esophagus. feasibility, duration of the different steps, and handling problems were documented. preliminary results: test series (box training on esophago-gastric explant) with prototype showed good feasibility. suturing was possible in out of attempts. median duration for single bite: min ( - ); knot-tying: min ( ) ( ) ( ) ( ) ( ) ( ) ( ) . test series (training in pig-model) with prototype showed improved feasibility with better flexibility of instrument shaft: median duration of double bite: min ( - ); knottying: min ( ) ( ) ( ) ( ) ( ) , overall duration intraluminal esophageal double bite suture and closing with knots: median duration: min ( - ). the new flexible endosuture instruments seem feasible to use and perform dependable intraluminal sutures. the training period and learning curve is short and the objective is to apply this system clinically for closure of perforations and fistulas. school of mechanical and aerospace engineering, nanyang technological university, singapore, singapore; general surgery, national university hospital, singapore, singapore; gastroenterology, national university hospital, singapore, singapore; surgery, chinese university of hong kong, hong kong, hong kong background: ideally, endoscopic suturing should mimic surgical closure as the latter is stronger than most endoscopic closure devices. however, endoscopic suturing is challenging due to the confined endoluminal space and lack of dexterity of current endoscopic instruments. we have developed a novel robotic suturing device to overcome these problems. aim: this animal study aims to demonstrate the feasibility of this device in closing perforations. method: the trial was conducted on an anaesthetized live pig. a double-channel colonoscope was first inserted into the rectum. following saline lift, a mm submucosal incision was created in the rectum to simulate a perforation. the robotic suturing device and grasper were inserted into the two colonoscope channels, allowing the endoscope to remain in position for tool exchanges or needle reloading. both the effectors were intuitively tele-operated by the user via a robotic master console. this robotic suturing device manipulated a curved, double-point needle (with a cm - vicryl suture) to penetrate tissues at desired orientations. the needle could be switched between both jaws of the device through a locking mechanism. this facilitated passing the needle through tissues to form stitches or through suture loops to form surgical knots. the articulated joints and five degrees of freedom allowed dexterous steering to reach targets and triangulation with other tools in a confined space. the robotic grasper facilitated handling of tissue and suture. result: a total of four running stitches were performed and secured with a surgical knot by passing the needle through suture loops. the suture was cut and the needle was removed by the robotic grasper through the channel. min and min were required to stitch and tie the knot respectively. there was no complication. conclusion: our novel endoscopic robotic device can suture perforations resulting from complex endoscopic procedures. as our suturing method is similar to laparoscopic and robotic suturing, closure using our device is expected to be as strong as a surgical through-and-through closure. when developed further, this device can be used to close full-thickness resection sites and orifices in transluminal endoscopic surgery. modelling a collaborative robot with the ieee sdc standard for combined focused ultrasound and radiation therapy j. berger, m. unger, l. landgraf, a. melzer medical faculty, university hospital leipzig, innovation center computer assisted surgery, leipzig, germany background: surgical robotics require a smooth integration into the operating room (or) . for this propose the ieee sdc(service-oriented device connectivity) standard has been developed in the or.net project. in preparation for a combined focused ultrasound and radiation therapy (fus-rt) we have shown concepts and evaluations to position ultrasound and interventional devices with collaborative kuka arms. however, the safe and intraoperative cooperation with multiple different or-devices (e.g. an irradiation unit) requires a more sophisticated exchange of the robot's information and functionality. aim: to realize a safe clinical integration, the aim of this work is to implement and evaluate a dynamic connection between the kuka robots and other devices using the vendor-independent sdc communication standard. project description: a kuka lbr iiwa r robot (kuka ag, germany) was modeled inside the sdc standard for medical device communication. the interconnection with other devices was implemented and evaluated on a mobile platform to position a clarius l wireless ultrasound transducer (clarius mobile health corp, canada). all necessary information of the robot was represented in the medical device description of the sdc standard to be shared via network. for each joint of the robot arm the position, torque, stiffness, damping, velocity and functional-states were represented, resulting in a total of parameters. the software was implemented in c ?? on a standard pc accessing the kuka controller cabinet with ros (robot operating system) via ethernet. the accessibility of each parameter, as well as activation commands for planning and movement were tested with an sdc-consumer application. preliminary results: the sdc-provider functionality of the robot was successfully implemented, allowing for dynamic changes of the robot state during interventions. all appliances (sdc standard compatible) in the robots network can react to state changes and send movement and planning commands to the robot via activations. after testing, % of the defined parameters are safely accessible. implementing the medical device communication for the kuka robot enables its integration into any networked operation room that supports the sdc standard. it is, therefore, ready to be set up and evaluated for the application of fus-rt in a clinical environment. background: assessment of perfusion of the left colon with fluorescence during anterior resections for cancer changes surgical decisions in up to % of cases. use of fluorescence has been shown to be associated with lower leak rates, and improved short-and long-term outcomes with reduced costs. given the high incidence of colorectal cancer, fluorescence-guided perfusion assessment could be of great importance in contemporary surgical practice. however, there is currently no standardisation of this technique which represents a significant limitation to widespread adoption. aim: to standardise fluorescence-guided perfusion assessment in rectal anterior resection through a computer vision algorithm. project description: videos were collected by a single surgeon in a referral centre for colorectal cancer treatment. perfusion assessment was used before proximal colon division to identify the best location for transection. a bolus of indocyanine green was injected intravenously and a near-infrared camera used to assess perfusion through fluorescence. photographs of fluorescent imaging of the colon were analysed using a non-supervised learning algorithm called 'k-means clustering'. the first step was to digitally subtract all background pixels, leaving only the area of interest of the colon. this area was then subsegmented into 'clusters' corresponding to perfused and nonperfused areas. a mathematical model was applied based on the sub-clusters centres to select the area for transection with optimal perfusion of the proximal colon. preliminary results: representative images of proximal colon under perfusion assessment were presented to expert surgeons. the optimal point for transection was selected based on their clinical judgement on previously delimited areas indicated by random letters. this was compared with the results from the automated segmentation using the algorithm ( fig. ). the area identified for section by the algorithm included the area selected by the expert surgeons in . - % of test cases. these results need to be further validated due to high risk of overfitting. next steps include the collection of multicentre data with a standardised fluorescence perfusion assessment. after robust training, the algorithm will be validated on real-time clinical data to ensure improved outcomes for patients, which is our ultimate goal. background: endoscopic submucosal dissection (esd) is a flexible endoscopic technique that allows for an en bloc removal of lesions of the gastrointestinal (gi) tract. these procedures are typically time consuming due to the difficult control of the tools, and they often require around min for removing lesions, that can reach - cm in diameter. the probability of intestinal perforation exceeds % and the hemorrhage risk ranges from . % to . %. a flexible robotic endoscope may offer a solution to overcome these limitations, by improving the degrees of freedom (dof) and operational efficiency. aim: within this clinical panorama, the aim of this project is presenting the development of a novel miniaturized robotic device to be coupled to the tip of a traditional endoscope for the surgical dissection of gi neoplasms. project description: the robotic platform consists of the miniaturized robot, the actuator housing (hereafter called external platform), the control unit and the master console (i.e.,two geomagic touch phantom) to allow the user driving and control (figure a ). during the operation, one surgeon stands close to the patient to maneuver the endoscope for exploring the gi tract and reaching the target area. another surgeon operates the miniaturized robot through the master console, carrying out the surgical procedure. the robot has been designed to be coupled to the tip of traditional flexible endoscopes of . mm in diameter. it exploits the flexibility of the endoscope for navigation through the intestine and integrates two-active robotic arms (i.e.,cautery and gripper) extending the dofs, and thus enhancing the efficiency during complex tasks such as manipulation and surgical tissue dissection. furthermore, the endoscope provides the optical system for visual feedback and one working channels for conventional instruments. preliminary results: firstly, a mock-up that faithfully reproduces the miniaturized robot has been realized using a d printer machine (projet mjp , d system, inc.) to verify the feasibility of the design solution. after verifying the potentiality of the d printed prototype, a final device, with the same features (i.e.,dof and geometry) of the d printed prototype, has been designed, fabricated and assembled ( figure b ). background: virtual and augmented reality has been widely used in many fields mainly for entertainment purposes. we think that it could be beneficial to use augmented reality in medical practice. aim: the aim of this study was to evaluate usefulness of d holographic images of patients anatomy displayed using augmented reality goggles during endovascular aortic repair (evar). project description: one of the major challenges during endovascular procedures is working on two dimentional x-ray images of three dimentional vascular anatomy. using d holograms of patients anatomy could be beneficial during the evar procedure and could make the orientation in vascular anatomy easier for surgeon. we performed two endovascular aortic repairs with the assists of microsoft hololens -smart glasses using augmented reality. we used carna life application created by polish company medapp. it was one of the first use of holograms during vascular procedures in the world (second and third stent-graft implantation using holographic imaging in the world). results: two patients with abdominal aortic aneurysms, -years old male and -years old female, were operated on. holograms of patient's anatomy made from preoperative angio ct scans by polish company medapp were displayed during the procedures using microsoft hololens. holograms could be displayed in any place and configuration using augmented reality, which means that the images did not interfere with the surgeon's field of vision. microsoft hololens use voice commends which permits the surgeon staying sterile. stent-graft implantations were successful. both patients were discharged three days after the procedure and the hospitalization was uneventful. seeing precise patient's vascular anatomy reconstructions in three dimention certainly helped us to navigate in a vascular tree. we believe that in the future this technology would enable to reduce the operation time and need for radiation. background: interaction with electronically controlled operating room (or) systems embedded in modern surgical environments is everyday practice for surgeons performing minimally invasive surgery (mis). while there is a non-sterile operating nurse available in the or, capable of interacting with these systems upon request by the surgeon, this indirect control is mostly slow, prone for error and disrupting surgical workflow. facing an unanticipated and unwanted outcome may cause distress emotions. distress emotions are undesirable when performing surgery, since they may impact available cognitive workload. furthermore, they may result in negative communication, hampering or-team empowerment and effective leadership. both factors are known to negatively influence quality and safety in the or. aim: the aim of the tedtrial is to investigate what setup best enables surgeons to interact with the endoscopic operating room setup during surgical procedures. as a result, disruptions of workflow, delays and errors may be reduced. outcome parameters will be objectified using medical data recorder (mdr) derived output and biometric analysis using hexoskinÓ. subjective evaluation of outcome parameters is done using questionnaires. project description: the tedcubeÓ system is a plug-and-play device enabling wearable sensors to act as a wireless alternative for a regular computer mouse, therefore enabling direct hands-free and sterile control of the or. the study is an observational trial with three different arms: intervention group ) direct interaction by surgeon with or environment using tedcubeÓ and myo tm armband, intervention group ) direct interaction of surgeon with or environment using tedcubeÓ and plantronicsÓ wireless microphone headset. the third arm is the control group using indirect interaction of surgeon with or environment using third-person computer interaction. main endpoint of study is the number of workflow disruptions due to the operation of laparoscopic or equipment. secondary endpoints are error rate, delay, team communication, subjectively reported frustration and satisfaction with the system and objectively measured stress as symptom of frustration and anger as distress emotions. preliminary results: primary and secondary endpoints of study are compared among groups. it is anticipated that reduction of miscommunication, error and delay may result in a reduction of distress emotions. trial start is expected q . anticipating the automated intraoperative tissue recognition: intraoperative tissue classification using hyperspectral imaging and machine background: iatrogenic injuries may occur despite a sound expertise in surgical anatomy. hyperspectral imaging (hsi) is an emerging optical method, combining the use of a camera system with a spectrometer. hsi analyzes optical properties of tissues and acquires d data sets with two spatial dimensions (x, y) and one spectral dimension (?). the data sets contain information about tissue physiology, composition, and perfusion. those spectral features coupled with machine learning algorithms might allow for automatic tissue recognition. aim: assessing the ability of an hsi-based machine learning to discriminate the hyperspectral features of different tissues during neck and abdominal surgical procedures. methods and procedures: fourteen pigs underwent laparotomy (n = ) or neck dissection (n = ). twenty data sets were acquired in vivo from abdominal organs and from neck structures by means of a customized hyperspectral camera (diaspective vision, germany). different anatomical structures were manually outlined by a surgeon using an image manipulation software (gimp). each pixel contained a hyperspectral curve and each curve was composed of bands (from to nm with a nm resolution). the curves were normalized using the standard normal variate method. a logistic regression machine learning (ml) algorithm was used to train the model to discriminate tissues, based on the hsi spectral features. the efficacy of the prediction model was tested using the k-fold (k = ) cross-validation. results: a large number of tissue-related hyperspectral curves could be extracted ( thyroid, vagal nerve, fatty tissue, cartilage, carotid artery, muscle, carotid vein, portal vein, biliary tract, gallbladder, hepatic artery, pancreas, duodenum, abdominal adipose tissue). the algorithm used min to 'learn' all data sets, and prediction was provided as an immediate output. overall, prediction accuracy was and % for neck and abdominal structures respectively. in particular, biliary ducts could be identified with a % accuracy and the vagal nerve with an % accuracy (see figure for details). background: a gaze-controlled robotic endoscope is innovative technology with myriad potential applications in the rapidly advancing field of flexible endoscopy. improvements to the current flexible device to allow examination of the gastrointestinal tract whilst minimising procedural discomfort and complications are desirable. aim: to use a gaze contingent framework to manipulate a flexible endoscope through a simulated upper gastrointestinal tract (ugit) model. description: a flexible gastroscope (karl storz pks) was attached to a ur axis robotic arm (universal robots), mounted onto a rail and placed on top of a surgical table. two cogwheel shaped dials were d printed and placed onto the up/down and left/right wheels on the head of the gastroscope ( figure ). robotization of these controls was achieved by using two motors (dynamixel rx- f) to steer the distal tip. this system allows users to operate a robotised flexible endoscope using gaze control. gaze interaction with the screen was based on a d gaze framework we developed with the synergy of conventional wearable eye-tracking, motion capture system and fixed in space rgb-d cameras for d reconstruction of the environment. users are able to control endoscope movements without handling the device. the distal tip of the gastroscope was controlled using eye gaze technology. the ur robot was used to enable shaft rotation (initiated by fixed head movements) and linear movements were triggered using a joystick handle (up for forward movement, down for endoscope withdrawal). pause and retroflexion of the endoscope are achieved by moving the joystick left and right respectively. users were asked to navigate an endoscope through an ugit model (chamberlain group) simulating a diagnostic gastroscopy using gaze control and targeting ten points scattered through the stomach. results: four expert endoscopists and one novice used gaze control to successfully navigate a gastroscope through a simulated ugit. all were able to intubate the oesophagus and accurately locate ten targets placed in the fundus, body, antrum and pylorus of the stomach. conclusion: gaze control endoscopy is a feasible concept. it allows ergonomic, user-friendly and intuitive control whilst maintaining the benefits of a flexible endoscope. background: image-guided needle biopsies and histopathological evaluation are the gold standard for the diagnosis of liver neoplasms. most often, however, these are reserved for suspicious, but not diagnostic, situations. radiomic may help to characterize tumor biology by correlating imaging features with relevant tumor-biology information. features derived from radiomic analysis may provide complementary information to support clinical decisions, especially in situations where tissue analysis cannot be performed or is inconclusive. aim: the goal of our technology is to exploit computational capabilities for image analysis in order to identify radiomic features useful for characterizing liver lesions and to identify relevant information related to patient prognosis. project description: patients derived from an internal database and patients randomly extracted from the cancer archive liver dataset were included in this study. lesions were extracted from those volumes using expert annotations ( secondary vs primary; well differentiated vs non-well differentiated). lesions were then split into training and testing sets. first order statistical features were computed and a lasso regression step was performed to reduce the number of features. both logistic regression and random forest models were built using cross-validation to predict the target classes on the test set. preliminary results: only features namely the energy and the volume of the lesion were sufficient, when combined in either model, to predict the differentiation grade on the test set with an f -score of . (± . ). we are currently working on the addition of higher order statistical features to the analysis in order to differentiate primary from metastatic tumors and identify complementary features that may assist clinical decisions in patients with inconclusive hepatic lesions. objective of the technology or deviceideally, the use of medical simulators could provide trainees with initial background information about indications for procedures, endoscopic technique, and early hands-on training experience that could shorten the initial critical learning curve. rationale for using ex vivo models is that in the beginning of the learning curve, the most important issue is having an initial exposure to the basic movements and maneuvers. our objective of is to create a stomach model from renewable polymer, which would closely simulate normal human stomach with gastric pathology for endoscopic diagnostic or interventional skill acquisition/evaluation. description of the technology and method of its use or application stomach model is based in several steps; the first one is in the in-silicodesign of the overall shape, after that we d print the positive two halves of it. the interior detail is obtained shaping the d printer parts with ceramic putty. once concluded, this elaborated part will serve as a template in order to build injection bleeding moulds. in the injection bleeding moulding a mesh is placed between layers in order to provide structural attachment points as stiches or several pathological models that will be incorporated after the casting process. we have developed for these instance polyp moulds, fistulae structures in order to attach endoscopic clamps. the two halves are closed once the pathological models are placed inside via a thermic-fusing and stitching creating a leak proof stomach model. preliminary results if available: our models were evaluated by international experts in ircar/ihu france in interventional endoscopy course and were favorable accepting for next trails in these prestigious institutions. conclusions: future directionsa new endoscopic training model of stomach was made and will be evaluated and validated for feasibility in mastering diagnostic and interventional endoscopic skills. clinical trials will be necessary to compare the ability of the simulator to perform training compared with traditional methods of training in endoscopic procedures. background: endoscopes are the eye of surgeons in minimally invasive surgery (mis). conventional endoscopes are mostly chopstick-like and are steered by the assistant. this limits the field of view and results in issues such as endoscope-instrument fencing, surgeon-assistant coordination. existing robotic endoscope holder enables solo-surgery, however endoscope remains blocking the instrument movement and impairs the operational safety. flexible endoscope such as the endoeye provides angulation at the tip and could enlarge the field of view. however, its steering the view is much more complex compared to the rigid endoscope. aim: to provide an intuitive robotic flexible endoscope with enhanced safety. project description: in this work, we present a robotic flexible endoscope for mis with enhanced safety. in the proof-of-concept system, it contains a flexible endoscope module and a robot manipulator. the endoscope contains a proximal rigid shaft and a distal flexible bending section. it is installed onto the patient side manipulator (psm) of the da vinci research kit (dvrk). visual servoing is adopted to achieve autonomous instruments tracking. during the tracking process, movements of the manipulator as well as the endoscope are minimized to save space for the operation and avoid instrument-endoscope fencing. the endoscope could also be controlled by the surgeon. a foot pedal is used to switch between the tracking-mode and control-mode. preliminary results: a prototype was developed and tested experimentally. in tracking a volume of * * mm , the spaces required by the flexible endoscope are . % (inside the trocar) and . % (outside the trocar) of that occupied by the rigid endoscope. evaluation with the fls tasks involved subjects. all of the participants completed the tasks under the tracking-mode without failure. in the ex-vivo test with porcine stomach, the endoscope successfully guided the detection, dissection and knotting autonomously. background: fluorescence imaging allows to visualize deep-seated anatomical structures, using a deeper tissue penetration of near-infrared (nir) compared to visible light. the most commonly used fluorescent substance, indocyanine green (icg), is not naturally excreted by the urinary system and requires retrograde stent placement and injection. lighted catheters have been proposed to help visualise the ureter. fluorescent dye-coated ureteral catheters could well represent a more effective and less expensive solution. icg is unsuitable for coating materials. aim: to develop a stable fluorescent coating for catheters to be used intraoperatively, working in the same nir window as icg, to facilitate its use with clinically available systems. project description: the coating was developed based on poly(methyl methacrylate) (pmma), a biocompatible polymer, and on specifically designed fluorescent dyes exhibiting icg-like optical properties. three nir dyes (substances a, b, and c) were tested in order to find the optimal one, in terms of fluorescence signal intensity, and were compared to icg in a polymer form and to an icg-based reference card (green balance tm ). the fluorescent coating was applied onto common ureteral stent materials: hydrophilic-coated ultrathaneÒ, silicone-coated latex, and pvc. the coating process involved cycles of immersion into the respective dyes blended in pmma polymer (icg, substances a, b, and c), followed by a drying phase. the various tubes were partly inserted into a porcine ureter, next to the icg-based reference card. images were taken in white light and nir modes using the d-light p camera system (karl storz), at a fixed camera-to-target distance. the fluorescence signal intensity was measured for the different regions of interest (each material/coating combination inside and outside of the ureter, reference card) using proprietary software and normalised against the reference card. preliminary results: the signal intensity was significantly higher for all new substances as compared to icg. substance a showed the strongest fluorescence signal intensity among the tested coatings in all tested conditions and materials and was identified as the ideal candidate to undergo further evaluation and in vivo testing. background: endoscopic resection(er) of early gastric cancers provides tremendous patient advantages. however, post-resection findings of deeper sub-mucosal(sm) and/or lympho-vascular invasion can necessitate a second, surgical intervention. we propose that pre-resection evaluation of the submucosal architecture under the tumour can provide critical information for staging and operative planning. we evaluate three techniques to assess the submucosal architecture underlying the gastric mucosa in a pig model. aim: to evaluate three needle-based methods of evaluating the sm before er. project description: acute pigs were used. a simulation of sub-mucosal tumours (endoscopically and eus visible bleb) by injecting the sm with cc of undyed nac. a linear eus was use for all procedures. the tumours were marked and labelled according to geography. methodology: after creating the tumours, anterior lesions were evaluated using the following g needle-based modalities: confocal microscopy(cm) using the through-the-needle cellvizio (mauna-kea) system; mini-biopsy(mb) using the micro-biopsy forceps moray (us endoscopy) and fine-needle biopsy(fnb). results: cm examinations were video recorded in all a positions. submucosal vascular visualisation was possible in all cases, excellent in / . mb was performed in lesions with a total of biopsies obtained from each lesion (total = ). fnb was performed once in the anterior lesions and twice in the posterior lesions with different needle brands. therefore, there was a total of biopsies collected. passes were performed in each biopsy (total = ). each pass constituted - insertion/withdrawal movements combined with fanning, slow pull technique, no suction and suction ( - cc air negative pressure) to collect the material. all material were sent to an animal anatomo-pathologist blinded to the acquisition method. mean time of confocal examination was min sec ( ' ' '- ' '') . mbtook a mean time of min and fnb was a mean of min for each biopsy. cm identified different patterns of vessels in relation to the probe position (superficial/reticular, middle cross-roads or deep/longitudinal). conclusion: eus-fnb, cm and mb are three potential methods to assess the sub-mucosal space underlying the gastric mucosa. cm offered the most architectural information but required more time to perform. these method's may have a role in better staging patients for appropriate er. background: the overall and disease-free survival of patients with rectal cancer is dependant on its staging, and adequate selection of the treatment strategy. mri has a proven efficacy in rectal cancer local staging and recognition of the adverse prognostic features. however, it can be difficult to utilise it as a navigation tool for surgeons, as it represents a complex three-dimensional pelvic space with a series of individual two-dimensional images. d image reconstruction has been successfully adopted in other surgical fields to overcome these limitations. aim: our primary aim is to develop a bespoke automated generation of patient-specific d pelvic models, which will improve surgical planning and navigation, patient interaction and surgical education. true-size, rotatable d models will offer a more realistic three-dimensional representation of the surgical space and its complex relationships, allowing for a more confident surgical rehearsal and potentially better utilisation of minimally invasive techniques in rectal cancer management. our secondary aim is to develop a large multipurpose database of the d models of male and female pelvis in health and in the disease. project description: our multidisciplinary team consists of colorectal surgeons, radiologists specialising in pelvic mri imaging and computer scientists. virtual d pelvic models are generated based on standard d dicom mri images routinely used for rectal cancer staging, which guarantees the high fidelity of cancer delineation. segmentation of the pelvic anatomy is performed with the use of itk-snap, an open-access, multi-platform software. machine learning technology is then employed to automate the d model generation, making it time-efficient, allowing for its clinical application. preliminary results: in the initial stage, using the manual segmentation, we have created ten models of normal male and female pelvic anatomy. a good inter-rater agreement level was found, which proves reproducibility of the approach applied. various machine learning algorithms are being explored to fully automate the process of d model generation, which will allow for their use in clinical practice and in development of the d colorectal database. the technology will be further implemented in creation of dynamic models of functional pelvic floor disorders. surgery, toho university omori medical center, tokyo, japan; surgery, neuchâtel hospital, neuch tel, switzerland background: laparoscopic gastrojejunostomies are time-consuming and require a specific training. alternatively, sutureless anastomosis can be achieved by means of endoscopically delivered magnetic rings. objective of the study: assessing the feasibility and reproducibility of an endo-laparoscopic gastrojejunostomy technique, using magnets coated with a fluorescent biocompatible polymer. methods and procedures: four pigs ( acute, survival models) and one cadaver were included in this study. the anastomotic device was composed of two magnetic rings ( x x mm; attraction force newton), each one attached to a cm long thread. the distal ring was inserted endoscopically into the first duodenum, and the extremity of the thread was clipped to the gastric mucosa. twenty-four hours later, a two-port laparoscopy ( mm, mm) was performed, using a near-infrared (nir) laparoscope (d-light-p; karl storz). the magnet's position in the jejunum was detected thanks to the transluminal fluorescence of the dye. magnetic interaction with the metallic tip of the laparoscopic grasper allowed to catch the ring and bring the bowel loop to the future anastomotic site on the gastric wall. simultaneously, the proximal magnet was delivered to the gastroesophageal junction using a flexible endoscope. the magnet was carefully advanced into the stomach allowing precise connection with the distal ring. in one cadaver the procedure was repeated. the sole variation was that, in order to reach the second jejunal loop, the distal magnet was placed using a gastroscope inserted through a transgastric port. in two acute animals, the distal magnetic ring was introduced into the jejunum via an enterotomy. the anastomotic procedure (from the distal magnet detection via fluorescence to the magnetic connection using a hybrid approach) was reiterated times. survival animals were followedup for days and underwent control endoscopies and ct-scans. results: the procedure was easy to standardize and reproducible, with a mean anastomotic procedure time of . ± . min. there were no technical problems and magnetic connection could be precisely directed in all cases, at both the anterior and posterior gastric wall. no complications occurred during the survival period and the anastomoses were patent by day . transluminal fluorescence allowed for a rapid detection of the magnet. colorectal cancer is the fourth most common cancer in high-income countries counting [ . deaths worldwide. survival rate reaches % in case of early diagnosis, falling down to % in case of advanced stage. conventional colonoscopy screening is limited by invasiveness, pain and often need of sedation. wireless capsule endoscopy enables inspection without discomfort, but passive locomotion often leads to incomplete and/or false negative results. the european endoo project (grant agreement ) aims to develop a novel system that overcomes most of the drawbacks of conventional colonoscopy, maintaining accurate and reliable diagnosis and therapy. the system is composed of an active robotic platform that magnetically drives a soft-tethered capsule; magnetic guidance is achieved through the magnetic localization of the capsule in combination with a closed-loop control that maintains an optimal and safe link between the capsule and the magnetic end-effector. a stereoscopic camera is integrated in the capsule for enhanced diagnosis though d reconstruction and automated detection of lesions/pathologies. the different modules of the endoo medical platform are illustrated in the figures. the robotic guidance systemconsists of an anthropomorphic manipulator that controls the capsule through an external permanent magnet. the robot, positioned on a dedicated trolley, is equipped with sensors for performing safe human-robot collaboration. the medical workstationincorporates: screens, buttons and pedals for visualization and command initiation, a joystick for system teleoperation and a back-end for fluidic control and data communication. the soft-tethered capsuleembeds an internal permanent magnet, magnetic sensors, an accelerometer, white and infrared illumination and an hd stereoscopic vision system with two wide-angle customized optics. a controller serves as the main control unitfor performing real-time communication and closed-loop control of the robot, localization system, capsule and physician commands. the synergistic cooperation of academic, industrial and clinical partners within the project allowed to develop and validate the system in in-vitro \/i [ , exvivoand preliminary cadaver sessions, performing comparisons with state-ofthe-art commercial colonoscopes. in conclusion, the endoo medical platform provides: reduced procedural pressures, user-friendly procedures, similar functionalities and performances of commercial devices, comparable procedural times and considerably lower costs with a new painless approach. background: this study is aimed at the comparison of the process of manual and robotic-assisted positioning of the electrode performing radiofrequency ablation with the usage of multifunctional robot-assisted surgical platform. under the control of the surgical navigation system. the main hypothesis of this experiment was that the use of a collaborative manipulator will allow to position the active part of the electrode relative to the center of the tumor more accurately and from the first attempt. we also check the stability of the electrode's velocity during insertion and consider some advantages in ergonomics using the robotic manipulator. methods: sphere-shaped tumor phantoms measuring mm in diameter were filled with contrast and inserted in cow livers. livers were used for the robotic experiment and an equal quantity for manual. the livers were encased in silicone phantoms. analysis of ct data gave the opportunity to find the entry and the target point for each tumor phantom. this data was loaded into the surgical navigation system that was used to track and record the position of the rf-electrode during the operation for further analysis. results: standard deviation of points from the programmed linear trajectory totaled in the average . mm for the robotic experiment and . mm for the manual operation with a maximum deviation of . mm and . mm respectively. standard deviation from the target point was . mm for the collaborative method and . mm for manual method. the average velocity was . mm/s for the manipulator and . mm/s for the manual method, but the standard deviation of the velocity relative to the value of the average velocity was . mm/s and . mm/s respectively.thus, in two criteria out of three, the manipulator is superior to the surgeon, and equality is established in one. surgeons also noticed advantages in ergonomics performing the procedure using the manipulator. conclusions: this experiment was produced as part of the work on the developing of the robotic multifunctional surgical complex. we can confirm the potential advantages of using robotic manipulators for minimally invasive surgery in case of collaborative practice for cancer treatment. surg endosc ( ) background and aims: laparoscopy has reduced tactile feedback compared to open surgery. in neuropsychological literature there is increasing evidence that visual and haptic information converge to form a mental representation of an object. through the combination of these inputs, this representation is believed to be more refined and robust. we investigated whether tactile exploration of a lifelike anatomical object before executing a laparoscopic action on this object in a laparoscopic box trainer improves performance of this action. description: a randomized prospective cohort study with two groups (a ? b) of ten laparoscopically naïve medical students was conducted. we compared the groups for baseline characteristics and performance, using a basic laparoscopic task (post and sleeve). to investigate the effect of haptic exploration, students performed ten repetitions of a laparoscopic needle action on a lifelike silicone caecum model (applied medical, rancho santa margerita, usa). group a did a pre-test visual exploration of the model. in group b manual exploration of the anatomical model was added to the visual exploration before executing the task. the box trainer was equipped with the forcesense tm (medishield, delft, the netherlands) system for skill assessment using objective force, motion and time parameters. results: baseline characteristics and-laparoscopic performance were comparable (p [ , ) . performances of trials on the anatomical model were captured and parameter outcomes were compared between groups. significantly less force (maximal force, maximal impulse, mean force and force volume) was exerted by the 'touch' group (p \ . ) (fig. ). this group also completed the task with less distance travelled by the instruments (p \ , ). there was no significant difference in time needed to complete the task (p = , ). conclusion: this study showed that, when performing a laparoscopic task on an anatomical model, pre-task haptic exploration of the model results in the use of significantly less force and less movement. adding haptic exploration to a laparoscopic training curriculum could therefore result in more efficient and more refined learning of laparoscopic actions. this, in turn, could lead to better, quicker and safer performance of laparoscopic operations. . esophagogastroscopy was performed before gabe and -week post-procedure assessing gastric abnormalities. weight and fasting plasma ghrelin were obtained at baseline, -, -, -and -months post-index procedure. after months, the sham group was unblinded and received gabe. both gabe and sham crossover to gabe groups were followed for months and received lifestyle therapy (behavioral-diet education). preliminary results: gabe was successful in all patients with no serious complications. significant, progressive weight loss was observed at and maintained at months. ghrelin in gabe group decreased by % ( . pg/ml) compared to baseline and months levels. weight-loss was approximately . % greater in the gabe group versus sham at months ( table ) . itt = intent-to-treat, pp = per-protocol analysis preformed using independent-sample t-test and à paired-sample t-test conclusions: gabe using eles is safe, accompanied by significant and so far maintainable weight loss. gabe using the eles demonstrated a reduction in ghrelin levels. aims: transanal total mesorectal excision (tatme) is the latest colorectal approach that continues to be in the spotlight. this study aims to describe the technique in depth by identifying and understanding technical advantages, errors and adverse events. methods: detailed video analysis using observational clinical human reliability analysis (ochra) was completed on clinical tatme cases performed by international surgeons. error frequency and error pathways leading to adverse events were described. tatme expert surgeons were interviewed and engaged in a workshop to elicit error-reducing mechanisms. results: overall technical errors and adverse events per procedure on average occurred ± . (range -- ) and ± . (range - ) times respectively. inadequate insufflation and poor camera optics were the most frequent set-up problems. instrument handling errors consisted most commonly of excessive grasper movement during the pursestring phase ( times total), inappropriate force applied ( times) with the energy device during the rectotomy, inappropriate force with the grasper ( times) and excessive movement with the energy device ( times) during tme dissection. incorrect dissection planes were created during tme dissection mostly due to insufficient retraction ( times) which didn't allow adequate exposure of the tissue planes. the most frequently occurring consequence was bleeding (mean: times per procedure). rectal perforation ( cases), vaginal wall injury ( cases), and prostatic injury ( cases) were also recorded. adverse events regularly occurred as a result of poor set-up/exposure, inappropriate retraction and/or instrument movement and incorrect plane surgery. error-reducing mechanisms and 'technical tips' describe specific steps and actions, both set-up/equipmentrelated and technique-related, that aim to prevent errors from occurring and avoid adverse consequences. ochra and individual feedback with error-reducing mechanisms developed by this study have been implemented into the national training programme for tatme. conclusion: tatme is an advanced complex procedure during which technical errors and their consequences are not infrequent. tatme requires knowledge of anatomy 'bottom-up', familiarity with its specialised equipment and technical skill working in a narrow space. appropriate structured training and mentorship are therefore recommended. surg endosc ( ) objective: insufficient vascular supply is one of the main causes of anastomotic leak in colorectal surgery. icg has been shown to provide information on tissue perfusion, identifying a well-perfused location for colonic and rectal transections and thus possibly reducing the leak rate. objective of this study is to evaluate the usefulness of intraoperative assessment of anastomotic perfusion using intraoperative indocyanine-green dye (icg) angiography in patients undergoing left-sided colon or rectal resection with colorectal anastomosis. methods: this randomized trial involved patients undergoing laparoscopic left-sided colon and rectal resection randomized : to intraoperative icg or to subjective visual evaluation of the bowel perfusion without icg (clinicaltrials.gov nct ). the primary aim was to assess whether icg angiography could lead to a reduction in anastomotic leak rate. secondary outcomes were possible changes in the surgical strategy and postoperative morbidity. results: after randomization, patients were excluded. accordingly, patients were included in the analysis; in the study group, and in the control group. icg angiography showed insufficient perfusion of the colic stump, which led to extended bowel resection, in cases ( %). an anastomotic leak developed in patients ( %) in the control group and in patients ( %) in the study group (p = n.s.). conclusion: intraoperative icg fluorescent angiography can effectively assess vascularization of the colic stump and anastomosis in patients undergoing colorectal resection. this method led to further proximal bowel resection in cases, however its role in reducing anastomotic leak rate should be studied in further research. endoscopic sleeve gastroplasty (esg) is a promising endoscopic bariatric procedure carried out with the application of transmural sutures resulting in a gastric reduction and gastric shortening. sutures are placed in u shape fashion, from the incisura to the fundus, which is preserved, using an over the endoscope suturing platform (overstitch, apollo endosurgery, austin, texas, usa). the choice of right lankmarks for suturing the gastric wall is extremely important for the efficacy and safety of the procedure. flexible endoscopy suffers from little anatomical reference points. correct spatial relation to precisely target the insertion of the helix device used for retraction and correct orientation of the full thickhness tissue bite require a good undrestanding of the anatomy of the stomach and sourrounding organs including vascular structures that could be inadvertently injured (left lobe of the liver, gallbladder, spleen, short gastric vessels, pancreas, transverse colon). surgeons by training can 'see' the anatomy beyond the gastric wall and undrestand whether they work in a safe layer or whether an underlying structure should be spared. this video illustrates all the potential risks realted with a wrong chioce of endoscopic landmarks when performing esg with respect to gastric and abdominal anatomy. introduction: central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are technically demanding procedures in minimally invasive approach because of difficult expoure and extensive parenchymal transection planes. with limited robotic instruments including absence of cusa, these procedures have been rarely perfomed by robotic approach. method: consecutive robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy were performed. patients were all males and were , , and -years-old, respectively. pathologic diagnoses were all hepatocellular carcinomas of each . , . , and . cm diameter. operative settings were identical for the three kinds of procedure. the patients were placed in supine with a reverse trendelenburg and right side elevation. umbilical -mm camera port, three -mm ports and additional -mm assistant port were used. glissonian approach and icg fluorescence image clearly demarcated the resection planes. parenchymal transection was performed using the maryland bipolar dissector and harmonic scalpel. the rubber band self-retraction method and third arm of robot system helped for stable and excellent exposure of surgical planes result: there were no conversions to laparoscopic or open surgery. the operative time was , , and min and estimated intraoperative blood loss was , , and ml. the pathologic surgical margin was . , . , and . cm. the length of stay after surgery was , , and days and there were no postoperative complications. conclusion: robotic central bisectionectomy, anterior sectionectomy, and posterior sectionectomy are still demanding procedures with long operative time. however, these procedures could be performed safely in regard to short-term perioperative outcomes. robot surgical system provided several benefits for anatomical hepatectomies including a stable and excellent operative field and clear surgical planes. suprapubic hernias (less than cm above the pubic arch in the midline) require important anatomical knowledge because of complexity of their repair and low incidence, by approximately % of all hernias. the problem to repair this type of hernias is that inferior margin of the defect is very close to pubic symphysis, consequently, mesh overlap is often inadequate. treatment of suprapubic hernias is controversial because of limited evidence in the literatura. this video shows the case of a -year-old female patient with suprapubic hernia with a defect of x cm. we performed a laparoscopic repair with a bilateral peritoneal flap of the groin region (as it is perfromed during tapp) for proper view of the pubic symphysis, cooper's ligaments, epigastric and major vessels, nerves and meticulous dissection the space of retzius. the defect was repaired by reconstructing the middle line with a running sutures. subsequently, titanium helical tacks were used to fix the mesh to the pubis and cooper and following the double-crown technique having special attention when fixing the mesh near to inguinal chanal, due to the possibility of causing chronic pain. the peritoneal flap was fixed over the mesh with abdsorbable fixation devices and seal with fibrin glue. laparoscopic repair of suprapubic hernias can be considered as the first option in treatment, because it endeavors to join the advantages of a minimally invasive approach and it is associated to low recurrence. the main advantages are that allows a proper visualization the anatomy and a proper fixation of the mesh. background and aim: thoracoscopic esophagectomy has been performed for two decades and becomes widely spread. we evaluate our cases who undergone the thoracoscopic esophagectomy and consider the future prospective of this operation.transient recurrent laryngeal nerve palsy after lymphadenectomy in this surgery is not rare and induces not only hoarseness but also aspiration or pneumoniae. new method to avoid this complication is desired. patients and methods: patients who received thoracoscopic esophagectomy in our institute from march to october were enrolled and studied retrospectively. operative indication is an all of the clinically resectable cases including with a neoadjuvant treatment or definitive chemoradiotherapy before surgery. overall survival rate of the patients with thoracoscopic approach and with thoracotomy until was analyzed. long term outcome of the patients with thoracoscopic esophagectomy was compared to the result from comprehensive registry of esophageal cancer in japan. short term results of the perioperative parameters were analyzed between left lateral decubitus position and prone position.we had introduced intraoperative nerve monitoring system for prone esophagectomy from . results: there was no significant differences of the survival rate between thoracoscopic group and thoracotomy group based on pathological stage. year survival without neoadjuvant treatment was . % (pstagei), . %(pstageiia), . %(pstageiib), . %(pstageiii), respectively. year survival rate of cstageii and iii with neoadjuvant chemotherapy was . % and year survival rate of the salvage esophagectomy after failure of definitive chemoradiotherapy was . %. every outcomes are as good as any reported results in esophagectomy. in the comparison of the lateral position with the prone position, total blood loss was significantly lower in prone position. inflammatory response after surgery was improved more rapidly in prone group, therefore, prone position is recommended as a minimally invasive procedure for thoracoscopic esophagectomy. transient recurrent laryngeal nerve palsy was observed % of patients. conclusion: thoracoscopic esophagectomy will develop further as a standard operation for esophageal cancer. nerve monitoring is useful for detecting recurrent nerve and avoiding nerve injury. background: laparoscopic total mesorectal excision (tme), in a wide female pelvis is usually technically easier than in a narrow male pelvis. however, this is not always the case, as the uterus and adnexae may obscure the views and hinder safe dissection, especially in obese patients. techniques such as graspers through additional ports or suspension with sutures through the broad ligament may potentially cause injury or need additional ports/assistants. aim: we present a novel technique using a self-retaining gynaecological uterine manipulator to improve access during deep pelvic laparoscopic surgery in female patients. technical tip: the operation is commenced in the standard manner for a laparoscopic rectal excision. once pelvic dissection is commenced, whenever it is felt that uterine retraction would be advantageous (depending on the level of the rectal tumour, size of the uterus and ovaries, obesity etc.) a self-retaining uterine manipulator (as shown in the video) is used. the tip of this disposable device is introduced into the uterus after dilatation of the uterine cervix. once the balloon at the tip has been inflated, the instrument is secure and hence there is no need for active manipulation by an assistant. the shaft can be rotated to allow anteversion/retroversion of the uterus to varying degrees as required to aid dissection. as the video depicts clearly, it acts as a self-retaining retractor for the uterus and is removed at the end of the operation. though the procedure is being demonstrated by a gynaecologist in the video, the instrument is quite easy to insert and some of our colorectal team have been trained as well. conclusion: the self-retaining uterine manipulator is an efficient tool for uterine retraction in laparoscopic rectal surgery and we have been using it routinely in tme in females for the past years, with no complications. this was previously published as a technical tip in the journal of minimal access surgerybut has never been submitted for peer review as a video. the authors present a video of two clinical cases treated by trans-axillary endoscopic approach. methods: a years-old male and a -year-old male presented with intermittent dysphagia and frequent reflux (class ii of lahey). one had a history of recurrent respiratory infections. the disease was characterized by oesophagogastroscopy (egd) and oesophagogram. trans-axillary approach with areolar port. step-by-step as follows: (i) dissection anteriorly to the pectoralis major muscle (ii) isolation of the anterior border of sternocleidomastoid muscle (iii) omohyoid muscle's isolation (iv) identification of the thyroid's upper pole (v) zd isolation (vi) myotomy of the cricopharyngeal muscle (vii) zd's resection with stapler and its withdrawn with sac. results: both cases progressed without complications. complete local recovery was verified in both cases one month after the procedure. conclusion: this technique seems feasible and reproducible, allowing zd diverticulectomy with a better cosmetic result and perhaps lower surgical site infections (ssi). in the authors' knowledge, this approach to dz has never been published. background: gastric leak occurs in - % of patients who undergo roux-en y gastric bypass (rygb) for morbid obesity. the pathophysiology may be related to gastric ischemia, fistula, or ulcer.gastric leak is a severe complication of gastric bypass (gbp) that is associated with significant morbidity and mortality. fistula may have several clinical impacts, depending on patientrelated factors, fistula characteristics, onset time, and therapy proposal. abdominal drainage, gastrostomy, and revisional surgery constitute the traditional approaches to dehiscence and fistula closure, with variable results. methods: we present a video of a clinical case of -year-old lady with body mass index of kg/m who underwent roux-en-y gastric bypass and h later presentedtaquicardia and right cuadrantum pain. the ctscan inform a apical leak at the gastric pouch level. the video shows the relevant aspects of a revisional surgery and the key points to drain the fistula and close de defect laparoscopically. results: after monts, the patient achieved succesful results, defined as a stabel clinical situation with image evidence of gastric fistula remision. conclusions: gastric bypass (gbp) is one of the most efficient bariatric interventions in morbidly obese patients. the most severe risk of this procedure seems to be the staple line leak, and the management of this complication can be very arduous. without any guidelines it is very difficult to determine the right procedure addressing the staple line leak after gbp. laparoscopic sleeve gastrectomy (lsg) has become the most commonly performed operation worldwide as a primary bariatric/metabolic procedure. however, conversion to other surgical procedures such as roux-en-y gastric bypass (rygb) or one anastomosis gastric bypass (oagb) have been described as treatment options for inadequate weight loss after lsg and unresolved co-morbidities or complications such as leak, stricture, and severe gastroesophageal reflux disease (gerd). we present two clinical cases of weight regain and severe gerd and dysphagia, which account for the main indications to reversal of lsg to either oagb or rygb. aims: we show in the video the surgical technique that we perform by laparoscopic aproach, in order to construct a roux-en-y polipropilene banded gastric bypass lrygb-b. methods: we are performing this procedures within a prospective randomized trial that is design to compare the long term results of lrygb-b versus the standard laparoscopic roux-en-y gastric bypass.the video shows our technique in a case of a years old female with a bmi of kg/m . first we create a vertical gastric pouch of about - ml, and a polypropylene mesh ( x mm) is placed - mm proximal to the anastomosis around the gastric pouch, with the help of a laparoscopic band retractor. after that a cm roux-en-y limb is constructed in an antegastric antecolic fashion, been the lenght of the biliary limb cm. a mm gastroyeyunal anastomosis is performed with a linear stapler, and the enterotomy and gastrostomy are closed with a / barbed running sutures. jejunojejunostomy anastomosis is constructed in similar fashion, but with a lenght of - mm. the petersen space and the mesenteric defect are closed with polipropilene / sutures. results: patients has been operated following this technique, and there has been no complications related to the polipropilene band. (the ramdomized prospective trial is still ongoing). conclusions:the video shows a reproductible easy way to perform a lrygb-b using a polipropilene mesh. introduction: a -year old female patient presented at our clinic two years after initial rouxen-y gastric bypass. she had had a preoperative bmi of , and had a significant weight loss which resulted in a bmi of , at two years postoperatively. she currently suffered from severe dumping with glycaemia levels dropping to mg/dl. pharmacological treatment with metformine, sandostatine and acarbose did not yield any results. on top of these problems she felt less restriction, could eat large portions and had gained kg in the last three months. objective: the usual approach for severe dumping-related hypoglycemia would be to undo the gastric bypass. this patient however was extremely anxious to regain weight, so we sought other options. we assumed that by adding more restriction and slowing down the emptying of the gastric pouch we could alleviate some-if not all-of the dumping related symptoms and prevent further weight regain. methods: in this video we present the banding of a gastric pouch for severe dumping after rouxen-y gastric bypass. results: although unconventional, the banding of the pouch yielded excellent results. the slower pouch emptying and reduced portions resulted in a near complete remission of all symptoms. as an additional benefit we found a slight weight loss of four kilograms six weeks postoperatively. conclusion: the usual treatment of severe dumping-related hypoglycemia would be an undo of the gastric bypass. in this case however the patient was extremely anxious to regain weight, being very pleased with the results her gastric bypass had yielded. in agreement with both the patient and treating endocrinologist we attempted a different approach. the slower pouch emptying and increased restriction offered another way to alleviate the dumping and deep hypoglycemia while concomitantly resulting in weight maintenance. aim: the aim of this video is to present a novel surgical technique to avoid stent migration after endoscopic placement in patients with leakage subsequent to laparoscopic sleeve gastrectomy (lsg) . methods: this video shows the case of a patient (bmi , kg/m ) who developed an upper gastric leakage days after lsg. a ct scan showed a small leakage at the eg junction complicated by intra-abdominal abscess. a ct guided percutaneous drainage of the abscess was performed. a stent placement was attempted endoscopically three times and failed for migration. we decided to place laparoscopically a non adjustable gastric ring (nagr) around the stomach, in order to avoid stent migration.first of all the stent is replaced endoscopically in order to cover the fistula tract. the patient is placed in a half sitting position and the pneumoperitoneum was obtained using a veress needle in left subcostal space. a port technique is used as in standard laparoscopic sleeve gastrectomy.the procedure starts with the mobilization of adhesions, the fistula is identified in the upper part of the tubule.the gastric tubule is isolated and the lesser omentum is opened. the blunt needle at the tip of the ring is passed retrogastrically, a tourniquet can be useful is the positioning turn out to be difficult. the nagr is then closed over the gastric tubule containing the stent. a drain is finally placed. results: the stent was removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of leakage. after months the patient was in a good condition with bmi , kg/m . the stent was endoscopically removed after weeks. a gastrointestinal ct scan with oral contrast showed a complete resolution of the leakage. after months the patient was in a good condition with bmi , kg/m . conclusions: this new technique is feasible and effective, as shown in this video; however the nagr can lead to complications, so a strict follow up is needed and if any complication appears, should be considered to remove laparoscopically the ring. introduction: in this case, we will discuss the case of a year old male patient who underwent a laparoscopic cruraplasty and gastric plication resulting in a weight loss of kg. other medical history reported insulin-dependent diabetes, reflux esophagitis and sleeping apnea with cpap. two years after gastric plication the patient presented with passage problems, gastro-esophageal reflux and epigastric pain. to this end a swallow test was performed revealing a large fundus with a restricted passage of contrast. due to the persistent complaints and the abnormal findings on barium swallow a surgical re-intervention was needed. objectives: despite the current bmi of and the age of the patient, conversion from a gastric plication to a roux-en-y gastric bypass was performed. several other surgical options were considered, including an undo of the gastric plication or a dilatation with a resizing of the fundus. methods: in the video we describe the laparoscopic approach for a conversion of a gastric plication to a roux-en-y gastric bypass. results: at months follow-up the patient showed a weight loss of kg and the resolution of his earlier symptoms. the patient had a normal oral intake without any gastro-esophageal reflux or epigastric pain. conclusion: after a gastric plication, partial loosening of the sutures and stenosis are both wellknown complications. as presented in the video, it is apparent that a laparoscopic undoing of gastric plication is not as straightforward as it seems. firm adhesions between folds can compromise the procedure and inhibit a complete separation of the tissues. we believe that in these cases the best surgical approach is to convert to a roux-en-y gastric bypass. laparoscopic sleeve gastrectomy (lsg) is a relatively new surgical approach in the weight loss surgeon's armamentarium. in literature there is a consensus about the importance of mobilizing completely the gastric fundus before transection. the resg (revised sleeve gastrectomyresleeve) may be a valid option for failure of primary lsg. we focused the attention on the consequences that can have an incomplete resection of gastric fundus during an operation of sleeve gastrectomy and how they can be solved by the repetition of this procedure. a sleeve gastrectomy was performed in an obese -year-old woman (bmi = ). three days after the operation, an upper gi x-ray with gastrografin did not show any abnormalities. three months after the surgical procedure, the woman referred frequent episodes of vomiting and a significant weight loss ( kilos). an upper gi x-ray with gastrografin demonstrated the presence of multiple communicating cavities of the gastric fundus. the esophagogastroduodenoscopy (egd) showed that the gastric tube close to the esophagogastric junction was separated from a recess ( - cm in diameter) by an incomplete septum. a severe hypokalemia and consequent ecg abnormalities were treated with intravenous infusion of potassium. then, we performed a laparoscopic operation. the gastric tube was completely released along the suture line of the previous operation and, especially, the posterior surface of the upper part until the left crus of diaphragm became evident. under the guide of the bougie, the recess was removed. results: the clinical course was regular, and the patient was discharged on third post-operative day after an upper gi x-ray with gastrografin which demonstrated the absence of leakage and a normal gastric tube. after year, the patient was very satisfied with the operation. conclusions: the complete mobilization of the gastric fundus allows to see clearly which part should be resected to obtain an adequate gastric tube and facilitate a correct placement of the stapler. in our experience, in patients with a residual fundus, an upper gi x-ray with gastrografin and an egd are needed to exclude the presence of stenosis. then, a resleeve gastrectomy is an efficient and safe procedure to treat this post-lsg complication. weight regain is one of the main problems in bariatric surgery. we have many surgical option but when we evaluate patients with long follow up and bmi of superobese patient before the first surgery, the weight recidivism can arrive up to - % at years.in most cases the first surgery is a restrictive procedure, and in many cases sleeve gastrectomy.here we present a case of weight regain after laparotomic super-magenstrasse (that we consider like a sleeve gastrectomy except for remnant removal) with a big incisional hernia. after a complete multidisciplinary re-evaluation we decided to perform an oagb (one anastomosis gastric bypass) but in this case we decided to create a functional exclusion to the duodenal transit by positioning a minimizer ring. this solution is effective in food diversion and guarantee gastric and duodenal endoscopic exploration in case of need. we think that this technique can represent an option to take in account for selected cases. at the end of bariatric procedure we perform a laparoscopic repair of incisional hernia with mesh in the hope to avoid future surgery and post operative small intestine herniation. patient rejected additional bariatric procedures and in fact she has gained kg two years later (bmi . ). conclusions: lagb gastric erosion is uncommon ( . - %) . intraoperative (such as perigastric approach) and patient related factors (smoking, alcohol…) have been described as risk factors. the most frequent clinical presentation is weight loss failure; band and port issues (such as infection) are also frequent. erosion is infrequent to present as an acute event (\ %: peritonitis, abscess…) or asymptomatically (\ %). diagnosis is mostly performed under upper endoscopy. the most common therapeutic technique is removal of the band (by endoscopy or surgery), repair of the stomach, if needed, and band replacement (at least three months later). some authors have performed immediate replacement but the incidence of recurrent erosion seems to be higher. other options are lagb removal alone or conversion to different bariatric procedure. for endoscopic removal, it has been advised to wait until the band buckle is in the stomach and is sometimes very difficult. replacement of the band is not associated with weight regain. she reports years of evolution presenting moderate intensity heartburn that was exacerbated during the night as well as submit occasional rejurgutation. the intensity of the symptoms is attenuated by maintaining a diet without irritants and improving feeding times. denies hematochezia, unintentional reduction of weight, dysphagia or early satiety. the patient has suffered from obesity since childhood, after pregnancy she had progressive weight gain and difficulty in controlling blood sugar, so she is scheduled a gastric bypass roux-en-y . preoperative endoscopy was performed, evidencing submucosal tumor in the gastroesophageal junction at of the dentary arch, approximately cm in diameter. an endoscopic ultrasound was performed, demonstrating subepithelial lesion of the gastroesophageal junction, hypoechoic, with well-defined borders, pseudobilobulated, . cm x . cm, and dependent on the external muscular layer. a fine needle aspiration is performed in which spindle cells are identified, leiomyoma is likely diagnosed. it is programmed for laparoscopic resection of submucosal gastric tumor, gastric bypass and laparoscopic cholecystectomy. a tumor at the level of the gastro esophageal junction of approximately . cm is identified in the surgery, which can be resected by laparoscopy without complications. the patient is discharged after days of postoperative stay. the final histopathological result: leiomyoma of . cm with free edges. cd (-) gog (-) caldesmon (?)s (?). background: fifty percent of patients who have undergone gastric bypass, posterior reversal and sleeve gastrectomy and finally complete hiatoplasty presents symptomatic gastroesophageal reflux disease. surgical reinforcement of the lower esophageal sphincter is necessary to prevent acid reflux. here, we describe ligamentum teres cardiopexy, a surgical technique that reinforces the lower esophageal sphincter and restores its competence with a new valve, in patients with previous conversion of sleeve gastrectomy to gastric bypass and hiatal hernia repair. methods: we present the surgical techhnique performed to a patient with initial gastric bypass who underwent sleeve gasterctomy for hipoglycemias and hiatoplastia for severe gerd. persistent gerd requested to undergo ligamentum teres cardiopexy. in this procedure, the ligamentum teres is released from its umbilical connection and the hernia reduced by manual traction, freeing the last - cm of esophagus in the abdomen. the distal ligamentum teres is fixed with one stitch to the apex of the angle of his, one at the gastroesophageal junction, and one joining the gastric fundus to the esophagus. the remainder of the ligamentum teres is fixed over itself with four to six stitches, forming a necktie cardiopexy. the procedure concludes with diaphragmatic crus closure. results: after months, the patient achieved successful results, defined as resolution of gerd, no protonpump inhibitor (ppi) use, and manometry measurement over mmhg after surgery. conclusions: ligamentum teres cardiopexy combined with closure of the gastric crus is a late alternative treatment for gastroesophageal reflux disease in patients with previous sleeve gastrectomy and hiatal hernia. general surgery, ponderas academic hospital, bucharest, romania introduction: as metabolic surgery techniques evolve during the years, we have to face more and more patients with complications ands uboptimal results after the older/initial procedures. vertical banded gastroplasty(vbg) is one of those procedures that gain momentum during the initial experience in bariatric surgery, but has proven to have dissapointing results and a lot of complications, nowadays surgeons having to deal with difficult revisional operations. aim in this video: we want to present from our experience the difficulties encountered during the revisional surgery, rouxen y gastric bypass (rygbp)aftervbg, and the tips and tricks that will make this a safer and easier procedure. objective: after thorough preoperative assessment and a review of the literature multiple treatment options were considered. the procedure of choice ended up being a laparoscopic adjustable gastric banding, with the objective to achieve optimal weight loss with the lowest risk for complications. methods: in this video we present the placement of an adjustable gastric banding in a patient with a cirrhotic liver and portal hypertension and the possible pitfalls. results: postoperatively there were no complications and patient had a satisfying weight loss both months and year postoperatively. in a short review of the literature we've found that bariatric surgery is feasible in patients with portal hypertension as long as the patient is not decompensated or has bleeding varices. conclusion: cirrhosis and portal hypertension are no absolute contraindication for banding, sleeve or rny gastric bypass as long as the patient is not decompensated or has bleeding varices. the type of surgery is dependent on patient and surgeon-related factors. the aim should be to achieve optimal weight loss with the lowest possible surgical risk in this type of patients. surg endosc ( ) :s -s introduction: in this case, we will discuss on a year old female patient who had undergone a laparoscopic nissen fundoplication years ago due to gerd grade b. because of morbid obesity a n-sleeve gastrectomy was performed year ago resulting in a weight loss of kg. at presentation she had regained all the lost weight, resulting in a bmi of , . the patient history also reported insulin-dependent diabetes and obstructive sleep apnea with cpap. gastroscopy was performed showing a large residual fundus but no esophagitis. on the subsequent upper gi series a relatively wide sleeve with an intact nissen-collar was detected. objectives: a laparoscopic conversion to a roux-en-y gastric bypass was performed. other potential surgical treatment options are a sadi procedure or a sleeve gastrectomy with transit bipartition (santoro procedure). methods: in the video we describe the laparoscopic approach for a conversion of a n-sleeve to a roux-en-y gastric bypass. results: at month follow-up the patient presented with a weight loss of kg. the patient had good restriction on oral intake and did not have any reflux-related symptoms or complaints. conclusion: conversion from a n-sleeve to a roux-en-y gastric bypass is a challenging procedure. the largest pitfall during the creation of the gastric pouch is to staple a double fold of the nissen fundoplication. we believe that in these rare cases of weight regain after n-sleeve, the best surgical approach is to convert to a roux-en-y gastric bypass. four years later, in , a laparoscopic conversion to roux-en-y gastric bypass was performed because of weight regain. she now presents with satisfactory and stable weight loss over the last few years. she was recently diagnosed with a brca- mutation for which she underwent a bilateral ovarectomy and mastectomy. the patient's brother was also diagnosed with this mutation and died of pancreatic cancer at the age of . genetic counseling advised a twoyearly follow-up because of an increased risk up to % of developing pancreatic cancer. control gastroscopy showed a normal esophagus and gastric pouch. control ct scan revealed hypertrophic stomach creases in the excluded stomach. these results prompted a laparoscopy-assisted gastroscopy of the excluded stomach which uncovered hypertrophic stomach glands and intestinal metaplasia on biopsy. methods: in this video we demonstrate the laparoscopic approach for complex revisional bariatric surgery. conversion from rny gastric bypass to a sleeve gastrectomy in a patient who already underwent a vbg. the focus of the video is on a manual gastro-gastrostomy with partial gastrectomy of the fundus and part of the stomach where the old vbg-band was placed. results: after , months follow-up the patient had no complaints and a stable weight. upper gi series shows a normal passage of contrast through the sleeve gastrectomy. conclusion: endoscopic surveillance of the remnant stomach and echo-endoscopy of the pancreas is no longer possible after rny gastric bypass. in cases where the need for such a surveillance arises after a rny bypass a patient-tailored approach is necessary. in our patient a laparoscopic conversion from a rny gastric bypass to a sleeve gastrectomy was performed. this approach keeps the patient's wish for weight loss intact while enabling further surveillance through natural-orifice endoscopy. a -year-old morbidly obese japanese woman with a body mass index of kg/m suddenly complained of swallowing difficulty months after laparoscopic roux en y gastric bypass surgery with retro-colic roux limb route. an internal hernia of the defect of the transverse mesocolon was suspected by computed tomography, and emergency intervention was performed. the surgery revealed no internal hernia. however, strong inflammation and adhesion were observed between the transverse mesocolon and the retrocolicroux limb. in addition, the roux limb on the oral side of the adhesion site was dilated and bent.the adhesion between the transverse mesocolon and the flexed roux limb was dissected, linearized and re-fixedby suturing to the transverse mesocolon. however, since the difficulty of oral intake persisted re-do surgery was performed again. after resecting the roux limb involved in the severe inflammation, a 'new' roux limb was lifted to the cephalad via the ante-colic route. finally, the gastric pouch and roux limb were re-anastomosed with - absorbable sutures in an interrupted full thickness single layer manner. in the present case, we experienced difficulty with both adhesiolysis and determining the accurate target line to resect at the 'old' gastrojejunostomy. however, blocking the blood flow of the 'old' roux limb facilitated the accurate recognition of the target line. esofagogástrica, cirugía general y ap digestivo, hospital regional universitario de málaga, malaga, spain; hepatobiliopancreática, hospital regional universitario de málaga, malaga, spain introduction: marginal ulcer is one a serious complications after a bariatric gastric bypass. tobacco, non-steroidal anti-inflammatory drugs (nsaids) and helicobacter pylori (hp) infection are known risk factors. methods: we present a -year-old women operated years before of bariatric surgery with a gastrojejunal (gy) bypass technique due to intraoperative dehiscence of the staple line after attempting a vertical gastrectomy (sleeve). she has persistent vomiting and epigastralgia from months after the intervention, affecting his quality of life. upper gastrointestinal endoscopy (uge) was performed, describing an ulcer in the gy anastomosis. she started hp eradication treatment, treatment with proton pump inhibitors (ppis), tobacco and nsaids were discontinued, but she had slight improvement. after months the uge was made again, which show peptic esophagitis and marginal ulcers. the plasma gastrin level was normal. due to the persistence of symptoms despite conservative treatment, we decided reoperation by laparoscopy. we found herniated bowel in petersen space, which were reduced and the space was closed. we proceeded to truncal vagotomy. the gy anastomosis was resected ( fig. ) and performed again. finally, we perform antrectomy. the pathological anatomy showed ulceration. she was diacharged home on the th postoperative day without any complications. results: a marginal ulcer after bariatric surgery appears in the jejunal mucosa of the g-y anastomosis. the symptoms are epigastric pain, nausea and vomiting. acid, tobacco, nsaids and hp infection has an important role in their development \ sup [ . \/sup [ the first treatment is medical, discarding out the risk factors, but if it is not effective, it will be surgical, resecting the previous anastomosis. the usefulness of vagotomy is debatable, but the percentage of success increases. in our case, we perform antrectomy to avoid retained antrum syndrome. the hernia through petersen space is a cause of intestinal obstruction and abdominal pain as the case presents. although we believe that the symptoms were mainly caused by the marginal ulcer, the internal hernia was probably a symptomatic cause. conclusion: the treatment of a marginal ulcer is medical, eliminating the risk factors, but if it is not effective, the surgery is indicated. results: bowel's measurements and confection of the gastric pouch are identical in both cases. in the first case, intestinal anastomosis is performed in the inframesocolic compartment once small bowel has been divided. in the second case, such union is made next to the gastrojejunal anastomosis with the bowel uncut, making the section once no leakage has been found conclusions: laparoscopic roux-en-y gastric bypass is currently considered one of the technique of choice in the surgical treatment of morbid obesity. there are variations and alternatives for its realization. to know them can allow to individualize the technique to each type of patient. we present a clinical case of a year old female. she had a vertical banded gastroplasty procedure (in another clinic) years ago with an initial weight loss of kg in a period of months. she was seen at our clinic because she was suffering from dysphagia to solids and general diffuse abdominal pain for the last month. at physical exam we found a bmi of and nothing else called our attention. we did an upper gi endoscopy and egd transit; we concluded that a gastric bypass would offer her the best results. therefore, we converted her vertical banded gastroplasty into a gastric bypass laparoscopically. she had an uneventful postoperative period and was discharged home without complications. aims: sadis emerged as a modification of biliopancreatic diversion with duodeno-ileal switch (bpdds) in which after sleeve gastrectomy (sg), the duodenum is anastomosed to an ileal loop in a billroth-ii fashion. sadis has promising outcomes for weight loss and comorbidity resolution in morbidly obese patients avoiding the high morbidity of biliopancreatic diversion with duodenal switch. clinical case: -year-old patient, subjected to bariatric surgery two years ago, including a sleeve gastrectomy (sg). despite this operation and dietary and hygienic modifications, the patient gained weight in recent months, reaching a bmi of kg/m and an overweight of kg. an endoscopy was carried out on her, which provided evidence of a gastric remnant of moderate size with flexible tissue, normal peristalsis, and fast disposal speed. the case was discussed in a joint session, leading to the decision to apply revision surgery. the decision was taken to apply sadis, a novel technique that had never been used before in andalucia. the rate of weight re-gain after the use of classical techniques such as sleeve gastrectomy (sg) or the roux-en-y gastric bypass (rybg) is considerable high. revision surgery due to weight re-gain is necessary in many of these cases. sadis emerged as a simplified alternative to the use of bpdds as revision surgery following a gv due to weight re-gain with good short-term results, in terms of both weight control and comorbidity control. since only one anastomosis needs to be applied, chirurgical time diminishes, as well as the rate of surgery-related complications. moreover, it could be used, through laparoscopy, for patients who have undergone previous, complex abdominal surgery. conclusion: sadis showed a promising short-term weight loss outcome and comorbidity resolution rate but long-term data are missing and there is currently a high level of technical variability. on the other hand, further studies are required to measure its cost-effectiveness compared to the currently popular bariatric procedures, sg and rygb. aims: the lps sleeve gastrectomy is the most common bariatric surgery technique because it has a low surgical complexity and acceptable weight loss results. however, - % of patients present with an insufficient weight loss, weight regnances, reflux or dysphagia. in these cases, it is recommended to perform a second bariatric surgery to combine a component of malabsorption such as gastric bypass or duodenal switch. the video describes the technique of a laparoscopic biliopancreatic diversion with duodenal switch with a previous laparoscopic sleeve. the objective is to describe the safety of the technique and the subsequent success of it. methods: a -year-old female patient presented morbid obesity with a bmi of after performing a laparoscopic sleeve gastrectomy in . initially, she presented a percentage of excess weight loss of %, reaching a bmi of after two years of follow-up. after this, she suffered a reganancia of all the weight lost despite diet and exercise, presenting a bmi . a study was made with tegd where no complications of the previous surgery or symptoms of gastroesophagical reflux or dysphagia were observed. the lps duodenal switch is proposed in the obesity unit committee in , without immediate postsurgical complications. the patient presented a favorable postoperative period and was discharged three days postoperatively. results: at the present time, the patient has achieved a % excess weight loss and has a bmi of . . presents good oral tolerance with stools a day without urgency. it doesn't present protein deficioncies. vitamin deficiencies are orally supplemented. the lps duodenal switch is a technique that can be performed after a sleeve gastrectomy safely in cases of insufficient weight loss or weight reganancia. the patients presented a greater weight loss after the duodenal switch than after the gastric bypass, observing a lost of excess weight of % compared to %. the differences being statistically significant. weight regain after gastric bypass is a challenging problem. a number of revisional surgical options have been reported. this is a case of a year-old woman years after lrygb. her initial bmi was , lowest after surgery- , at presentation- . the video shows a robotassisted laparoscopic conversion of rygb to loop duodenal switch. the roux limb is transected and dissected to the gastrojejunostomy. the gastrojejunostomy is resected and the gastric pouch is recreated over a bougie. the gastric blood suply is confirmed with icg. a gastro-gastrostomy is created to restore gastric continuity and a sleeve gastrectomy is performed. the duodenum is devided and a duodeno-ileostomy is created cm from the ileocecal valve. the remaining roux limb is resected. the patient recovered uneventfully. conversion of rygb to loop duodenal switch requires creation of as little as two anastomoses, in comparison to standard ds, which requires four. it is a safe option for patients with weight regain after lrygb. methods: irb approval and informed consent have been obtained. a dissection is conducted to separate the descending mesocolon of the gerota's plan from the medial aspect to the peritoneal lining to the left parietal gutter. the peritoneal layer is incised parallel to the vessel and close to the colonic wall. the dissection is continued anteriorly up to reach the resected parietal gutter. a passage into the mesentery of the upper rectum is created for the allocation of the stapler and the dissection of the rectum. these maneuvers permit to straighten the mesentery simplifying the identification and cutting of the sigmoid arteries. a caudal-to-cranial dissection of the mesentery is performed from the sectioned rectum to the proximal descending colon by a sealed envelope device. it can be very useful to mobilize the colon in any direction: laterally, medially, or upward. the dissection is performed along the course of the vessel up to the proximal colon, with progressive sectioning of the sigmoid arterial branches. the specimen is extracted by a pfannenstiel incision. the anastomosis is performed transanally with a circular stapler according to knight-griffin technique. results: we performed a laparoscopic segmental colectomy using this approach for patients with benign sigmoid lesions: diverticulitis, flat polypoid lesions (no lift-up sign), and bowel endometriosis. the mean operative time and blood loss were . ± . min and ± ml, respectively. there were not a single conversion to open surgery and no any leakage or stricture. only cases of intraluminal bleeding and case of wound infection (treated conservatively) were observed. conclusion: we consider this approach to be safe and useful for segmental colectomy to be performed sectioning the sigmoid artery close to the colonic wall. aims: to show a clinical case with a video of a patient was operated for colon cancer in hepatic angle by a single suprapubic incision (ssilrh). methods: a -year-old male assessed for abdominal pain and weight loss. on physical examination: a painful mass was detected in the upper right quadrant. the colonoscopy revealed an ulcerated lesion in the hepatic angle and the biopsy revealed a moderately differentiated adenocarcinoma. in the abdominal ct a mass of x cm was observed (figure). the patient was operated with ssilrh technique, as shown in the attached video. results: the patient was placed in the supine position and with the legs separated. the surgeon is placed between the patient's legs. a transverse incision of the skin was made in the middle line of . cm to cm above the pubis. the underlying fascia was divided transversely, the rectus abdominal muscle was exposed, a purse-string suture placed in the fascia. an mm reusable trocar was inserted for the chamber, a mm reusable flexible trocar was placed at the o'clock position and another trocar was placed at the o'clock position. the ileocecal valve was released from the peritoneal parietal foil, as well as the mesocolon right by a lateral to medial approach to the second portion of the duodenum. the hepatic angle was also dissected from lateral to medial. for the anastomosis, the mm trocar was replaced with a mm trocar and a stapler was placed. a mm °chamber was inserted through the mm flexible trocar. the small intestine was divided as well as the proximal transverse colon with endogia. an intracorporeal ileocolic anastomosis was performed. the piece was removed through the suprapubic incision. he was discharged after days without complications. the histological studies confirmed a differentiated adenocarcinoma of x x cm. the surgical margins were free, without infiltrated lymph nodes ( / ) with stage pt n . the ssilrh technique allows a complete resection of the mesocolon and complies with the oncological principles. they can present with abdominal pain, nausea, acute abdomen, symptoms of intestinal obstruction or asymptomatic with incidental diagnosis. their diagnosis can be difficult. the objective is to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology. material and methods: we present a video of the surgical intervention of a -year-old patient, with functional dyspepsia, with a casual diagnosis of a pseudocystic mass of the right colon after performing a ct scan: giant diverticulum of the hepatic colon angle with fecaloid content inside it under tension the patient goes to the emergency room for acute abdominal pain, pending colonoscopy, antibiotic treatment is established, and a laparoscopic approach is decided upon after the patient's evolution. results: intervention: complete laparoscopic approach, trocars. large size tumor in the right colon, diverticular in appearance, with stony content inside, with locoregional adenopathies, oncological radical right hemicolectomy, manual intracorporeal anastomosis, correct postoperative, hospital discharge. on the th day. definitive pathological anatomy: giant diverticula on areas of intense mucosal ulceration, free edges. conclusion: the laparoscopic approach of the symptomatic diverticula of the right colon is safe and effective. introduction: minimally invasive transanal surgery (tamis) is a surgical technique whose established indications are the complete exeresis of rectal polyps that are not resectable endoscopically or early rectal neoplasms with good prognosis criteria. transanal devices with gel platform facilitate dissection in this field. however, one of the drawbacks of this approach is the oscillation of the right nerve, which hinders dissection and prolongs the surgical time. material and methods: we present the case of a patient with a central depression neoformation, located cm from the anal margin in the posterior aspect of the rectum in a male patient. the lesion occupies % of the circumference and was considered unresectable endoscopically. the endoscopic biopsies showed a tubulovillous adenoma with moderate dysplasia. results: an exeresis of full thickness of the rectal wall is performed, with subsequent suture of the defect. we show in the video the use of a glove interposed in the pneumoperitoneum gum to maintain the stability of the neumorectum and the technique of dissection and suture, as well as the stability of the neumorectum with this technique throughout the procedure. the use of a glove as a reservoir to stabilize the nemorectum is an economical and easy-to-use method that can safely replace extra devices. aims: endometriosis is a gynecologic disorder defined by the presence of endometrial glands and stroma outside the uterine cavity. deep infiltrating endometriosis (die) invades mm to the retroperitoneum of the pelvic sidewalls, the rectovaginal septum, or the muscularis of the bowel, bladder or ureters. the rectum is being the most common bowel site of involvement. for symptomatic die, medical therapy should always be the first-line treatment. therefore, a minimally invasive approach using laparoscopy is considered the gold standard option and challenging aiming at complete disease excision. also, there are several advantages of natural orifice specimen extraction when compared with abdominal incision that may directly impact the postoperative results of these young patients. methods: we report a case of a -year-old female with a -month history of chronic pelvic pain, dyschezia and rectal bleeding. these symptoms were refractory to hormonal, antispasmodic and opioid therapy. magnetic resonance imaging reported a nodule x cm invading the rectal wall cm to the dentate lane. we performed a laparoscopy and we found the nodule at the uterine posterior wall invading the rectal anterior wall. the nodule was invading into the rectum in a large area so we proceeded with segmental resection and added hysterectomy and salpinguectomy because it was the preference of the patient. the anastomosis was created intracorporeally and the specimen was removed through the vagina performing in this way a totally laparoscopic procedure with natural orifice specimen extraction. results: the total operative time was h, the postoperative stay was uneventful and the patient was discharged on day four. the pathological report showed an endometrioma cm length predominantly involving colonic muscularis propria. conclusion: laparoscopic surgery is a safe and feasible approach for the surgical management of deep infiltrating endometriosis of the rectum and the gold standard for female young patients that often need multiple surgeries. in addition natural orifice specimen extraction avoids potential complications of abdominal incisions. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy; urology, clinica mater dei, rome, italy aims: we describe a case of a patient affected by a mass in the left kidney and a diverticular stenosis of the sigma. methods: a years old woman complained abdominal pain in the left flank of the abdomen and in the left iliac fossa radiated to the hypogastrium, with fever and no passing flatus. contrast enhanced computer tomography scan (ct-scan) showed a cm mass of the superior pole in the left kidney and a colonic diverticulitis with thickness of the wall and a microperforation of the sigma. she underwent to medical therapy with resolution of the diverticulitis. after weeks a laparoscopic nefrectomy and sigmoidectomy was planned. patient was positioned on the right flank. this position was kept for both the procedures. we performed four trocar accesses along the left subcostal region and a periombelical incision for the specimen extraction. results: post-operative course was uneventful. patient was discharged in post-operative day. istopathological exam showed a renal cell carcinoma confined to kidney with no positive lymph nodes and a diverticular stenosis of the sigma. laparoscopy allowed to perform two fine procedures in a critical situation using few trocar incisions and obtaining good results. background: hartmann procedure consists in a sigmoidectomy followed by a terminal colostomy. stoma is associated with complications and suboptimal quality of life, so the restoration of colonic continuity should be at least considered in any case. open restoration has been associated with significant morbidity and mortality. many authors have described the advantages of laparoscopic hartmann reversal. we want to go a step further showing our experience using a combined laparoscopic and transanal approach in an attempt to improve the surgical technique in a patient with previous abdominal surgeries and a rectovaginal fistulae. methods: the transanal and laparoscopic team work simultaneously. by the abdominal approach a pericolostomic incision is made, the distal affected colon is resected and a purse string suture is performed around the anvil of the eea mm single-use stapler with . mm staples (autosuture, covidien). a mm umbilical trocar is located for a °camera and a gelport laparoscopic system (applied medical) with two mm trocars is introduced through the colostomy wound. hard pelvic adhesiolysis was performed and splenic flexure was also mobilized.the gelpoint path transanal access platform (applied medical) is introduced through the anal canal with three trocars in a triangle position. the proximal rectum and mesorectum are dissected until the peritoneal reflexion. the previous stapler line with the resected tissue is then exteriorized throught the anus. the distal rectum is prepared with a circumferential purse string suture. the vaginal defect was sutured transanally. the proximal colon and the anvil are extracted through the rectal stump and connected to the circular stapler, performing an end-to-end anastomosis. results: the total operative time was h. the postoperative stay was uneventful and the patient was discharged on day . conclusions: as in patients with rectal cancer, dissection of the stump in hartmann reversal procedure may be better and associated with shorter operative time. as with any new surgical procedure, it is probably too early to draw conclusions but nowadays transanal combined with laparoscopic approach seems to be a safe and feasible technique to perform a hartmann reversal, especially in challenging cases. intravenous and endoluminal contrast enhanced ct revealed the presence of a large retroperitoneal fluid and gas collection, due to diverticular perforation, extended from pelvis to iliac bifurcation, involving the left urether. no hydrosoluble contrast media leakage or massive pnuemoperitoneum were present. after an initial conservative treatment without significant improvement an emergency laparoscopic left colectomy with primary anastomosis and laparoscopic retroperitoneal collection drainage was performed. the laparoscopic approach was very challenging due to the obesity of the patient and the presence of the abscess. the patient was discharged on pod after requiring re-intervention for dehiscence of the left iliac mini-laparotomy on pod . conclusion: diverticular perforation in obese patients adds a further challenge to its laparoscopic treatment and deserves an aggressive surgical approach since its outbreak. although intracorporeal anastomosis has been demonstrated to be safe and effective after right colectomy, limited data are available about its efficacy after left colectomy for colon cancer located in splenic flexure. there are few studies comparing patients who underwent laparoscopic left colectomy with intracorporeal anastomosis or with extracorporeal anastomosis. anyway literature shows that there is no significant difference between intracorporeal anastomosis and extracorporeal anastomosis about oncological result. as for right hemicolectomy, intracorporeal anastomosis seems to show a trend towards a faster recovery after surgery due to the shorter time to flatus and lower post-operative pain expressed in the mean vas scale. laparoscopic left colectomy with intracorporeal anastomosis is associated with a lower rate of post-operative complications as for right colectomy. literature results could suggest that a complete laparoscopic approach could be considered a safe method to perform laparoscopic left colectomy with the advantage of a guaranteed faster recovery after surgery. as usual further randomized clinical trials are needed to obtain a more definitive conclusion. we show a video of a years old patient with a pure splenic flexure colon cancer who underwent to a laparoscopic left hemicolectomy with intracorporeal anastomosis. case presentation: here we describe a case of a year old asthmatic and hypertensive lady with an asa score of iii who presented to emergency after a right knee replacement with a four day history of lower abdominal pain. she was septic upon arrival to the resuscitation roomimmediately prompting the hospital's local septic management protocol. a ct scan of her abdomen showed a rectosigmoid perforation with free intra-abdominal air and fluid. the patient underwent laparoscopic hartmann's procedure within h of admission. after an uneventful postoperative recovery the patient was discharged home after a total of days of hospitalisation. she was followed up at surgical outpatients with no adverse events over the course of the subsequent months. conclusion: this case exhibits the feasibility of laparoscopic hartmann's procedure as a surgical modality for hinchey stage iv diverticulitis. the positive outcome supports the claim that for experienced surgeons laparoscopic hartmann's procedure remains a safe and viable option for elderly comorbid patients in the emergency setting. introduction: mesenteric cysts are a very infrequent pathology, they usually present an anodyne clinic, and their diagnosis is reached casually. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with intra-abdominal cysts of benign etiology, using material with mini-instruments, reducing surgical aggression, maintaining its safety and efficacy.material and method: clinical case: a -year-old man with no personal history of interest. in the last two months he presented episodes of pain in the right hypochondrium, exploration without findings, us-ct scan: a cystic tumor of cm. in hepatic colon angle compatible with uncomplicated benign mesenteric cyst, tumor markers and normal colonoscopy. evidence of interest is exposed. given the evolution it is decided tto. elective surgical. result: intervention: laparoscopic approach, trocars, two of . mm, optics of mm °, benign cystic tumor, with colloid content of more than cm. of diameter in antimesenteric border of colon, which is not possible to separate, mobilization and resection is carried out by endo-gia, including a portion of the colonic wall, appendectomy, extraction in a pocket. good postoperative course, alt to nd day. definitive ap: mesenteric cyst, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of benign intra-abdominal cystic pathology, the use of mini instruments reduces surgical aggression, favoring the recovery of the patient. male, yr, wuth doblue post-operative coloanal stenosi and women, with ultralow rectal neoplatis stenosis ( cm from anla verge). both patients were discharge ater days from prosthesis positionins without pain and complications. the first patient, with protection ileostomy, showed fecal incontinence before the operation and was performed prosthesis positioning because rectal losses of infected material and fever. fecal incontinence was showed also after procedure but he had not fever. second patient, yr, with ultralow rectal tumor, after prosthesis positioning was submitted to radiotherapy and she decided for not to be operated and she survives after months in ful well-being. conclusion: endoscopic prosthesis positioning is a consolidated procedure for treatment of bowel obstruction. this study demonstrated that this procedure is safe and this kind of prosthesis is suitable for correct positioning. results: we present a case of a years old man with faecal occult blood test positivity that was diagnosed by colonoscopy of a villous lesion at cm of anal verge. biopsies were taken showing a tubulovillous adenoma with high grade dysplasia. a rectal mri was done showing the lesion fixed to the postero-lateral left side of the lumen at cm of anal verge. no pathological lymph nodes were reported. extension study was negative. the case was presented in multidisciplinary committee agreeing in local excision. in october the procedure was done without incidents. the patient was placed in lithotomic position finding a lesion occupying of the lumen. resection was done without incidents and posterior suture with continuous barbed sutures. he presented an uneventful recovery being the patient discharged in rd postoperative day. definitive pathological findings showed a ptis with negative margins. after three months of followup the patient remains with good functional results and waiting for the first endoscopic revision. conclusions: tamis is a safe and feasible technique with low morbidity that gives us an alternative for early rectal cancer or big rectal lesions much less invasive than techniques used until now. complete mesocolon excision and d lymphadenectomy are two fundamental points in the oncological surgery of right colon cancer. most of the adenopathic recurrences of colon neoplasia in tumors located in the hepatic angle and the ascending colon are located near the head of the pancreas and the vascular axis of the superior mesenteric vein due to an alleged incomplete dissection. we present a case of right colon neoplasia where we performed a laparoscopic right hemicolectomy associated with a d lymphadenectomy. we use medial to lateral dissection of the mesocolon focused on the dissection of the superior mesenteric vein with the identification of ileocolic vascularization, right colic vessels and henle's trunk. this approach is safe and facilitates a correct resection of the mesocolon, which is approached following the embryological plans and a vascular ligature near the bifurcation. the performance of an extended lymphadenectomy allows a wider resection of the mesocolon and the excision of a greater number of lymph nodes, all of which can contribute to a greater survival. the efficacy of pc treatment is related with a properly preoperative imaging diagnosis of the disease, but the poor sensitivity for identifying small peritoneal metastasis are the major obstacle to achieve a complete resection and that leads to peritoneal recurrence. imageguided surgery using icg, could represent an advance in the detection of small peritoneal nodules. there are only a few clinical studies that have analyzed the role of icg for the staging of pc, specially from ccr, and nearly in all of them the selected approach were exploratory laparotomy. this study presents a laparoscopy case, as a non-invasive way of cs in selected patients with limited pc. a new category, tis, was created for low-grade appendiceal mucinous neoplasms (lamns) that invade or push into the muscularis propia by ajcc cancer staging th ed. management of these tumors depends on stage and histology. traditionally, laparotomy was the most recommended approach, however, if laparoscopy is safe, it could be used. the laparoscopic appendectomy should be done with 'not touch' technique and a radical approach has been recently proposed for its treatment. the laparoscopic radical appendectomy should start by exploring complete abdominal cavity. grasping of appendix should not be done. complete resection of mesoappendix is obligated. cequectomy with stapled endogia is necessary. the specimen must be extracted in an endobag. methods: we report a case of a year-old female patient with a personal history of three caesarean sections. this patient was studied due to chronic abdominal pain. a computerized axial tomography was performed, showing an appendix increased in size and a thick wall. the colonoscopy evidence a lesion that protrudes from appendiceal base which is biopsied. results: a laparoscopic way was used and large and width appendiceal was viewed ( x cm). furthermore, a rounded right anexial tumor was also found. a radical not touch laparoscopic appendectomy with stapled cequectomy was done. the intraoperative study was mucinous appendiceal tumor without serose affection. the final result was ptisnx (lamn) without resection margins affected. after h of admission, the patient is discharged without incidents. conclusion(s): minimally invasive surgery in lamns is possible if it is performed with enough experience, following specific rules and tips to manage this tumors. a correct follow-up should be carried out using tumor markers and computer tomography (ct). introduction: resection of both benign and malignant colovesical fistulae can be particularly challenging and carry with it specific surgical considerations. often there is a large inflammatory mass sat within a narrow pelvis, limiting specimen mobility and consequently access to dissection plains. additionally, with the underlying inflammatory process, the ureters may be displaced anatomically and be at risk of injury. aim: to demonstrate a streamlined and reproducible approach to the laparoscopic management of both benign and malignant colovesical fistula, with specific emphasis on the different modalities for bladder repair. method: the following method portrays an overall technique which is adapted dependant on the clinical scenario and specific intra-operative findings: approach to abdominal cavity in standard fashion.identification of right ureter.poster-medial mobilisation of the mass to facilitate delivery out of the pelvis followed by visualisation of the left ureter on the medial and lateral sides before division of the fistula.division of the fistula in benign disease or resection of the bladder dome in malignant disease.transverse laparoscopically sympathetic suprapubic skin incision.vertical incision through linea alba to deliver bulky specimen.intra/extracorporeal repair of bladder dome. results: all of the considered cases were successfully completed with a laparoscopic approach, irrespective of the malignant status of the disease in question. conclusion: both benign and malignant colovesical fistula disease can make the laparoscopic approach to resection challenging, especially when encountering a bulky mass in a narrow male pelvis. the stepwise and streamlined approach considered here can help facilitate successful and safe laparoscopic completion without the necessity to convert to open. background: primary neoplasms of the retrorectal space are very rare. they are located in anatomically difficult area to be addressed, hence a complete evaluation of the lesion is required to determine the extent of resection and the appropriate surgical approach, which include posterior, abdominal and combined abdominoperineal, depending on the characteristics of the lesion. objective: to show a combined laparoscopic abdominoperineal approach of retrorectal tumor. method: we present a video of a combined laparoscopic abdominoperineal resection of a lowlying retrorectal tumor in a -year-old female without prior abdominal surgery. conclusion: retrorectal tumors are infrequent. their anatomical location can make difficult the surgical approach. preoperative imaging can provide useful information for surgical planning. in the recent years, minimally invasive surgical approach has been proposed. laparoscopic approach is feasible and safe, but it is important to select adequately the patients. background: adult intussusception is a rare clinical event representing only - % of all bowel obstruction cases and % of all intussusceptions and the occurrence of adult intussusception due to colonic cancer is even more rare. aim: we present this case of malignant colo-colic intussusception and literature review to increase the awareness of the incidence of colocolic intussusception due to colonic cancer. case report and literature review: our patient is a years old female was admitted to our hospital due to central abdominal pain, cea level of , she was further investigated with ct scan of the abdomen and pelvis which raised the suspicion of mid transverse colon intussusception due to large polypoid lesion. she was further assessed with urgent colonoscopy which confirmed mid transverse colon tumour with biopsies confirmed adenocarcinoma. laparoscopic extended right hemicolectomy with lymph node dissection was performed. upon laparoscopic exploration it was found that the colocolic intussusception was evident as described on the ct scan and as clearly shown on the video. histologically, the transverse colon carcinoma was a moderately differentiated adenocarcinoma, with no lymph node involvement ' out lymph nodes', tnm staging of pt pn pm and r resection. intussusceptions of the colon in adult are frequently found in the ileocecal portion or sigmoidal colon but rarely in the transverse colon. only two cases of adult intussusception of the transverse colon caused by colonic cancer have been reported. overall cases on literature review reported showing colo-colic intussusception due to colonic malignancy. conclusion: colo-colic intussusception due to colorectal cancer is a rare clinical event, however it should be included in the differential diagnosis of colonic obstruction. laparoscopic surgery is safe in malignant colocolic intussusception. aims: single-incision laparoscopic colectomy (silc) aims to achieve better cosmetic outcomes, less pain, and faster recovery compared to multi-port laparoscopic colectomy, but it also has several limitations, especially the technical difficulties. we report our experience with singleincision robotic right hemicolectomy via video presentation. methods: we arranged robotic-assisted single-incision right hemicolectomy for a -year-old female patient with ascending colon tumor. the operation was performed with gloveport singleport device and a three-arm da vinci robotic surgical system through a small midline umbilical incision. colectomy was proceeded by a medial-to-lateral approach along with one or two accessory instruments for maintaining sufficient bowel traction or surgical field exposure. after vessel ligation, complete colon mobilization and right side omentum division, the robotic arms were undocked to perform anastomosis extracorporeally. results: the operation was performed successfully without drainage tube placement. the total operative time was min. the bowel movement returned on post-operative day ,and the patient tolerated normal soft diet on post-operative day . she was hospitalized for days after operation. the pathology report revealed colon adenocarcinoma (t n m , tumor size . cm), and lymph nodes were harvested. conclusions: single-incision robotic colectomy (sirc) approach seems feasible and safe in treatment of ascending colon cancer. this surgical option provides less pain and wound scar for the patient. moreover, it also achieves further benefits for the surgical procedures compared to silc. reasons being, first, it has better instruments flexibility and precision with endo-wrist, as well as less instruments clashing. second, the improved camera stability achieved through the use of the robotic arm is unattainable through manual hand-controlled methods. third, roboticassisted approach gives us an ergonomic environment, which enables the operator to control the arms while sitting by the console, and also to reassign them whenever they cross each other or block the surgical view. in spite of the advantages above, we still need to sincerely consider each patient's situation for proper management. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify sentinel lymph node, for studying its prognostic value on nodal status, to facilitate vascular dissection when vascular anatomy of the tumor site is unclear and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.when tumor is in difficult site, as hepatic or splenic flexure, ml of icg solution ( , mg/kg) is intravenous injected. in about - s a real-time angiography of tumor area is obtained; on this guide, vascular dissection and pedicle ligation is performed.after anastomosis, another ml of icg solution is injected to confirm anastomotic perfusion. if there is an ischemic area, a new anastomosis is performed. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and resections of splenic flexure. in ten cases, intraoperative angiography led to the identification of vascular anatomy. in two cases the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to reduce the incidence of anastomotic leak, to facilitate the assessment of vascularization in order to perform oncological resections, and to perform the slnb to study its clinical role on nodal status and for the sln ultrastaging in order to identify the micrometastases. background: surgical emptying of lateral pelvic lymph nodes (llnd) is a strategy used differently when compared the approaches to rectal cancer in the west and eastern countries. there is evidence that = mm lymph nodes in lateral compartment should be removed, even in the setting of neoadjuvant chemoradiation. minimally invasive surgery with nerve-sparing technique and sharp dissection with minimal bleeding may help overcome the significant complexity of the procedure that may have been a technical obstacle to implementation in the past. the standardization of the technique may help implementation with shorter learning curves and excellent surgical outcomes. methods: a -year-old male with distal rectal cancer underwent neoadjuvant crt for a mrt cn m mremvi ? mrcrm ? disease. there was one left obturator node of mm prior to crt. following weeks of crt completion, the patient underwent tatme for the primary disease followed by left lateral node dissection by laparoscopy. results: the present video illustrates the most relevant surgical steps to perform lateral node dissection. the procedure has been didactically divided into steps. the left ureter is identified and retracted using a vessel loop (step ). identification of the common iliac vein and dissection with subsequent identification of psoas and internal obturator muscles (step ). identification and dissection of accessory vessels. ( step ) identification of obturator nerve and obturator vessels (step ). blunt dissection of obturator nerve (step ). identification and ligation of obturatory vessels. (step ) umbilical artery is skeletonized to allow identification and clearance of fatty tissue along superior vesical arteries, internal iiliac artery/vein, inferior vesical artery and internal pudendal artery (step ). postoperative course was uneventful. conclusion: standardization of lateral-node dissection for rectal cancer has paramount importance. laparoscopic lateral-node dissection for rectal cancer provides optimal anatomical view and allows safe dissection of the nodes of interest. aims: the aim of this video is to describe our technique using fluorescence to assess the lymph flow to ensure a complete mesocolic excision and central vascular ligation in order to provide expertise to contribute to the standardization of this new tool. methods: laparoscopic right colectomy with total excision of the mesocolon was proposed in all cases. for the detection of lymph flow, we injected indocyanine green dye ( milliliter of milligrams dye dilution in milliliter of distilled water) into the subserosal to submucosal layer around the tumor at point with a -gauge injection laparoscopically after trocar insertion, and observed the lymph flow using a near-infrared system (visera elite ii, olympus) after injection. we also performed a total mesocolic excision with central vascular ligation in the region where the lymph flow was fluorescently observed. results: ( %) patients were included. no intraoperative or postoperative complications presented. no adverse effects were reported due to the infusion of indocyanine green. the lymph flow was visualized intraoperatively in a satisfactory way helping the surgeon in decision making to determine an appropriate separation line of the mesentery. the section line of the mesocolon was modified in ( %) case based on the findings obtained by fluorescence. the mean operative time was ( ) min. the morphometric laboratory data of the specimens to audit the correct complete mesocolic excision were satisfactory according to the oncological standards. conclusion: fluorescence lymphography during colorectal surgery was feasible and reproducible with a minimum of added complexity. fluorescence-guided surgery may be a helpful technique for determining an appropriate total mesocolic excision in colon neoplasms. aims: this video shows our technique for complete mesocolic excision (cme) during right colectomy for cancer. methods: in this video, a years old patient underwent a laparoscopic right colectomy with cme for a cancer of the ascending colon diagnosed with a colonoscopy performed after positivity to fecal occult blood test (fobt). after ct scan staging we obtained d printed models to clarify patient's vascular anatomy. patient was placed in supine position, trocars were inserted in left quadrants as for standard right colectomy. cme is performed by sharp dissection between the visceral fascia that covers the posterior lay of the mesocolon and the parietal fascia that covers the retroperitoneum (toldt's fascia). the ileo-colic vessels are used as landmark to identify the right anterior surface of the superior mesenteric vessels. with a caudo-cranial approach, the mesocolon is sharply dissected and the root of tributaries venous is ligated, up to the inferior margin of the pancreas. the gastro-colic trunk is dissected out with ligation of the right colic vein, while the gastroepiploic vein is preserved (harvesting the sixth group lymph node). the pancreas-duodenum fascial plane is entered and all the lymphoid tissue around the vessel surface is harvested. procedure is completed with ileo-transverse intracorporeal stapled anastomosis. results: in our experience, between april and december , laparoscopic right hemicolectomies with cme were performed. we had no major intraoperative vascular lesions. no patients needed intraoperative blood transfusion. compared to our series of standard right colectomies we did not notice any significant difference in post-operative complications. the follow-up is too short to demonstrate if the cme approach has a better oncological outcome compared to standard right colectomy. conclusions: laparoscopic cme is feasible, although it requires a higher expertise level of surgical know-how. the quality of evidence is limited and does not consistently support the superiority of cme as compared to standard right colectomy. better data are needed before cme can be recommended as the standard of care for colon cancer resections. h. bando gastroenterological surgery, ishikawa prefectural central hospital, kanazawa, japan aim: in case of right-sided transverse colon cancers, it is necessary to dissect the lymph nodes around the root o f the middle colic vessels. but in this area there are dangerous organs, for example : pancreatic head, duodenum, and gastrocolic trunk. it is the point of our technique that we resect the accessory right colic vein and middle colic vein, and then dissect pancreas head and duodenum at early step of the operation. method: we perform the operation by five trocars. the first step is to transect the great omentum, and confirm the lower edge of pancreas.there are much adhesion between mesocolon of transverse colon and stomach, great omentum. it is very important to dissect the adhesion accurately. secondly, the mesocolon is incised at lower edge of pancreas. it is possible to detect the lower edge of pancreas in obese people. the anterior surface of superior mesenteric vein is exposed. the accessory right colic and middle colic vein are resected. and then front face of surgical trunk, pancreas, and duodenum is dissected caudally as possible. the superior mesentery artery is resected below the mesocolon after flip up of transverse colon. this approach is safe and feasible, because the dangerous organs are handled by direct vision. by that, extraction of intestine is easy from small incision. afer flip up of transverse colon, the mesenteric of ileum is incised. the root of ileocecal vessels is exposed and these are resected. the peritoneum of the front of superior mesenteric artery is incised, and the lymph nodes around the surgical trunk are dissected. this dissected area is easily connected with the one done beforehand. uniquely we resect the mesocolon and major omentum from the root of dissected vessels to resected side of transverse colon. and then right-side colon is dissected medial approach. conclusion: we dissect the dangerous organs in advance. that prevent major injury of them. background: good visualisation of the operative field is a fundamental requirement for safe laparoscopic colorectal surgery. over the past years of the senior author's experience, camera systems have evolved from single to three chip, high definition (hd) and most recently, the k system. in parallel, the rest of the infrastructure such as cables, processors, monitors etc. have also undergone improvements, resulting in improved image quality. aim/methods: we present a video of a case of laparoscopic total mesorectal excision (tme), performed with strict adherence to our previously published 'stepwise approach to laparoscopic colorectal surgery' which places particular emphasis on safety aspects. tme was performed in a year old male patient with history of previous abdominal as well as robotic prostatic surgery. the procedure was filmed with all components including the camera head, cables, processing unit, screens as well as the recording/mixing decks being k. multiple external k cameras were also used. live transmission to a remote audience as part of our masterclass was achieved using appropriate bandwidth and projection on to k screens. results: feedback from the operating team as well as from the live audience was that the image quality was far superior to hd systems. the k system accorded a degree of clarity well beyond usual expectations. the depth of field also appeared to be different initially, but within a few minutes of starting the procedure and acclimatisation, the effects were appreciable. the clarity of the image which showed the fine details of the dissection planes and anatomical landmarks as well as the vibrancy of the vasculature gave a distinct three-dimensional effect to the picture. this excellent visualisation added one more layer of safety and complemented our stepwise approach for a successful procedure. conclusion: the laparoscopic k system, in our practice, proved to bea beneficial visualisation tool to enhance the accuracy of dissection. vital structures appeared to be more vivid and clearer with dissection planes being more easily apparent. in our opinion the laparoscopic k system when combined with a systematic approach enhances safety, especially in complex laparoscopic colorectal surgery. accumulating evidence suggests that laparoscopic surgery for colon cancer has feasibility and efficacy equal to or over conventional laparotomy. for cases with pasthistory of laparotomy, especially history of colon resection, however, there is almost no evidence for laparoscopic recolectomy for metachronous colon cancer. since , we have been used submucosal local injection of indocyanine green (icg) around primary colorectal cancer by using intraoperative endoscopy, and complete mescolic excision (cme) have been convincingly carried out, which was clarified by completely resected icg positive area. although evidence on the oncological efficacy of icg guided surgery has not yet been clarified, since it can be easily judged whether cme is performed clearly, it is considered that icg guided surgery for primary colon cancer is useful for education. recently, we are applying this to ensure convincing cme for patients with colorectal cancer who had a history of colic resection. the representative case is as follows. a -year-old female was diagnosed as advanced sigmoid colon cancer, and laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery was performed years ago. then she was diagnosed as the metachronous descending colon cancer. the feeding artery of the new tumor should be the left colic artery, however, the left colic artery was already resected and genuine feeding artery was not identified by preoperative examination. by injecting icg into submucosa endoscopically during operation, it was clearly observed that the lymphatic flow from the tumor was directed to the inlet portion of the inferior mesenteric vein (imv). re-cme was performed by ligating the inlet of imv. intraoperative icg was also useful for clarifying the borderline for adhesion detachment of pastoperation between the mesentery and retroperitoneum (figure) . interestingly, icg flow in the mesentery direct to of the anus side was disrupted clearly at the past anastomotic site. we believe that laparoscopic surgery under icg guidance is potential useful tool that can confirm evidence to date more intuitively in real time. further studies, ideally randomized controlled trials, are required for define the oncological usefulness of icg guided surgery for re-do colectomy. the operation movie will be presented at the meeting. background: laparoscopic lateral pelvic node dissection (llpnd) is a minimally invasive alternative to open surgical therapy for advanced low rectal cancer patients. in this video, we demonstrate the technique of llpnd for rectal cancer patients with suspicion of lln metastases after neoadjuvant chemo-radiation. methods: the principle of this approach is en bloc resection with bilateral peritoneum. the peritoneum is incised lateral to the ureter following the line between external and internal iliac vessels. in the next step, llpnd dissection of the regional lymph node and high ligation of inferior mesenteric vessels were performed. a contralateral llpnd was performed in the same manner as a mirrored technique. after extracting the specimen, an end-to-end double-stapled circular anastomosis was performed. results: the procedure was done safely without any complications.the surgical duration was mins, and the blood loss was ml. the number of harvested lateral pelvic lymph nodes was . the tnm stage was ypt an m . conclusion: this approach enables extended resection during lymph node dissection, allowing autonomic nerve preservation. it is maybe a helpful approach in the treatment of locally advanced rectal cancer with a lateral lymph node metastasis. aims: the aim is to present an inspection method where the anastomosis vascularity is testing simultaneously using the indocyanine green fluorescent angiography intraluminal and intraperitoneal. methods: sixty-five year old female patient underwent standard laparoscopic-assisted low anterior rectal resection for rectal carcinoma. the proximal end of the bowel and the stump of the distal rectum were checked using near-infrared fluorescence imaging with d-light camera. after making sure of adequate perfusion of the bowel, the end-to-end stapled anastomosis was performed under the laparoscopic visualisation. the d-port proctoscope was inserted into the anus. the second icg injection was administered. the perfusion of the anastomosis in transabdominal way and viability of the mucosa in transanal way was evaluated with two d-light cameras simultaneously. the anastomosis was determined cm from the anal verge. an air-water leak and tension of the bowel tests were performed. after evaluation of anastomosis viability with fluorescence imaging, after negative air-water leak and tensions testing, the decision was made by surgeon not to perform preventive ileostomy. results: the patient had no complains for the first three days postoperatively. nevertheless, crp level was growing and was . mg/l on the second postoperative day, and . mg/l on the th postoperative day. the patient complained of the pain in the right iliac area and below symphysis on the th postoperative day. the abdominal and pelvis computed tomography scan with oral contrast was performed which denied our thoughts about the anastomotic leakage. intravenous cefuroxime and metronidazole antibiotics were prescribed. the crp level was . mg/l on the th postoperative day. the patient was discharged on the th postoperative day without preventive ileostomy. conclusion: using the original, standardized colorectal anastomosis inspection method we can determine which patient doesn't need the preventive ileostomy after low colorectal anastomosis. the important causes of anastomotic leak are local ischemia and staple line defect. the purpose of this study was to investigate the combination of methods aimed to reduce the risk of anastomotic leak after anterior resections for rectal cancer. methods: we retrospectively analyzed perioperative outcomes of the first patients, who underwent modified laparoscopic anterior resection with partial mesorectal excision for rectal cancer without preventive stomy. operative technique was modified and included routine preservation of the left colic artery (fig )(aimed to improve anastomotic blood supply), manual suture invagination of the 'dog ears' (fig ) (aimed to reduce the risk of staple line defects), transperineal pelvic drainage and pelvic peritoneum reconstruction (aimed to reduce the risk of reoperation in case of leakage). anastomotic leak rate, reoperation rate, left colic artery preservation rate, additional operative time (time required for left colic artery preservation, 'dog ears' invagination and pelvic peritoneum reconstruction), blood loss, morbidity and mortality were analyzed. results: ( . %) patient developed an asymptomatic leakage, which was managed conservatively. there was no postoperative mortality and no reoperations. median additional operative time was min for the first procedures and min for the last procedures. left colic artery preservation was successful in ( . %) patients. median blood loss was ml. conclusions: additional techniques used in our modification of laparoscopic anterior resection are safe and may lead to improved perioperative outcomes. however, they are associated with increased operative time, which may be reduced with a better learning curve. introduction: parastomal hernias are a significant cause of post abdominal ostomy morbidity with an overall life-time incidence exceeding %. the complications can range from a bulge resulting in stoma bag leakage, to life threatening bowel obstruction. the prevent-trial sought to determine if prophylactic utilisation of polypropylene mesh would decrease the incidence of parastomal hernias, with initial results demonstrating that it was safe to use in permanent end stomas. aim: to demonstrate a reproducible and streamlined technique for laparoscopic parastomal hernia repair with intraperitoneal funnel mesh, and assess the outcomes with the clavien-dindo (cd) classification tool. method: parastomal hernia repairs ( colostomy, ileostomy) were considered, with the following approach adopted for each: swab sutured in stoma orifice to prevent wound contamination.sharp dissection of the stoma using parachute technique.stoma end refreshed followed by change of gloves and instruments.lateral stay sutures placed to tighten sheath later on.pneumoperitoneum temporarily created to assess/divide adhesions.funnel mesh placed in-situ, orientated in the optimal intraabdominal position, and sutured to the peri-colic fat to prevent slip.medial suture placed to narrow the sheath further.pneumoperitoneum re-created and mesh fixed in place with double crown laparoscopic tacks.redundant portion of end stoma excised and stoma formed. results: at median follow up of months: no recurrence.no reported symptoms of pain or decreased stoma functionality.one superficial wound infection treated with drainage at bedside (cd = grade ) conclusion: laparoscopic parastomal hernia repair with intraperitoneal funnel mesh for permanent end stomas yielded good outcomes in our patient cohort. a streamlined and reproducible approach ensures that the technique can be adopted for both prophylactic, primary and recurrent repair. parastomal hernias are common and can be associated with significant morbidity. when taking this into account, in conjunction with the recommendations of the initial results of the prevent-trial, one may consider prophylactic utilisation of a mesh in patients receiving a permanent end stoma. general surgery, rambam medical center, haifa, israel year old, female patient referred to our institution with common bile duct stricture, caused by iatrogenic injury during laparoscopic cholecystectomy. during last year, patient suffered from recurrent episodes of ascending cholangitis. recently, she underwent ercp and severe stricture of middle cbd was diagnosed. plastic stent was inserted through the cbd. mrcp also showed severe stricture of cbd with dilatation of biliary tree, proximal to the stricture. due to severe and resistant (did not resolved by recurrent dilatation) structure of middle cbd, she was referred to operation. patient underwent da vinci robot-assisted excision of the cbd stricture, hepaticojejunostomy and extracorporeal jejunojejunostomy of roux-an-y limb. total operating time was min. day three after operation patient started regular diet and was discharged home on day four. final pathology has shoved part of cbd with severe inflammation. aims: extrahepatic biliary duct resection for the treatment of bismuth i and ii stage klatskin tumor is the standard surgical technique [ ] . methods: a years old patient present at emergency room (er) with right upper abdominal pain with an elevation of the inflammatory markers at the blood exams and fever. the patient was submitted to a computer tomography (ct) that shows a tumor involving the lower tract of the principal bile duct. an endoscopic retrograde cholangio pancreatography (ercp) with biopsy (intraductal papillary neoplasm of the bile duct,ipnb with high-grade dysplasia) and stent placement was performed. considering the good general conditions of the patient and an absence of vascular and nodal invasion at the preoperative imaging, a minimally invasive surgical resection of the biliary tract with cholecystectomy was performed. results: a four port laparoscopic biliary tract resection with cholecystectomy was performed with lymphadenectomy of the hepatic hilum. no vascular or liver infiltration was found. the hepatic hilum was completely skeletonized. the resection of the biliary duct was performed with adequate free margin. a biliary reconstruction with roux-en-y technique was performed and a fully laparoscopic hepatico-jejunal anastomosis was done. and abdominal retro anastomotic drain was placed. the operative time was min. the postoperative course was complicated by a low rate biliary leakage that was treated conservatively. the patient was discharged at post operative day in good general conditions. the histological examination revealed a moderately differentiated in situ cholangiocarcinoma of the principal bile duct with the involving of the cystic duct with free resection margin (pt bn r ). conclusions: laparoscopic resection of the biliary tract is a challenging procedure that allows, in expert hands, to achieve in selected cases negative pathological margin, complete linfonode retrieval and entero-biliary bypass. injury to the extrahepatic bile duct during bile duct or hepatic surgery can be reduced by better real-time visualization. recently, indocyanine green (icg) fluorescence imaging has been used in laparoscopic hepatobiliary surgery. we applied icg fluorescence imaging in patient with huge hepatic cyst which severely deviated extrahepatic bile duct. the patient had received laparoscopic cholecystectomy and huge hepatic cyst stuck firmly with peri-hepatic structures including bile duct. icg fluorescence imaging correctly identified the common hepatic duct and remnant cystic duct and allowed for more meticulous and easier dissection. therefore, icg fluorescence imaging may guide a safe and accurate dissection and excision in hepatobiliary surgery. results: total patients who underwent ercp were , and . percent ( cases) had a first failed ercp and of then were unsuccesfull in the second intent of ercp. intrahospitalary stay was more than days in the percent, in the . percent was to days, with and average of days. conclusions: before, during or after lcbde, ercp remains the gold standard for manegement of choledocolitiasis confirmed by clinics, laboratory and imagenology. lcbde is a very good option that requires experience and specific skills, and especialized equipment. in years the rate of sucess in our hospital was . % and there were no posoperatory complications such as: biliar peritonitis, pancreatitis or liver abscess. aims: easier intraoperative recognition of the biliary anatomy may be accomplished by using near-infrared (nir) fluorescence imaging after an injection of indocyanine green (icg). neither radiological support nor additional intervention such as opening the cystic or common bile duct is required, making it an easy and real-time technique to use during surgery. the aim of this video is to describe our experience in fluorescence-guided cholangiography in different clinical situations. methods: intravenous injection of icg is used to illuminate extrahepatic biliary anatomy. however, the simultaneous enhacement of liver parenchyma can disturb the visualization of clinical details. the key is in the used dose of icg, the route of administration and the time since its infusion. in the first case, a scheduled cholecystectomy is shown in which a dose ( ml of mg dye dilution in ml of distilled water) administered intravenously h before the intervention was used. the second case shows an urgent cholecystectomy in which the dose ( ml of mg dye dilution in ml of distilled water) was administered intragallbladder during surgery. all patients underwent laparoscopic cholecystectomy with traditional four-port technique. all procedures were performed using a -degree mm laparoscope with nir imaging capability (visera elite ii, olympus). results: there were no intraoperative or postoperative complications. there was no increase in operative time due to the use of icg. in the first case, a clear identification of the cystic duct and the main bile duct was obtained thanks to the biliary excretion of the icg and the intravenous clearance. in the second case, the identification of the cystic duct, the main bile duct and the cystic artery occurred due to the intravesicular absorption of icg. conclusion: fluorescence-guided cholecystectomy clarifies the dissection plane. it can be considered to increase the safety of laparoscopic cholecystectomy. being aware of the doses, times and possible routes of administration is basic to universalize the technique and give it utility in different scenarios. introduction: mirizzi syndrome type is an uncommon cause of obstructive jaundice caused by an inflammatory response to an impacted gallstone in hartmann's pouch or the cystic duct with a resultant cholecystocholedochal fistula. the obstructive biochemical changes can be caused by direct extrinsic compression from the impacted gall stone or from the fibrosis caused by advanced chronic cholecystitis, or for the established fistula. objective: we present a case of a mirizzi type syndrome with choledocholithiasis which was solved by laparoscopy approach. material and methods: a -year-old female patient with no past medical history. the history of present illness begans with the presence of icteric dye since the last days; she received symptomatic treatment with poor improvement. a liver and biliary tract ultrasound was performed with report of a mm coledochus, mm wall gallbladder. then an endoscopic retrograde clolangiopancreatography was performed with successful endoscopic sphincterotomy and removal of gallstones. but the patient jaundice persisted after the procedure. the patient underwent cholecystectomy and laparoscopic common bile duct exploration, where the findings were a mirizzi type according to the csendez classification, chronic cholecistitis and choledocholithiasis. results: in this laparoscopic approach we performed a partial cholecystectomy, bile duct exploration with removal of residual gallstones. the closure of the choledocotomy was performed with simple knots using vycril . . a subhepatic drainage was left. the patient showed adequate clinical evolution. after days the patient was discharged. conclusions: it is important to properly identify the anatomy at the time of surgery to avoid injury of the common bile duct. operative treatment of mirizzi syndrome type includes either laparoscopic or open subtotal cholecystectomy or placement of a t-tube or choledocoplasty. near-infrared fluorescent cholangiography (nirf-c) is an innovative intra-operative imaging technique that allows a real-time enhanced visualization of the extrahepatic biliary tree by fluorescence. thanks to the development of laparoscopes/endoscopes with light sources emitting infrared frequencies, it is possible to visualize anatomical structures (vessels, ureters, bile ducts, etc.) through the luminous intensity of substances (fluorescein, blue of methylene, indocyanine green) which are injected into the patient. this technology may be considered as an important teaching tool for laparoscopic surgery, especially for young surgeons in their surgical learning curve and it could lead to reduce the risk of iatrogenic bile duct injuries during laparoscopic cholecystectomy. the following video is characterized by a series of intra-operative images of biliary anatomy by fluorescence, having an important educational interest, while also detecting anatomical variations of the cystic duct. a. umezawa, minimally invasive surgery center, yotsuya medical cube, tokyo, japan aims: laparoscopic cholecystectomy(lap-c) for cholecystolithiasis has become standard. however, serious bile duct injury has been reported as a complication. repeated colic and chronic inflammation in cholecystolithiasis lead to the so-called difficult gallbladder conditions, such as dense fibrosis and scarring of the tissue. dissection of calot's triangle includes the risk of bile duct injury. critical view of safety (cvs) is the most well-known land mark for safe cholecystectomy. in the revised tokyo guidelines (tg ), important land marks and bailout procedures had been proposed. those are for the difficult gallbladder which are not able to achieve cvs. methods land marks: baseline of segment of the liver and sulcus rouvier should be confirmed. the gallbladder wall itself is also useful landmark. bailout procedure: when the dissection of calot's triangle is considered impossible, bailout procedures should be considered. subtotal cholecystectomy which leave the neck is one of option. the fundus first technique is another approach. however, because fundus first technique has a possibility of leading to serious bile duct injury, it should stop by the neck. in this video, first case shows the importance of landmarks from near miss cases of misidentified injuries. second case shows bailout procedure, subtotal cholecystectomy with fundus first technique. result: in the atrophic gallbladder (case , near miss), it is liable to misidentify the junction of common bile duct as the gallbladder neck. the neck and common hepatic duct were lifted together easily. with confirming the landmark, misidentification was corrected and bile duct injury was avoided.in the case , since the calot's triangle was obscured due to repeated cholecystitis, dissection of gallbladder was performed from the bottom to the neck, and was excised with the cervical portion remained. the remaining neck was reconstituted.in each case, intraoperative cholangiography was performed, and it was confirmed that there was no bile duct injury. without postoperative complications, those patients were discharged pod as usual lap-c. conclusion: during lap-c for difficult gallbladder, the most annoying part is bile duct injury. confirming landmarks and switching bailout procedures can be contributory to avoid bile duct injury and to achieve safe lap-c. aims: choledocholithiasis is an important cause of morbidity and is present in about % of patients submitted a cholecystectomy. his treatment should be done in the same operative time, avoiding the morbidity and hospitalization time and costs of multiple procedures.the transcystic approach is preferable to prevent morbidity associated to choledochotomy.large stones can preclude this procedure. the use of laser lithotripsy to stone fragmentation is an option to provide transcystic extraction. methods: we present a video of laparoscopic transcystic common bile duct (cbd) exploration for choledocholithiasis. results: female patient, years old with a previous hospitalization for acute cholangitis with choledocholitiasis.submitted to laparoscopic cholecystectomy with intraoperative cholangiography that showed the presence of stone in distal cbd with cm size. the use of holmium laser lithotripsy made the stone fragmentation and provided his extraction by transcystic route using a basket.the patient was discharged at th postoperative day, with no complications. conclusion: the use of laser lithotripsy for large cbd stones is safe and effective, making possible the transcystic approach and preventing the choledochotomy morbidity. surg endosc ( ) :s -s gallbladder adenocarcinoma is rare and extremely aggressive. its' incidence is higher in elder females and its progression is rapid and silent with a dismal prognosis if diagnosed at advanced stages. we present the case of a years-old female with dyspeptic complaints. the abdominal ultrasound revealed a cm solid lesion of the gallbladder suspect for malignancy. the ct confirmed the presence of a vegetant mass on the free border of the gallbladder fundus with x mm. we performed a radical cholecystectomy with lymphadenectomy and liver bed excision. the post-operative period was complicated with a urinary tract infection, with full recovery after antimicrobial treatment. the histological sample revealed an adenocarcinoma of the gallbladder (t bn m ) and the patient remains asymptomatic and tumour free months after the surgery. gallbladder cancer treatment depends of the stage and clinical presentation of the disease. complete surgical excision is the only curative treatment and should include a limited hepatectomy and portal pedicle lymphadenectomy. laparoscopic surgery might be an option in early stages, although it is challenging and requires both expertise in hepato-biliary and laparoscopic surgery. seen at the emergency room for a two month history of abdominal pain associated with jaundice. she is evaluated by the surgical team and diagnosed with acute cholecystitis and moderate risk for choledocholithiasis. the initial surgical plan was cholecystectomy with intraoperative cholangiogram. during surgery, firm adhesions are found from the gallbladder to omentum. friable tissue with edema and easy bleeding. difficulty is encountered during the dissection of calots'triangle. an intraoperative cholangiogram is done through hartmans'pouch without identifying correctly the biliary tract. therefore, an endoscopic retrograde cholangiopancreatography (ercp) is done to visualize the correct anatomy. during the ercp, a stenotic common hepatic duct is found and no stones are visualized. a biliary endoprosthesis is placed. she is discharged asymptomatic. a month later, the patient is back in the emergency room with abdominal pain. after an abdominal ct scan, we found that the endoprosthesis had migrated to the th portion of the duodenum. a second ercp is done and this time we found a big stone ( . - cm) aims: when training in the residency you watch your teacher perform laparoscopic cholecystectomy with ease, and even yourself perform several steps. but as a young surgeon, when confronted with a patient with acute cholecystitis, you're filled with emotions, and you do not know where to start the gallbladder dissection. the aim of this presentation is to show to young surgeons that you can, and must achieve, critical view of safety when performing laparoscopic cholecystectomy for acute cholecystitis. methods: we present the case of a years old female patient, bmi of . , who presented with a grade ii (moderate) acute cholecystitis. following tokyo guidelines, we initiated antibiotics and general supportive care, but without clinical improvement. the patient was proposed for laparoscopic cholecystectomy. results: at initial exploration we identified a cm long gallbladder, with a thick wall, difficult to manipulate. we opted for an anterograde cholecystectomy, in our opinion the best option in acute cholecystitis. the dissection was started with hook electrocautery and then continued with a combination of blunt dissection with the aspirator and with the hook. when reaching the pedicle, blunt dissection was used in order to appreciate the anatomy of the cystic duct and cystic artery. after correct identification of these structures they ware clipped and cut. a drainage tub was then placed, and the abdomen deflated. conclusion(s): as a young surgeon, when dealing with acute you must maintain your calm, and try to achieve critical view of safety before transecting the cystic duct and cystic artery. this can be achieved with a combination of blunt and sharp dissection, keeping your camera clean and with a good collaboration with the assisting surgeon. conclusions: here, an easy and reproducible method is described for future macroscopic analysis by the surgeon following a cholecystectomy. in addition, we depict several frequent macroscopic abnormalities in order to provide surgical colleagues with some cases of abnormal macroscopic gallbladders. the left hepatectomy is a demanding and difficult procedure, still limited to reference centers. the caudal approach and exposure of the middle hepatic vein is a reliable way to achieve a safely and reproductible left hepatectomy. with this technique, exposing the middle hepatic vein, we believe that we can perform a safe and feasible laparoscopic left hepatectomy increasing the quality of this hepatectomy. we present a -year-old woman with an intrahepatic and common bile ductlithiasiswhich was previously submitted to an ercp. with an unsolved intrahepatic lithiasis the patient was proposed to alaparoscopic left hepatectomy. the minimally invasive approach for alpps in a patient with a large hepatocellular carcinoma in a liver with severe steatosis is shown. during the first stage a partial alpps is performed. pve is performed in postoperative day one. after days from the first stage both liver volume and function (by hida scan) are re-assessed. right hepatectomy (second stage of alpps) is then conducted by laparoscopic aproach. hepato-bilio-pancreatic, centro hospitalar são joão, porto, portugal a year old woman with a previous history of anxiety and catheter ablation to treat heart arrhythmias, was studied for for multiple pancreatic cysts incidentally discovered on a routine ultrasound. an mri was performed showing multiple cystic tumors throughout the pancreas, the largest of which was mm. this led to a suspicion of multi-focal, side-branch intraductal papillary mucinous neoplasm (ipmn), with minimal dilatation of the main pancreatic duct. an echo endoscopy was subsequently performed indicating probable multifocal ipmn. a fna was carried out during this procedure, with aspiration of cystic content which was sent for cea analysis and cytology. cytology was compatible with mucinous neoplasm with mild atypia and cea u/ml. a splenic preserving total laparoscopic pancreatoduodenectomy was proposed. the procedure was uneventful and the patient was discharged on the th post-operative day. pathology revealeded a mm ipmn, with severe dysplasia and foci of microinvasive ductal adenocarcinoma of mm-pt n r . indocyanine green immunofluorescence guided laparoscopic partial hepatectomy y. tai obtaining negative tumor margin during laparoscopic hepatectomy has always been a very challenging topic for surgeons in that the surgeons are not able to palpate the tumor during laparoscopic surgery. although intraabdominal echo is available, but it demands great experiences and skills. with the guidance of icg immunofluorescence, surgeons can avoid failure of not obtaining enough negative margins nor resect too much healthy liver. icg is often used to estimate the liver function prior to hepatectomy traditionally. it binds to plasma protein and has a peak absorbance at nm and emits fluorescence with a wavelength of approximately nm. icg is preferentially retained in or around biliary malignancies due to impaired biliary excretion of hepatocytes in the affected area. we performed icg immunofluorescence guided laparoscopic partial hepatectomy on a years old male who suffers from hcc located at segment and . icg was injected days prior to the operation day. while evaluation of liver is performed, it also allowed us to use a high-end laparoscopic camera system equipped with integrated filters for detection of near-infrared fluorescence. during the surgery, we were able to clearly locate the borders of malignancies through the use of integrated filters combine with icg injection. the pathology study also confirmed that the adequate tumor free margin ([ . cm) were obtained in both tumors and the patient's condition was stable as well. icg immunofluorescence guidance enables surgeons to obtain optimum result in tumor resection through laparoscopic surgery. it also has the ability to detect bile leakage. with the use of icg immuofluorescence, surgeons will have higher chances to achieve adequate negative margins. background: parenchymal sparing hepatic resection has the advantage of preserving valuable tissue in chemotherapy-treated livers, assuring an adequate future remnant volume without compromising long-term survival. moreover, the laparoscopic approach offers the decreased postoperative morbidity of minimally invasive surgery. whenever technically feasible, this kind of procedure should be considered a suitable alternative to the classic major hepatectomy for the treatment of multiple colorectal liver metastases. methods: -year old male with a previous history of laparoscopic sigmoidectomy in november for a pt n m sigmoid adenocarcinoma. a control scanner three years later showed liver metastases in segments v, viii, ii and caudate lobe. after chemotherapy (xelox), control mri and pet scans showed a good response. he was proposed for a laparoscopic parenchymal-sparing liver resection. results: total operative time was h and min with no intraoperative complications. patient presented a right atelectasis as the only postoperative complication and was resolved with respiratory therapy. he was discharged in days. pathology report showed that lesions on segment v and viii had no viable tumor ( % fibrosis) and lesions on segment ii and caudate lobe had moderately differentiated adenocarcinoma. margins were free in all the lesions. after a month follow up, the patient has no recurrence and normal liver function tests. conclusion: minimally invasive liver resection is possible in patients with multiple bilobar liver metastases and allows to perform parenchymalsparing surgery safely. difficult localization of lesions such as the caudate lobe are not a contraindication for this type of surgery. laparoscopic approach for perihilar cholangiocarcinoma is still poorly reported in the literature due to technical challenges secondary to the combination of major hepatectomy, lymphadenectomy and biliary confluence resection. despite this, in selected cases it can be a good option to provide a short term benefit to patients. the video reports the case of a perihilar cholangiocarcinoma with involvement of left bile duct and therefore requiring left hepatectomy. komagome hospital, bunkyo-ku,tokyo, japan aims: segmentectomy is an anatomic liver resection, in which the tertiary branches of the glissonean pedicles are selectively transected. however, the branching pattern of the tertiary branches varies depending on the case, particularly in segment (s ) and segment (s ). the extrahepatic approach to the glissonean pedicle from the hepatic hilum is very difficult depending on the branching pattern. furthermore, the distance of exposing the secondary branches that are to be preserved becomes longer, and there is an increased risk of biliary leakage and delayed biliary stricture due to excessive traction in laparoscopic surgery. therefore, laparoscopic s and s segmentectomy are considered technically difficult. we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. methods: we standardized the intrahepatic glissonean pedicle approach for laparoscopic s and s segmentectomy. we identify the targeted glissonean pedicle intrahepatically after the parenchymal transection along the major hepatic vein or its branch running on the intersegmental plane, referring to the preoperative simulation by d imaging. (a)s segmentectomy; after the mobilization of the right lobe, the glissonean pedicles of s (g ) can be approached from the dorsal side by transecting the parenchyma between the ivc and the right hepatic vein. after the division of the g , the parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. (b)s segmentectomy; first, the parenchyma is transected along the middle hepatic vein (mhv) from the root side to the peripheral. g is typically detected on the right dorsal side of the mhv. after the division of the g , the liver parenchyma is transected along the demarcation line and the rhv from the root side to the peripheral side. results: we have experienced cases of laparoscopic s segmentectomy and cases of laparoscopic s segmentectomy. conclusion: our approach to the g and the g is safe and very useful. laparoscopic anatomical segmentectomy of right anterior section is technically demanding because it is difficult to dissect the deep tertiary branches of right anterior portal pedicle (rapp). we present three cases of laparoscopic anatomical segmentectomy using the extrafascial and transfissural approach: ) anatomical resection of segment , ) anatomical resection of the ventral area ) anatomical resection of segment dorsal area. the extrafascial and transfissural approach means that the liver parenchyma along the fissure lines is opened, then the surgeon can confirm the glissonean pedicles and territory directly. the extrafascial and transfissural approach in laparoscopic anatomical segmentectomy of right anterior section is feasible and effective because this technique can easily be approached to the deep tertiary branches of rapp. repeated liver resection has significant role in patients with recurrent hepatocellular carcinoma (hcc) in several situations. laparoscopic redo surgery is becoming safer along with advance in surgical technique. we have performed laparoscopic re-resection for limited intrahepatic hcc recurrence. the aim of the present study was to investigate its significance comparing with first laparoscopic liver resections. subjects: patients with limited intrahepatic hcc recurrence after open hepatectomy underwent laparoscopic liver re-resection (n = ). methods: adhesion between abdominal wall and visceral organs was carefully divided, after the first laparoscopic port was safely inserted. adhesion between diaphragm and liver surface or between previous liver cut surface and colon or duodenum was also minimally dissected. approach to the glisson's pedicles at the hepatic hilum was often difficult due to previous surgical procedure, thus pringle's maneuver was generally applied. dissection of hepatic parenchyma approaching to the target glisson's branch was often preceded under the ultrasound-guidance. liver resection was performed using lcs, biclamp, and cusa using intermittent block of the hepatic inflow. operation time, intraoperative bleeding, morbidity, mortality, and postoperative hospital stay were compared with those in patients who underwent first laparoscopic liver resection during the same period (n = ). results: operation time was significantly longer in the re-resection group, possibly due to the adhesiolysis. meanwhile, no significant difference was detected in intraoperative bleeding, morbidity, mortality and postoperative hospital stay between the first and the redo surgeries. methods: the donor was a -year-old gentleman who decided to donate part of his liver to his wife suffering from viral liver cirrhosis and hepatocellular carcinoma. his bmi was . kg/m and the preoperatively estimated donor's right liver volume was ml, representing . % of his entire liver. with the recipient's weight of kg, the graft to recipient weight ratio (grwr) was . %. the liver had classic hilar anatomy except that the right posterior intrahepatic duct seperately joined to the left main hepatic duct. after isolation and clamping of right hepatic artery and portal vein, indocyanine green of . mg was injected intravenously. results: the total operation time was min and the estimated blood loss was ml without transfusion. indocyanine green fluorescence image clearly demonstrated the anatomical demarcation between the lobes and visualized the running of the biliary tree. his postoperative course was uneventful and discharged postoperative day . conclusion: real-time indocyanine green fluorescence image may be particularly helpful to delineate anatomical surgical plane and to determine the appropriate division point of hepatic duct during laparoscopic living donor hepatectomy. surg endosc ( ) the correct management of intraoperative volemic status is essential in laparoscopic liver resection in order to control bleeding and to perform even complex procedures with a good profile of safety. central venous pressure is not really reliable in laparoscopy, due to presence of the pneumoperitoneum and patient position. monitoring of haemodynamic parameters via vigileo system is a minimally invasive method to control stroke volume variation, cardiac output, cardiac index and oxygen delivery in order to optimize the anaesthesiological management by controlling venous bleeding and avoiding tissutal ischemia. introduction: non-hydatid liver cysts represent a heterogeneous group of disorders that differ in their etiology, prevalence and clinical manifestations.within them, the simple hepatic cyst is the most frequent.the majority of simple cysts are an incidental finding during the performance of an imaging test for another unrelated cause and few of them are symptomatic or are associated with complications, and surgery is not necessary in most of them. described various therapeutic approaches so far there is no consensus about the optimal treatment of simple symptomatic, complicated or growth-showing liver cysts during its follow-up. currently the laparoscopic approach is widely used for the management of cysts hepatic, with results similar to open surgery but with the advantages of laparoscopy. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the approximation of complicated simple hepatic cysts.material and method: clinical case: a -year-old female patient with a history of: giant hiatus hernia intervention with laparoscopic nissen, fibromyalgia, previous ischemic colitis. hospital admission due to pneumonia and right pleural effusion with us: simple cyst x x mm in segment v hepatic, with dilatation of biliary radicals adjacent to the cyst, distended gallbladder with irregular walls in the hepatic side. ct: cystic lesion in segment iv-v of the liver, which has increased in size, with small microabcesses adjacencies to the lesion, thickening of the gallbladder wall, to assess cholecystitis. antibiotic treatment is established with good evolution, deciding surgery. results: intervention: complete laparoscopic approach, trocars, edematous cholecystitis, large retroyuxta vesicular cyst,with thickened walls with serous content. cholecystectomy maintaining the cyst wall, puncturing and taking samples for cytology and biochemistry of the contents, resection of the cyst wall, partial flare of its internal surface, negative intraoperative biopsy, epipoplasty, with drainage placement.correct postoperative course.pathological anatomy: simple biliary cyst with negative cytology, ck ?, ck -, calretina-. conclusion: the treatment of choice of complicated simple hepatic cysts is laparoscopic.we recommend performing an intraoperative biopsy of all resected liver cysts to confirm its nature,we propose cyst enucleation as the best surgical treatment. objective: the objective of the following case is to present a patient with symptomatic polycystic liver disease, which was solved by laparoscopy approach and the management of its complications. material and methods: the case reported is about a years old female patient with abdominal pain in upper right quadrant associated to asthenia, adynamia and hyporexia. ct scan reported heterogeneous liver with multiple ovoid images with regular edges defined which the biggest one measure x x mm with volume of cc on segment and , which comprises stomach, and the other one in segment with a volume of cc and others small sized located in segment , and b. results: in this laparoscopic approach, we performed a cyst unroofing of the two biggest cysts as well as cholecystectomy because of firm and lax adhesions. the patient evolved with fever in the th day postsurgical day and biliary leaking in a volume of cc in hrs. an ercp (endoscopic retrograde cholangiopancreatography) was asked for that was carried out by finding leak at the intrahepatic biliary duct therefore; esphinterotomy with placement of plastic endoprotesis was performed. the patient evolved without complication and was discharged at the th day. conclusions: only symptomatic polycystic liver disease needs to be treated. the choice of treatment is not yet standardized, for voluminous cysts the unroofing ideally by laparoscopy is the gold standard and the ercp is the elected treatment when the biliary leak appears as a complication. introduction: laparoscopic liver resection (llr) for tumors located in the posterosuperior segments of the liver (segments (s) or ) is a challenging procedure. especially, llr for s is difficult because the access of instruments is limited, bleeding control is not feasible, major llr is sometimes required, and obtaining sufficient resection margin is not easy. to overcome this obstacles, we performed llr in s with a lateral approach using intercostal trocars. to obtain competent resection margin, llr through right hepatic vein (rhv) first approach was performed for . cm mass located near the rhv in a year old female. case: after full mobilization of right liver including all short hepatic veins and caudate lobe, rotate the whole liver completely to the left side to approach to the root of rhv. one intercostal trocar was inserted to access the lesion. parenchymal transection started from the confluence of hepatic vein and then, followed along rhv with ligating several small branches from rhv. resection margin was demarcated after localization using laparoscopic ultrasonography. after completion of parenchymal dissection using cusa and ultrasonic shears, hemostatic agents were applied and drain was inserted. operation time and estimated blood loss were mins and ml. the patient was discharged without any complication on postoperative day . final pathological assessment confirmed clear resection margin (safety margin : . cm). conclusion: laparoscopic s segmentectomy with hepatic vein first approach technique is safe and recommended to obtain better resection margin. aims: simple liver cysts are the most common cystic lesions of the liver. most are diagnosed casually in image tests such as ultrasound or computerized tomography, most of which are asymptomatic and do not require treatment. in symptomatic patients (abdominal distension with palpable mass, abdominal pain, dyspnea, jaundice, etc.) the clinical manifestations are usually due to the growth of the cysts or the compression of neighboring structures. liver function tests are usually not altered. intracystic complications occur in less than % of cases and malignancy is exceptional. in this video, we present the case of a symptomatic patient with polycystic liver disease including a large size hepatic cyst. material and methods: -year-old woman with a personal history of arterial hypertension, saos, partial hysterectomy due to endometrial cancer, who was referred to our department complaining of supraumbilical pain and abdominal distension with palpable mases. abdominal ultrasound showed cholelithiasis and multiple simple hepatic cysts. in ct scan, multiple hepatic cysts were found, the largest one of about cm of larger diameter. echinococcus granulosus serology test was negative. there was also no evidence of cancer disease in pet scan. results: a laparoscopic approach was performed with four trocars, three of mm and a hasson trocar inserted thought a umbilical small incisional hernia. aspiration and wide unroofing of the large size cyst and smaller accessible ones was done. the patient also underwent cholecystectomy with intraoperative cholangiography and umbilical eventroplasty. the patient recovered uneventfully and is asymptomatic one year after surgery. conclusion: simple liver cysts rarely require treatment. in some cases, especially in large, complicated and symptomatic simple liver cysts, surgery is indicated. laparoscopic fenestration treatment is the best choice. aims: liver resection is the preferable initial treatment option for solitary or limited multifocal hepatocellular carcinomas. surgical indications for laparoscopic liver resection (llr) are the most important consideration, like liver function, tumor size (diameter less than cm) and location (easy technical access like in the left lateral section or on the surface of the inferior region). partial liver resection or left lateral sectionectomy are the typical procedures for such tumors and are considered the best way to begin llr. with accumulating experience and technical advancement, llr has been performed for tumors larger than cm and for others locations. some requirements to perform llr are to have experience in liver surgery and laparoscopic also, adequate technology and intraoperative ultrasound. methods: a -year-old male smoker, ex-parenteral drug users with chronic hcv liver disease child-a stage. he is diagnosed with a single lesion of cm in segment iii of the liver, biopsied twice without conclusive diagnosis and with a three-phase ct suggestive of hepatocarcinoma li-rads with data of portal hypertension (pht) and mild ascites. after the study is commented on tumor committee deciding surgical intervention. results: a laparoscopic resection of segment iii was performed with trocars. liver is explored by intraoperative laparoscopic ultrasound. vascular control was performed using the pringle technique. liver transection was done with sonostar until identification of intraparenchymal segment iii vascularization, which is sectioned with endogia ( mm) with seamguard. after the resection, we perform hemostasis control with electrocoagulation and hemostatic material. intraoperative bleeding of ml. favorable postoperative evolution, high on the th postoperative day. ap: cm trabecular hepatocarcinoma moderately differentiated pt b, r resection. conclusions: llr allows major liver resections with low morbidity and mortality and the advantages of laparoscopic surgery. an efficient learning curve can be achieved by a parallel evolution of procedures and indications (according to modified bclc staging system and treatment strategy). studies suggest that llr results in less blood loss, shorter postoperative hospital stays, lower abdominal wall trauma and lower incidences of ascites accumulation and postoperative liver failure. with respect to oncological considerations, tumor margins are adequately maintained during llr. v. drakopoulos, s. voulgaris, i. iliadis, k. botsakis, p. trakosari, v. vougas st department of surgery and transplantation unit, district general hospital of athens « evangelismos » , athens, greece introduction: laparoscopic surgery is gaining acceptance in the treatment of liver metastasis. laparoscopic treatment of liver metastasis often presents technical difficulties and requires an extensive learning curve. material-method: we present the case of a year old woman presented with a liver metastasis in section of the liver. the patient had been submitted to a laparoscopic low posterior resection in february . patient underwent laparoscopic left lateral hepatectomy, with the use of three trocars (umbilical mm, and two in the midclavicular line bilaterally.) left lateral hepatectomy was conducted with the use of a linear stapler. the postoperative period was uncomplicated and the patient remains in good condition three months after surgery. conclusion: laparoscopic approach seems to be safe for treatment of liver metastasis, offering better surgical field view and less postoperative complications. year survival rate after laparoscopic hepatectomy is compared to the open approach. general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan purpose: laparoscopic hepatectomy is a quickly growing method for liver tumor because of modern technology. but for the ihd thrombosis, it is still technique dependent. the video was tried to share our experience for special case. material and method: one y/o female patient suffered from fever episode and image show s cm hcc with right anterior ihd obstruction r/o tumor thrombosis, hilum ln enlargement, double right portal vein, hilum adhesion with duodenum, no ascites . lab data : no-b, no-c child a, afp , icg clearance rate . %, plt . heart, lung function exam normal. the laparoscopic right total hepatectomy and hilum ln dissection was conducted. results: laparoscopic approached was performed. the hilum ln dissection was done with vessel and bile duct isolation. hilum ln frozen show negative malignancy. hemi-vessel control was done with resecting the vessel. right hepatectomy was done with preserving middle hepatic vein. the right anterior and posterior ihd was opened and tumor thrombosis was removed from right anterior ihd carefully. the stump of ihd was closed by suture separately. the total op time was min with cc blood loss. post op minimsl bile leakage was found in the drain at day . the patient discharged at day with drain. conclusions: laparoscopic hepatectomy may be a feasible method for hcc even with ihd tumor thrombosis. surg endosc ( ) introduction: the progressive laparoscopic learning in gastric surgery and the great development of instruments and laparoscopic material that facilitates the realization of advanced procedures, has led to an increase in the use of laparoscopy in the treatment of gastric cancer. material and methods: we present the case of a -year-old man without amc with a history of ischemic heart disease who enters our surgery department for cholangitis secondary to choledocholithiasis. ercp is requested during his admission that describes a gastric lesion from which a biopsy is taken, making it impossible to access vater papilla to perform sphincterotomy and lithiasis extraction due to the existence of duodenal diverticula. the result of pathological anatomy of the gastric lesion was compatible with adenocarcinoma. negative extension study. the clinical case is presented in a committee of multidisciplinary tumors and it is decided to perform surgical intervention of both pathologies. a subtotal gastrectomy was performed with a roux-en-y reconstruction. surgical time of min. choledochotomy was performed with lithiasis extraction, as well as intraoperative exploration of the bile duct and main conduits by means of a choledochoscope. results: income of days, with a clavien ii. the definitive pathological anatomy was an ai stage with a total of isolated nodes without evidence of neoplasia in any of them, therefore it does not require adjuvant treatment. the patient is asymptomatic, with nutritional supplementation with follow-up in ccee of surgery. conclusions: in our case, there were no serious postoperative complications when performing gastric resection and bile duct exploration with drainage of the same. from the oncological point of view, the number of lymph nodes extracted and the surgical margins are similar to those obtained in patients in whom we perform open surgery; therefore, although it is a single clinical case, laparoscopy in expert surgeons is a safe and effective technique. the puestow procedure was initially proposed to alleviate the pain in patients with chronic pancreatitis and dilated wirsung duct. its objective is to provide an efficient drainage of the pancreatic fluids and, in the meantime, to preserve the pancreatic tissue and minimize the risk of endocrine and exocrine pancreatic insufficiency. aims: to describe the particular technical aspects and the efficacy of totally laparoscopic puestow procedure in patients with cystic duodenal dystrophy. methods: a years old patient presenting diffuse epigastric pain, vomiting and weight loss was diagnosed at endoscopic ultrasound and biopsy with cystic duodenal dystrophy. a conservative treatment was decided with octreotide and opioids. however, due to the persistence of symptoms surgery was performed. results: due to the association of a dilated wirsung duct, the patient was submitted to a puestow procedure. the surgical procedure was completed in a minimally invasive manner; after dissecting the anterior surface of the pancreas an intraoperative ultrasound was performed in order to identify the wirsung duct. therefore, the pancreatic parenchyma was transected along the wirsung duct, a totally laparoscopic pancreato-jejunostomy on roux en y limb being performed. the early postoperative outcome was uneventful, the patient being discharged in the sixth postoperative day. at one month and six months follow up the need for opioid treatment significantly diminished. a kinking of the enteral anastomosis required a laparoscopic intervention one year after with a very good evolution after. conclusions: totally laparoscopic puestow procedure seems to be a safe and efficient method in order to treat symptomatic patients with cystic duodenal dystrophy in whom a dilated wirsung duct is present. aims: the approach to the intraductal papillary mucinous neoplasm (ipmn) is various, from a radiological follow-up with magnetic resonance (rm) to the surgical treatment with a pancreatic resection [ ] . the surgical approach is various and depends on the localization of the lesion and on the surgical skills [ ] . methods: a years old patient was admitted at the chi possy-saint germain-en-laye with an acute pancreatitis. at the ecoendoscopy was found a pancreatic cystic at the junction of the pancreatic body and tail with a wirusng diameter of mm. a second episode of acute pancreatitis occurred a few months later. after that episode the patient was submitted to a computer tomography (ct) that found a cystic lesion of cm with an increasing dilatation of the wirsung duct. the serum ca - was ui/ml. a laparoscopic sils distal pancreatectomy with spleen conservation was performed. results: a trans-umbilical incision was performed with the positioning of the gelpoint sils platform with the placement of trocars. a distal pancreatectomy with a spleen preservation and without a standard linfadenectomy was performed. the pancreatic stump was closed with an endo-gia mm with seamguard device. any drain was placed. the post-operative course was uneventfull. a ct scan was performed in …. post-operative day which didn't show collections. the patient was discharged in -…… post'operative day. the histological examination shows an ipmn with low grade dysplasia. no invasive carcinomatoses cells were found. the distal pancreatic sils resection with spleen conservation is a feasible and safe technique that combine all the advantages of the minimally invasive laparoscopic approach with the esthetic advantages of the sils approach. pancreato-duodenectomy is a complex surgery, requiring several anastomoses to reconstruct the digestive tract. due to its technical complexity, the laparoscopic approach is not yet the goldstandard and there remains some controversy about its oncological safety. worldwide experience is limited, and its safety and effectiveness are yet under evaluation.we present the clinical case of a years-old woman with a prior history of epilepsy. she was studied due to painless obstructive jaundice and a cm pancreatic head tumour was diagnosed on imaging, causing cbd and wirsung channels' dilatation. the tumour was considered locally resectable and she was proposed for a radical pancreato-duodenectomy.we present the main steps of the surgery including the oncological resection with lymphatic basin clearance and totally laparoscopic reconstruction.the post-operative was uneventful, and the histologic sample revealed a ductal adenocarcinoma (t ) with an r resection and / lymph nodes invaded. although technically demanding, laparoscopic pancreato-duodenectomy is safe and effective requiring teams with experience both in pancreato-biliary and laparoscopic surgery. chronic pancreatitis is characterized by a progressive pancreatic fibrosis with loss of endocrine and exocrine function. one of its main symptoms is debilitating pain. surgical drainage of a dilated pancreatic duct is an option to consider in cases of refractory pain. longitudinal pancreato-jejunostomy allows an effective decompression of the pancreatic channel and a significant improvement in the quality of life. we present the clinical case of a years-old lady with a prior history of gallstones. she was treated for an acute pancreatitis in may , followed by recurrent relapses of pain and enzymatic elevation. she required opioid use for partial pain control and a significant kg decrease on body weight due to 'fear of eating'. the endo-ultrasonography and the mri revealed a chronic pancreatitis with an mm wirsung duct with ductal stones and an atrophic body and tail. we proposed a laparoscopic longitudinal pancreato-jejunostomy. the surgery was performed with trocars, with the surgeon on the right side of the patient. we performed a trans-mesocolic cm pancreato-jejunostomy. the post-operative was uneventful, and the patient was discharged on the th post-operative day, asymptomatic. laparoscopic longitudinal pancreato-jejunostomy, although effective is a technically demanding surgery but brings the benefits of a minimally invasive approach. background: preservation of spleen in distal pancreatectomy is also useful from the maintenance of platelets and the prevention of overwhelming post splenectomy infection. we have performed laparoscopic spleen preservation distal pancreatectomy: lspdp to benign and low-grade tumors of the pancreatic body tail. the aim of this study was to report our surgical experience with the method of svp: splenic vessel preservation and wt: warshaw technique of lspdp, describe our techniques with videos. method: there are three points of our surgical technique. , precede pancreatic dissection, improve the mobility of the pancreas. , confirming the courses of splenic artery and classified them into two major types. , preserving the left gastro-epiploic vessels and short gastric vessels.the postoperative cases of lspdp which performed from april to september was retrospectively studied. result: of consecutive patients were performed lspdp at our institute, were svp and were wt. ages, gender and bmi were similar for two groups. there were no significant differences in operative time, blood loss and length of stay after surgery. comparing pathological finings, wt was associated with a slightly large tumor lesion (median mm vs. . , p = . ). among the median observation period of months, splenic infarction was observed in case in svp and cases in wt. however, they were focal splenic infarctions, they did not need surgery or drainage. there were no cases in which late onset of splenic artery occlusion or esophageal / gastric varices. conculusion: after performing lspd, the function of the spleen was good in all cases. both svp and wt were safe and feasible procedures. this is the case of a -years-old lady presenting with recurrent abdominal intractable pain she has been suffering from for the last years. msct revealed pancreatic calcifications from mm to - mm and dilatation of the main pancreatic duct in the body of the pancreas up to mm. the patient underwent laparoscopic local resection of the head of the pancreas combined with longitudinal roux-en-y pancreaticojejunostomy-a technique known as frey's procedure. it is recognized as an effective therapeutic option for the surgical treatment of patients with persistent pain caused by chronic pancreatitis.after performing the posterior wall of the pancreaticojejunal anastomosis we've faced an intraoperative complication such as volvulus of the roux limb causing serious ischemia of the limb. we were forced to remove all previous sutures in order to untwist the roux limb, thereafter the pancreaticojejunostomy was started anew.the purpose of this video is to demonstrate that frey's procedure can be performed in a minimally invasive fashion, which provides all the well-known advantages of this approach. we demonstrate that even such serious intraoperative complication as volvulus of the roux limb can be managed without conversion. our center has an experience of over laparoscopic frey's procedures, however this is the first case where we encountered with such complication and we believe this is an experience worth sharing.yet we would like to underline that this approach should be used by highly skilled minimally invasive surgeons experienced in intracorporeal suturing which is the most challenging stage in frey's procedure. v. tomulescu, i. hutopila, c. copaescu spleen preserving distal pancreatectomy (spdp) is commonly applied in patients with benign or low-grade malignant tumors in the body and tail of the pancreas. two surgical techniques for spdp have been described. the first technique was described by kimura (spleen preserving distal pancreatectomy with splenic vessel preservation-spdp-svp) and preserves the main splenic artery and vein and excises the tail of the pancreas and those small, short vascular connections to the body;the second technique was described by warshaw and involves resection of the splenic vein and artery before distal pancreatectomy, and conservation of theshort spleno-colic and gastric vessels to keep normal blood flow for the spleen (spleen-preserving distal pancreatectomy with splenic vessel resection-spdp-svr). we present the case of a years old female with / mm tumor of the pancreatic tail on ultrasonography. ct scan confirmed the tumor and endoscopic ultrasonography with fna have shown a solid pseudopapillary tumor. due to the low grade malignancy we have decided to perform a laparoscopic spleen preserving distal pancreatectomy with splenic vessels preservation (lspdp-svp). for lspdp-svp the difficulty is related with the splenic vessels dissection and manipulation. primary dissection and control of main trunk of splenic artery and vein will help to quickly control bleeding during vascular rupture in small vessels dissection. optimal stapling of any tissue requires an adequate tissue compression time to allow elongation of the tissue being compressed, smooth firing of the instrument, consistent staple line formation balanced against the risk of increased tissue tearing and excessive tensile strength. this is why, for pancreatic division, we prefer choosing a cartridge loaded with higher staplers. the pancreatic stump transection line is evaluated for bleeding and when it is needed, hemostatic clips are applied. histology report confirmed a solid pseudopapillary tumor t nomxl v r at this moment with month good follow up. in conclusion lspdp-svp is safe, reproductible and demonstrated very good outcomes when certain indications are respected. surg endosc ( ) aim: advances in minimally invasive surgery has permitted to perform complex techniques by this approach, being the laparoscopic duodenopancreatectomy (lpd) one of these. the aim of this communication is to present a surgical technique video for a complete laparoscopic pd, showing the most important steps of the resective and reconstructive phase, with the anastomosis realized completely by laparoscopy. methods: a surgical technique video is presented showing the main steps for the lpd and a complete laparoscopic reconstruction with an hepaticojejunostomy, duct-to-mucosa pancreatic-jejunostomy and a gastrojejunostomy. results: an years old woman with past medial history of arteria hypertension, dyslipidemia, type ii diabetes mellitus and a breast cancer treated in with lumpectomy and axillary lymphadenectomy plus radiotherapy, recently diagnosed of and adenocarcinoma of the head of the pancreas. the ct scan showed a neoplasia localized in the head of the pancreas without extension to other organs. a laparoscopic pd was indicated after a multidisciplinary committee evaluation. a supraumibical hasson trocar was used for the pneumoperitoneum, three mm trocars and two mm trocars were used. lpd was performed. the resective phase was done following the conventional steps of the open whipple procedure and for the reconstructive phase, a child limb was used for a termino-lateral hepatico-jejunostomy with an absorbable / monofilament; a duct-to-mucosa pancreatic-jejunostomy with an absorbable / monofilament and finally a latero-lateral mechanical gastro-jejunostomy was performed. surgical time was min. postoperative course without complications and the patient was discharged on the th postoperative day. definitive anatomopathological exam: intraductal tubulopapilar neoplasia, x x mm, with wide high grade epithelial dysplasia. free margins. ptisn ( / ). conclusion: laparoscopic pd is a feasible procedure with a high technical requirement which should be performed in specialized centres with high experience in hepatobiliary surgery and in advanced laparoscopic procedures, because of its high morbidity and mortality. conclusions: robotic assistance in whipple may overcome limitations of laparoscopy and offer a minimaly invasive approach to this procedure potentially resulting in lower blood loss and less morbidity. we need further prospective randomized trials in order to determine the exact role of robotics in pancreatic surgery. aims: distal pancreatectomy is the standard curative treatment for symptomatic benign, premalignant, and malignant disease of the pancreatic body and tail. the most obvious benefits of a laparoscopic approach to distal pancreatectomy include earlier recovery and shorter hospital stay. spleen-preserving distal pancreatectomy should be attempted in case of benign disease. laparoscopic spleen-preserving distal pancreatectomy (lspdp) is expected to be less invasive than laparoscopic distal pancreatectomy with splenectomy. however, there are few reports regarding the details of the procedure for lspdp, and its safety remains unclear. this study aimed to evaluate the feasibility and safety of lspdp. methods: retrospective analysis of surgery treatment of patients was made. lspdp was conducted in the period from to in the department of laparoscopy surgery of state institution o.shalimov national institute of surgery and transplantology. the average age was : . years, the body mass index (bmi) was . ± . results: laparoscopic distal pancreatectomys was performed in % of cases, were attempted in female and male patients. postoperative pathological examinations revealed cases of serous cystadenoma in the body and tail of the pancreas, case of serous oligocystic adenoma, case of mucinous cystadenoma, case of neuroendocrine tumor (insulinoma), and case of solidpseudopapillary neoplasm. complications related to the surgery were like acute pancreatitis with -fold increase normal plasma amylase confirmed by ct- cases, fluid collection- cases, pancreatic fistula (grade a)- cases. the operation time was . min, (range - min) blood loss of . g (range - g), mean hospital stay was . days (range - days). conversion to laparotomy was in case. mortality was . conclusion: laparoscopic spleen-preserving distal pancreatectomy is minimally invasive, safe, and feasible for the management of benign pancreatic tail tumors, with the advantages of earlier recovery and less morbidity from complications. aims: a pancreatic pseudocyst is an encapsulated, mature fluid collection occurring withing the pancreas that have a well-defined wall minimal or no necrosis secondary to pancreatic injury and mediated by the enzimatic and inflammatory disruption of pancreatic tissue. it is a common complication of acute and chronic pancreatitis. we present the case of a pancreatic pseudocyst located within the body of the pancreas due to recurrent necrotic pancreatitis. the objective of this video is to show the minimally invasive surgical approach of this entity. methods: a -year-old man without medical history was admitted to hospital in the digestive service on times for acute necrotizing pancreatitis. after study in which is evidenced cholelithiasis and pseudocyst in pancreatic body of cm maximum diameter and formation of two peripancreatic collections without signs of superinfection, cholecystectomy is indicated. magnetic control cholangiography was performed after surgery and it showed an increase in the size of the pancreatic pseudocyst, suspecting wirsung's duct disruption. therefore, endoscopic retrograde cholangiopancreatography (ercp) was performed by placing a plastic pancreatic prosthesis and performing a sphincterotomy. after hospital discharge, the patient is re-admitted due to recurrent abdominal pain without analytical alteration. tc abdominal observed an increase in the pseudocyst from to cm. this case was discussed in a multidisciplinary committee and surgical intervention was decided. results: laparoscopic approach is decided and four trocars were placed. initially, a gastrostomy was performed with liquid outlet. an aspiration of the liquid and quistogastrostomy with mm endogia was made. the patient progresses favorably, being high on the tenth postoperative day, without complications. conclusions: almost every pancreatic pseudocyst improves spontaneously and needs no specific treatment. draining is indicated when secondary symptoms to compression, complications or rapidly enlarging are found. depending on the complexity of the pseudocyst, its communication with wirsung's duct and the existence of ductal injury, it may perform a percutaneous, endoscopic or surgical drainage. the goal of pancreatic debridement is to excise all dead and devitalized pancreatic and peripancreatic tissue while preserving viable functioning pancreas, controlling resultant pancreatic fistulas, and limiting extraneous organ damage. only the surgical procedure is definitive. case: a y old male presents with intermittent low retrosternal pain and progressive dyspnea with exercise since a couple of months. cardiac investigation was negative and gastroscopy showed a grade b esophagitis. he was treated medically but with only partial response. on a thoraco-abdominal cat-scan the diagnosis of a left sided bochdaleks' hernia was made. the hernia includes the left kidney (with blood vessels and ureter), transverse colon and small intestine which are positioned in the left lower thoracic cavity with the left lung considerably compressed. method: given the clear correlation between the patients' complaints and these anatomical findings, he was referred to our service of abdominal surgery. we performed a laparoscopy with the patient in lithotomy position and the surgeon between the legs. the patient was tilted to his right side. mobilization of the spleen was necessary to gain maximal access to the hernia. we were able to reduce all the herniated content, freed the margins of the defect, reduced the hernia sac and repositioned the kidney intra-abdominally. the defect was manually closed with non-resolvable stitches and covered with a mesh which was secured with tackers. result: postoperatively the patient recovered well with adequate pain relief and pulmonary support. he could leave the hospital after days. control cat-scan on day postoperatively shows an intact lining of the diaphragm with normal positioning of the intra-abdominal organs. on follow-up weeks after surgery the patient had regained normal activities and was symptom free. conclusion: a symptomatic left sided bochdaleks' hernia in adults with an ectopic intrathoracic kidney is extremely rare. we hereby state that, during a laparoscopic repair, the kidney can also be safely reduced, which has almost never been described in literature yet, enhancing pulmonary recovery, improving access for mesh placement and thus diminishing recurrence rate. aims: large incisional hernias repair involves an actual problem for surgeons to face. anterior component separation has been an important method allowing to close the fascia defects without tension while also having underlay mesh reinforcement.therefore, we present a case of incisional hernia reparation performing endoscopic anterior component separation with advantages compared with open approach. method: we present the case of a -year-old woman, bmi kg/m , with previous laparoscopic gastric sleeve and posterior reintervention using open approach. the patient presented a cm size incisional hernia m w . a ct scan was performed, confirming a midline incisional hernia containing colon, with an herniary defect of cm. full minimal invasive abdominal wall repair was proposed. a cm size incision was made in left iliac region to reach the aponeurosis of external oblique muscle. we placed a balloon trocar and subcutaneous pneumo-dissection with mmhg pressure was performed; then, we placed a mm trocar in left lumbar space. the aponeurosis of external oblique muscle was incised and anterior component separation from inguinal to subcostal area was achieved. an extensive intermuscular dissection was performed to achieve complete midline closure. we performed the same procedure on the right side. then, with laparoscopic approach using v-loc n° suture, we completely closed the midline. eventually, we placed a x cm ptfe-c mesh fixed with a double crown of tackers and fibrin glue. results: postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. the patient has been followed up for months without any complication or recurrence in ct scan, confirming the correct minimally invasive reconstruction of the abdominal wall. conclusions: trends in abdominal wall reconstruction and complex-hernia repairs have advanced rapidly in recent years. the goal is to perform a complete abdominal wall repair with no tension in midline incisional hernias. endoscopic anterior component separation and laparoscopic eventroplasty with closure of the defect, leads to a complete wall reconstruction without tension and avoids drawbacks due to primary close defect in those patients with herniary defects wider than cm. aims: endoscopic technique is a valid and safe approach for the treatment of abdominal wall defects. to combine the advantages of complete endoscopic extraperitoneal surgery with those of sublay mesh repair we propose totally endoscopic sublay anterior repair (tesar), a safe and feasible approach for the treatment of ventral and incisional midline hernias. methods: from may to september patients were referred to our unit for clinical and radiological diagnosis of midline ventral or incisional hernia and selected for tesar. exclusion criteria were: complicated ventral or incisional hernia (i.e. incarcerated hernia), maximum defect width [ cm, contraindications to general anesthesia. the procedure consisted of suprapubic access with trocars, complete endoscopic pre-aponeurotic dissection, isolation and reduction of the hernial sac, bilateral incision of the medial rims of recti aponeurosis and dissection of retromuscular plane to create the retromuscular space, sublay non-absorbable mesh positioning and anterior aponeurosis reconstruction. one drain was always placed in the retromuscular space and one drain in the subcutaneous space. results: all procedures were completed with endoscopic approach, with no conversion to laparoscopy or open surgery. no intraoperative complications were registered. total mean operative time was ± . min. no post-operative major complications were registered. only one subcutaneous seroma was registered ( . %), and treated conservatively. the mean postoperative stay was . ± . days. at post-discharge clinical checkups drains were checked and removed when indicated. no wound complications nor recurrence were registered to date. cosmetic and functional results were successful in all patients. conclusions: tesar is a safe and feasible technique for the extra-peritoneal sublay repair of ventral hernias with totally endoscopic approach. it provides accurate hernia repair with good outcomes in terms of resolution of symptoms and post-operative complications. r. mizuno, m. kondo backgrounds: abdominal incisional hernia is found in more than % after abdominal surgery, and risk factors such as wound infection, obesity, elderly, high abdominal pressure are pointed out. laparoscopic hernia repair using intraperitoneal onlay mesh (standard ipom) is becoming widespread in japan since the insurance release in , and our hospital is actively working on it. recently, ipom plus procedure which also carries out fascia suture in addition to laparoscopic mesh placement has been introduced. aims: we report the clinical results of laparoscopic abdominal incisional hernia repair in our hospital. methods: we performed hernia repairs using a mesh for cases from january to september . of these, cases were standard ipom and cases were ipom plus. there was no significant difference in the patient background such as gender, age, bmi, etc, and in the intraoperative findings such as hernia orifice diameters and adhesions. surgical time, postoperative hospital stay, and the rate of complications such as seroma, mesh bulging, postoperative pain, hernia recurrence were compared and examined between the two groups. results: as a result, in ipom plus group, the operation time was longer and the incidence rate of postoperative pain was higher, but the incidence of mesh bulging was significantly lower. also, in some cases since , the ' u reverse stitch method ' is used as an ingenuity of fascia suture in ipom plus. conclusions: laparoscopic abdominal incisional hernia repair has the advantage of being able to reliably confirm the hernia orifice from the intraperitoneal side?it is excellent in the identification of the fragile part of the abdominal wall and in the visibility of the restoration range. with regard to the ipom plus procedure which has been introduced in the last few years, although the operation time is extended, it has usefulness such as reduction of mesh bulging. from the viewpoint of cosmetic surgery, usage of ipom plus will increase in the future. introduction: incisional hernia is one of the most common complications after abdominal surgery. several methods have been introduced, and yet, there is no consensus on the best method of repair. we present a novel method for hernia repair which uses the retromuscular sublay mesh repair through a single incision at the pubic area to improve cosmesis. methods: medical records of patients who underwent single-port retrorectal incisional hernia repair from may to december were reviewed. patients were placed in supine position and a cm incision was made in the pubic area below the panty line. a flap is made upwards until the defect is found and bilateral rectus sheathes are dissected. a mesh is then placed between the posterior rectus sheath and the muscle. results: a total of patients with midline incisional hernia underwent single-port retro-rectal incisional hernia repair. mean age was . ± . years with an average bmi of . ± . . all the patients had midline hernia defect with an average of . ± . cm. mean operation time was . ± . min and estimate blood loss was . ± . ml. there was no postoperative complication, and ( %) patients were discharged on the day of surgery. conclusion: the single-port retrorectal incisional hernia repair is safe and effective while providing good cosmesis to selected patients with incisional hernia. aims: closing hernia defect during laparoscopic hernia repair is a vast extended technique nowadays. however, this technique is associated with mesh placemnt intraabdominally in contact by the abdominal content. nowadays there is a trend to recontruct the midline and to avoid a mesh intraabdominally in those cases suitable for it, as a new step forward of minimally invasive abdominal wall reconstruction. laparoscopic sublay approach with retromuscular placement of a mesh without mechanical fixation after reconstruction the linea alba migth be considered an option in primary hernias of the midline. methods: we present a case of a year old male with an umbilcal hernia of centimeter in diameter associated with rectus diastasis. a laparoscopic approach was performed, using one and two millimeter trocars placed on the left flank. the first step was to open the lateral side of the posterior fascia of the left rectus muscles, dissecting the retromuscular plane until we reach the linea alba getting into the preperitoneal space where the sac was diseected preserving the integrity of the peritoneum. the contralateral posterior fascia was also dissected all the way to the semilunaris line. the midline was closed, including th hernia defect, using a running double loop suture (maxon-loopÒ). a self gripping mesh (progripÒ) is placed in the retromuscular space in a sublay position ( cm long, cm wide). last, we close the fascia of the left rectus muscle using a barbed suture (v-locÒ). results: surgical time was min, being discharged of the hospital on postoperative day . pain was controlled with conventional analgesia and no postoperative complications, nor seroma was detected. conclusions: sublay approach for ventral hernia can provide a midline reconstruction, reestablishing abdominal function and avoiding the use of intraabdominal meshes and traumatic fixation, decreasing postoperative complications and pain. aims: lumbar hernia is one of the rare cases that most surgeons are not exposed to. hence the diagnosis can be easily missed. this is often related to previous surgery as lumbotomies or primary in the superior lumbar triangle. this leads to delay in the treatment causing increased morbidity. we report a case of adquired lumbar hernia in a middle-aged woman repaired by laparoscopic approach. methods: a years old woman with surgical history of a myelomeningocele surgery by posterior approach over years ago, a laparoscopic left nephrectomy years ago with a left colostomy due to a left colon injury during this procedure. a hartmann reversal by laparoscopic approach months later. patient showed a large lumbar mass over cms in the left lumbar region and a large scar near to spinal cord. it was soft in consistency, reducible and expansible on coughing and straining with defined borders. computerized tomography showed a large defect in the superior lumbar fascia over cms in the grynfeltt-lesshaft triangle with the left colon inside. results: patient was placed in a full lateral decubitus position. in order to optimize exposure, a lumbar roll was placed under the lumbar region. a capnoperitoneum ( - mmhg) was built up. one mm and two mm trocars were used and positioned in the left mid axillary line. a optic was used. adhesions were removed and toldt fascia was opened in order to expose the hernia defect bounded by quadrates lumborum, erector spinae muscles, rib and serratus. hernia content was carefully extracted from the sac using a ligasure maryland (covidien medtronic-usa). hernia defect was measured and an intraperitoneal mesh (dinamesh-ipom feg textiltechnik mbh, aachen, germany) was positioned and sutured by tackers to the margins included the bone. patient was discharged in h with a low pain rate and without complications. there is not recurrence in months follow-up. conclusion: laparoscopy might be a safe and feasible approach for repairing lumbar hernias, either primary or adquired, with a low rate of pain and complications s surg endosc ( ) after pneumoperitoneum is done, three mm trocars are placed on the left flank. the defect is delimited by drawing it over the skin of the patient with aid of an intramuscular needle and intraabdominal vision. posterior fascia is opened longitudinally at its medial edge and the retromuscular space is dissected. the arcuate line of douglas and the epigastric vessels are identified. from this point, transversus abdominis fascia is sectioned cranially cm medial to the semilunar line, preserving the neuro-vascular pedicles that reach the rectus abdominis laterally. at supraumbilical level, transversus abdominis fibers advance behind rectus abdominis, so they need to be sectioned to access to the space below the ribs. lateral dissection of this space enables a tensionfree closure at midline. once the procedure is repeated on the contralateral side using two mm and one mm trocars on the right flank, a continuous suture of the posterior fascia is performed with a barbed suture. the anterior fascia is closed with a slowly-absorbable monofilament loop-type suture. finally, a double-layer polypropylene mesh is placed at retromuscular level without any suture and fibrin glue is applied. results: the patient was discharged hous after surgery. no recurrence has been presented to the moment. conclusions: the section of the aponeurotic plane from the arcuate line of douglas enables a more accurate dissection of the retrotransversus plane without sectioning its fibers except for its cranial end, preserving the innervation and vascularization of the abdominal wall. this technical modification aims to simplify a complex laparoscopic procedure allowing its estandarization. aims: the authors present a video with their standardized laparoscopic ventral hernia intraperitoneal mesh (ipom) hernioplasty procedure but introducing a novel laparoscopic technique for tension releasing while hernia gap closure and midline anatomical restoration. methods: a years old male patient with a bmi presents a symptomatic ventral hernia recurrence after a sigma colic cancer open surgery. a ct scan study showed a cm transverse diameter midline ventral hernia. a laparoscopic ipom hernia repair procedure is performed using mm instruments and a mm camera. when checking tension while midline restoration suturing, we decide to add a tension-releasing maneuver: a totally laparoscopic transverse abdomini muscle release (taltar). this maneuver allow right rectus posterior sheath to advance some distance to the midline, in order to provide a tension-free midline closure. a double-faced ready-to visceral contact mesh is now placed and fixed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, lateral asymmetry, umbilical or abdominal wall complications have been reported with this technique. conclusions: depending on the patient characteristics, anatomical hernia factors and surgeon mini invasive experience, a taltar maneuver could be a safe and feasible option for releasing tension when midline anatomical laparoscopic closure. more studies are needed in order to standardized this approach. aims: when primary ventral hernia and simultaneous diastasis recti are diagnosed, there is no consensus among the international surgical community on the surgical treatment regarding indications or surgical technique. however, if diastasis recti is symptomatic of or is associated with midline hernias, the corrective surgery of both pathologies at the same time could be the most recommended option. when we only correct the herniary defect, we risk performing a reparation on an anatomically weak tissue, so the rate of hernia recurrence may increase. we propose a minimally invasive access using totally endoscopic retromuscular hernioplasty. by developing this technique, several advantages are provided, such as no peritoneal opening without intraabdominal access, no mesh fixation needed and simultaneous solving of both pathologies. method: we present the case of a -year-old man, with bmi kg/m and no previous medical history complaining of ventral hernia with associated recti diastasis. a cm size umbilical hernia was diagnosed with a cm size supraumbilical diastasis recti associated. full endoscopy retromuscular hernioplasty was proposed. a cm size incision was made in left hypocondrium, openned the anterior rectus sheath and retracted the rectus muscle. we placed a balloon trocar and open the homolateral retromuscular space after placing two mm trocars in left lumbar space and epigastric position. we crossed-over the linea alba and achieve contralateral retromuscular space. after this step, the hernia sac was reduced and we extended the dissection cm caudal to the hernia ring. both medial posterior rectus sheaths were sutured with running barbed suture n° and a x cm size light-weight, big pore, polipropilene mesh was placed in retromuscular space and unrolled properly with enough overlap. a drain was placed and the anterior rectus sheath incision was closed. results: the patient was discharged h after surgery without remarkable events during his postoperative stay. he has been followed up for months remaining asymptomatic. conclusions: totally endoscopic retromuscular ventral hernia repair in men with umbilical hernia and diastasis recti associated, is feasible and reproducible procedure with several advantages compared to traditional laparoscopic ipom in terms of pain and mesh position. aims: parastomal hernia (ph) is one of the most frequent long-term complications of stoma formation, occurring in %- % of patients. surgical treatment for parastomal hernia is the only cure but a fairly difficult field with a recurrence rate ranging from % to % of cases. due to its advantages, the number of laparoscopic mesh repairs for parastomal hernia has gradually increased over the past decade. according to this common complication, we report a case of laparoscopic reparation of ph using the sugarbaker technique. method: we present the case of a -year-old patient with surgical antecedent of laparoscopic low anterior resection due to rectal cancer, presenting in postoperative period an anastomosis leakage with severe peritonitis was identified and a laparotomy with end colostomy was performed. the postoperative course was uneventful. during the follow-up the patient showed a centimetres size paraestomal hernia, being a m w incisional hernia confirmed with ct scan.the patient underwent full laparoscopic hernia repair, performing a sugarbaker technique, exposing parastomal hernia completely to measure the hernia ring size ( centimetres) and the midline associated defect ( centimetres). a x cm size ptfe-c was selected to allow a -cm overlap over two defects. results: using this approach, the bowel loop was pushed into the abdominal wall and appropriate place between the mesh edge and the abdominal wall is left to allow the bowel loop to pass through. postoperatory course was uneventful and the patient was discharged h after surgery without any remarkable event during his postoperative stay. he has been followed up for months without realizing any clinical signs or alterations in ct scan. compared with traditional open surgical repairs, laparoscopic repair has certain advantages including its safe operation, postoperative rapid recovery, fewer complications, and lower recurrence rate. however, it still faces challenges regarding parastomal hernia treatment, and there is a need to improve existing surgical techniques. aims: nowadays, the principal disadvantages of laparoscopic approach in hernia repair are the use of intraabdominal meshes and traumatic fixation. first, intraabdominal meshes involve the contact of the prosthesis with the intestinal loops with the consequent risk of adhesion and fistula. also, using helicoidal sutures in prosthetic fixation produces adhesions to the tackers and a non-negligible incidence of chronic pain. when it comes to lead to better results, placing the mesh in retromuscular space avoids the drawback of contact with the loops, and using self-fixation meshes may decrease the rate of acute and chronic pain. accordind to this facts, we present a case of laparoscopic ventral hernia repair with transabdominal retromuscular mesh placement without traumatic fixation. methods: we present a -year-old patient with a cm diameter hernia showed in preoperative ct scan, m w , with diastasis recti associated. the patient underwent laparoscopic surgery using transabdominal retromuscular route. one mm and two mm trocar were placed in left flank. the posterior rectus sheath on the left side is opened starting cms far from the left egde of the defect. once the retromuscular space is dissected, the hernia ring is dissected and the hernia sac reduced, we continue with the dissection in retromuscular space on the side. craniocaudal dissection is achieved cm distal to the defect margins. the hernia defect with the anterior rectus sheath and the diastasis recti were closed using v-loc running suture. self-adhesive mesh was subsequently placed. the mesh should be overlap cm from the margins of the defect, covering the defect widely, with grips facing upwards. finally, we closed the posterior rectus sheath with peritoneum on the left side with v-loc running suture. results: the postoperative course was uneventful and the patient was discharged h after the surgery. after months of follow-up no clinical or radiological recurrence was showed. conclusions: the combination of laparoscopic approach, retromuscular mesh placement and the use of self-fixation meshes, seems to be an actual useful solution, combining the advantages of each item and avoiding the use of intraabdominal meshes and helicoidal sutures. aims: laparoscopic ventral hernia repair has clear advantages over open repair, including less post-operative pain and earlier return to normal activity. however, a prolonged surgeon learning curve is necessary to perform this technique effectively. robot assistance may improve outcomes of minimally invasive ventral hernia repair with improved three-dimensional visualization and enhanced dexterity with articulating instrumentation. we report a case of robotic rives-stoppa epigastric hernia repair in order to demonstrate the feasibility of the robotic approach. methods & results: a -year-old man came to our attention for the presence of a palpable mass in the epigastric region. the abdominal ct scan showed the presence of an epigastric hernia with herniation of omental content, and the presence of diastasis recti. the patient was then submitted to a rives-stoppa robotic hernia repair under general anesthesia. the da vinci-si surgical system (intuitive surgical inc., sunnyvale, ca, usa) was brought into position over the head of the patient and docked after placement of the ports. three trocars were placed in the hypogastric region along the transtubercular line. a fourth trocar was placed in the left iliac fossa and used by the assistant. the operation started with an extended adhesiolysis and hernia reduction. then, the retromuscolar dissection began by incising the posterior sheath starting from cm above the pubic symphysis. an extended dissection of the rives space was performed to create a correct housing for the mesh. the hernia defect and the diastasis recti were closed using a - absorbable barbed suture. a phasix st tm mesh (bard inc./davol inc., warwick, ri) was positioned in the retromuscular plane, and was anchored with absorbable sutures and glue. the midline incision was closed using a - absorbable barbed suture. the operative time was minute. the postoperative period was uneventful, and the patient was discharged home on the second post-operative day. conclusions: robotic rives-stoppa ventral hernia repair is feasible, safe, and effective when a standardized approach is performed. whether robotics may improve the outcomes of minimally invasive ventral hernia repairs, including lower recurrence rates, decreased post-operative pain, or shorter surgeons' learning curve, will require careful prospective investigation. aims: the authors present a video with a left chronic bochdaleck hernia classical hernioplasty repair but performing a mini invasive thoracoscopic approach and mm instruments. methods: a years old female patient come to hospital due to chronic left dorsolumbar pain. a ct scan study showed a chronic left diaphragmatic bochdaleck hernia. a lateral right decubitus thoracoscopic repair is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with no pain and a clean chest x-ray. in a year time follow-up, not an anatomical or clinical recurrence has been reported. neither chronic pain or respiratory complications happened, with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, left bochdaleck hernia mini invasive thoracoscopic hernioplasty repair using mm instruments could be a safe and feasible option. more studies are needed in order to standardized this approach. surg endosc ( ) abdominal wall surgery has expanded exponentially in the last decade. many techniques have been developed, mainly in minimally invasive surgery. laparoscopic ventral and incisional hernia repair (lvihr) has become a common procedure because of its feasibility and safety but unfortunately, it is not free of complications. chronic postoperative pain and bleeding are frequent complications, prolonging hospital stay and altering quality of life of the patients. absorbable or non-absorbable tacks are the usual method of mesh fixation and sometimes combined with transfascial sutures to secure the mesh. these mechanical fixations pierce the abdominal wall causing nerve or vessel injuries. some studies showed no differences between absorbable tacks, non-absorbable tacks or transfascial sutures concerning postoperative remarkably high pain. some authors consider that a non-penetrating fixation of the mesh getting an effective mesh-abdominal wall interface will reduce significantly the postoperative pain after a laparoscopic ventral hernia repair. tissue glues are used in different medical treatments and also have been used successfully for extra peritoneal mesh fixation in laparoscopic inguinal hernia repair, open ventral hernia repair but not so in laparoscopic ventral hernia repair in spite of good results published in the literature. cyanoacrylate and its derivatives are 'synthetic glues' and classified as medical devices with stronger adherent properties than fibrin glues. experimental studies have reported good results compared with suture fixationand also tissue toxicity doesn't lead to an increased foreign body reaction. some authors have studied the use of cyanoacrylate in laparoscopic inguinal hernia repair but unfortunately, clinical trial reports in ventral and incisional hernia repair were not found in the literature because the lacking of experimental studies that guarantee the safety of intra-abdominal mesh fixation and the interaction of the glue with the intra-abdominal tissue. our group developed an experimental study demonstrating the feasibility, safety and effectiveness of the cyanoacrylate using for intraperitoneal mesh fixation and after this conclusion, started a clinical study. this video shows the methodology for laparoscopic mesh fixation with only glue in our first cases. aims: small epigastric hernias, associated or not with the rectus abdominis diastasis, and small umbilical hernias are common in middle-aged women, particularly with past history of pregnancy. the aim of this video is to illustrate a new extraperitoneal approach to these clinical situations. methods: patients between the ages of and years old, with epigastric hernia orifice up to cm, with or without associated umbilical hernia (up to cm), were chosen for this procedure. the surgery begins with a vertical umbilical incision for the umbilical hernia's correction, and dissection of the pre-aponeurotic plane. two mm trocars (mini-laparoscopy instruments) are introduced at both flanks to enlarge the pre-aponeurotic plane towards the xiphoid appendix. in this way epigastric hernial defects are isolated. the surgery proceeds with defect suturing with braded suture, midline invagination and mesh placement if necessary. results: all patients had an eventful post-operative period and were discharged home at postoperative day . the aesthetic and functional results are optimal conclusion: for selected cases with high aesthetic motivation this technique seems to be feasible and with optimal cosmetic results. this technique allows the mesh placement both in-lay and onlay, protecting it from surgical site infections often present at the classical approac bochdalek hernia is a rare entity in adults. fewer than have been reported in medical literature, the majority of which were incidentally diagnosed. as such, the optimal repair of a symptomatic hernia is unknown. we present a case of adult bochdalek hernia repair. methods: a -year-old obese male patient with a years of chronic dry cough and left lung opacity in chest x-ray. a large posterior and lateral bochdalek hernia with herniation of intestinal loops and fat to the left hemithorax was seen in chest and upper abdominal ct scan. the hernia extended to mid-thorax, caused significant atelectasis of left lung. eighteen months later, due to appearance of chest and abdominal pain following a recent motor vehicle accident, a repeat chest ct was done and a slight enlargement of the hernia was shown. results: the patient was operated laparoscopically, positioned in a semi-right lateral decubitus with double lung intubation. a large left posterior and lateral diaphragmatic hernia which contained transverse and descending colon with omental fat was seen. they were pulled in to the intraperitoneal space carefully. the defect was measured to be * cm. it was reduced to * cm by suturing with a non-absorbable v-loc suture . advancing the camera to the thoracic cavity showed the left lung to be severely atelectatic. after selective recruitment lung was well expanded. a symbotex composite cm mesh was fixed to the defect area by suturs and laparoscopic tacker. the operation and post-operative course were uneventful. chest x-ray demonstrated the bowel below the diaphragm. the patient was discharged on pod . at -month follow-up, chest x-ray was normal. objective: to demonstrate the safety and efficacy of the standardized laparoscopic approach in the treatment of large parastomal hernia. currently, this approach is recognized as the one of choice in parastomal hernia pathology, being controversial which is the best technique of choice: keyhole vs sugarbaker. material and method: clinical case: a -year-old woman with a history of laparoscopic abdominoperineal amputation due to rectal neoplasia (pt n ), a year ago, with symptomatic parastomal hernia with incarceration episodes and inflamation changes in the stomal orifice.tac: large hernia parastomal with intestinal content inside. surgical treatment is decided. result intervention: complete laparoscopic approach, right lateral partial decubitus, trocars, dissection of the hernia defect and reduction of the content, partial mobilization of the pre-stomal colon, with bleeding at the level of the vascular origin, requiring careful hemostasis to avoid ischemia of the colostomy, herniorrhaphy with stitches with extracorporeal knotting, placement of polypropylene/pvdf mesh,fixed with irreabsorbable tackers with administration of biological glue at the edges of the mesh. correct postoperative, discharge at the rd day. asymptomatic and without hernia recurrence at one year of follow-up. conclusions: the technique of sugarbaker using a laparoscopic approach is a safe and effective alternative in the treatment of parstomal hernias. objetives: laparoscopic ventral hernia repair provides advantages in term of low infection rates and postoperatory stay when is compared with open repair. trends in laparoscopic abdominal wall surgery is to complete defect closure without tension in midline. closing the defect in ventral hernias wider than - cms creates high tension in midline and postoperatory pain. it's proposed different techniques to solve this drawback. laparoscopic posterior component separation makes the defects closure easier with no tension and placing the mesh extraperitoneally. methods: years old woman with previous total hysterectomy, a m m w midline incisional hernia was clinically diagnosed and confirmed with ct scan. full laparoscopic abdominal wall repair with defect closure was proposed. trocars in left side were placed and posterior rectus sheath right side in the defect margin is freed. once the lateral edge of the rectus sheath is reached, the posterior rectus sheath is incised, dividing the posterior aponeurotic sheath of the internal oblique muscle. this allows access to the plane between the internal oblique and the transversus abdominis muscles. it's is made the same steps in the left side with trocar on the right flank. the posterior rectus sheath both side is reapproximated in the midline and cms polipropilene mesh is placed and unfolded properly. it's fixed using cyanocrilate glue. one drain is left in retromuscular position and mm trocar wounds are sutured. results: postoperatory course was uneventful. hospital stay h. the drain was removed in day after surgery. after months follow-up no complication or recurrence were identified. methods: this video will show the evidence of gangrenous jejunal segment due to superior mesenteric vein thrombosis in a patient with history of breast ca on hormonal treatment.in this video, the gangrenous segment was resected and primary anastomosis was done using endogia mm. results: a second look after h revealed to be negative for any further ischemic bowel. conclusion: therefore, laparoscopy in acute abdomen is diagnostic and for treatment. introduction: gastric pseudo-volvulation is a rare entity of paraesophageal hernia that is characterized by migration of the stomach into the posterior mediastinum. this clinical-radiological picture has severe complications so in certain cases should be operated urgently. another small group of patients are asymptomatic, although the current literature recommends their regulated surgical intervention. we present a gastric pseudo-volvulation in the mediastinum, with a laparoscopic approach, showing that by systematizing the surgery, it is possible to perform this type of intervention with relative ease and safety material and methods: we present a video of an urgent laparoscopic approach in a female patient of years with a personal history of hypertension, smoking and dyslipidemia. with a hiatus hernia diagnosed more than ten years ago. he went to the emergency department due to significant symptoms of heartburn and reflux, as well as incoerctable vomiting and difficulty feeding one week of evolution. a simple abdomen and postero-anterior chest radiograph was performed, showing a paraesophageal hiatus hernia with almost the entire stomach included in the mediastinum. a thoraco-abdominal axial tomography corroborated giant hiatus hernia with pseudovolvulation and incarceration data. urgent intervention was decided by laparoscopic approach in which hiatus hernia reduction and esophageal abdominalization were performed. closure of pillars and reinforcement with bioabsorbable mesh. gastric and gastropexy toupet of anterior face to anterior peritoneum of abdominal wall. results: the patient had a post-operative h without incident, discharged with a crushed diet. the follow-up and evolution has been acceptable without notable complications. conclusion: the laparoscopic approach, in extreme cases of paraesophageal hiatus hernia with incarceration of the stomach and pseudovolvulation of it, is a correct, safe and effective alternative in experienced groups. surg endosc ( ) case report of incarcerated hiatal hernia. years old female was admited to the hospital due to severe chest pain and vomiting for about six h. physical examination and lab test showed no abberations. chest xray revaled incarcerated stomach above the diaphragm. she was rushed to the or. laporoscopic approach was used, the stomach was removed from the chest and nissen fundoplication was performed. day after surgery patient was asymptomatic, got full oral diet. she was discharged on postoperative day two, without a need of any analgetics. gastroduodenoscopy was performed weeks after surgery and showed proper image of oesophagus, stomach and duodenum, neither signs of hiatal hernia nor inflamation were present. laparoscopic approach is good way to treat incarcerated hiatal hernias and is related with shorter lenght of stay, lesser postoperative pain and better patient comfort. and it should be procedure of choice in this kind of cases. she was operated open technique using a cm long incision in right iliac fossa and the appendix was phlegmonous. the patient began feeling bad from the second day postoperative having temperature over °c, pain and increasing crp. the general condition worsened the next day when the temperature went up till . °c, extreme generalized pain and crp: . the ct abdomen control indicates signs for generalized peritonitis and rises the suspicion for a forgotten large gauze. the patient is operated using laparoscopy technique: identifying and taking out the foreign body, doing adhesiolises, extensive lavage and in the end inserting one drain in douglas. the video is presenting what king of special graspers can be used but also tips and tricks when speaking about identifying the anatomy but also dissection in acute and inflamed environment. postoperatively the patient began to feel better and in the th day was released home. conclusion: this case illustrates that even after open surgery, laparoscopy is a viable solution with the condition that there is available experience in minimally invasive surgery. introduction: foreign bodies can enter inside the human body by different mechanisms such as ingestion, aspiration, trauma or in some cases due to medical procedures. they are potentially life-threatening events, the diagnosis could be challenging and its management depends on their location. case report: a -year-old male was referred to our hospital due to chronic abdominal pain. he had cholelithiasis, medical history of acute pericarditis and past surgical history of left adrenalectomy, left nephrectomy, distal pancreatectomy and colon resection due to an adrenal adenocarcinoma (stage t n m ).abdominal radiograph showed a foreign body in the left lower quadrant of the abdomen, as an incidental finding. this was not detected in ct scans during ten years of oncology follow-up. ct scan revealed an extraintestinal metallic curved object in the right lower quadrant. this finding was not related to any surgical intervention or trauma. diagnostic laparoscopy was performed: the foreign body seemed to be a guidewire, it was included into the omentum and almost stuck to the abdominal wall. the guidewire was reached and carefully extracted through a mm trocar without any evidence of intra-abdominal organ injury. then an elective cholecystectomy was also performed due to his medical history of symptomatic cholelithiasis.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: is extremely rare to discover a guidewire that had migrated into the peritoneal space without abdominal injuries.this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive laparoscopic removal. aims: the authors present a video with their standardized laparoscopic groin hernia transabdominal preperitoneal hernioplasty (tap) procedure but using mm instruments and mm camera approach. methods: a years old male patient with a bmi presents a symptomatic bilateral groin hernia for months. us study showed an indirect bilateral inguinal hernia. a laparoscopic tap hernia repair procedure is performed using mm instruments and a mm camera. a selfgripping mesh preperitoneal hernioplasty and peritoneal flap barbed-sutured hermetic closure was performed. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic pain, anatomical recurrence, umbilical or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, a laparoscopic bilateral hernia repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. results: during tapp approach a direct hernia relapse was identified, the previous mesh was included on preperitoneal space and some non-absorbable sutures to inguinal ligament were identified. stitches and nearly total mesh removal (only the part surrounding cord elements was left in place) were performed. x heavyweight polypropylene mesh was employed fixed with gubran Ò and the flap was closed with running sutures. patient was discharged uneventfully the same day. seven months later he did not need analgesics and had no physical impairment. conclusions: post inguinal hernia repair chronic pain can be severe and disabling, and is becoming more prevalent. the origin is complex and meshes and sutures could play a role. the management is multimodal and demanding. for refractory patients, surgery may be an option. laparoscopic, open and mixed approaches have been employed. they usually combine mesh removal and substitution (often in different planes) and groin nerve therapies. nowadays, triple neurectomy seems to be the most effective treatment (more than % pain relief). generally, removal of mesh alone does not lead to lasting pain relief or has worse outcomes compared with associated neurectomy. introduction: mesh repair of inguinal hernia is sometimes followed by adverse effects such as mesh migration, chronic groin pain or recurrence. removal of the mesh is necessary in selected cases. we affront this cases by tapp intervention. methods: we present a video with two intreventions of inguinal recurrent hernia by laparoscopy (tapp). we remar the points to decide explant the mesh or not to explant. the conditions to decide the explant were the proximity to the main vessels in inguinal area (espigastric and femoral vessels) and the plication of the mesh. results: and conclusion as we show in the video, the explant of the mesh is only conditioned by the plicature of the mesh for its migration and recurrence, accompanied usually with pain. we don't remove any time the mesh or the plug if it is in the triangle of doom with firm adhesions to the main vessels. we cover the previous mesh with a new ligthweigth d mesh and closing at the end the preitoneum over the new reparation. introduction: tep technique isn't a controversial area in surgical practice for inguinal hernias anymore, but a fully accepted method. the use of general anesthesia has been the mainstay of laparoscopic hernia repair, but epidural anesthesia is not a contradiction to properly selected patients. material-method: the approach of the extraperitoneal area achieved without use of a dilation balloon, but via the indroduction of the camera and the dissection of the regional structures. trocars ports were used: a mm trocar through the umbilicus for the camera, exactly as in sils (single incision laparoscopic surgery), another one mm is placed in the midline between the umbilicus and pubis, the last mm trocar is placed in the midclavicular line ipsilateral with the hernia. the key for every operation was the tension free technique with placement and fixation of a mesh x cm. in / cases the mesh was placed with tacks on the inside of the inferior epigastric artery-vein complex. all patients were dismissed from the hospital in h, no drain was placed and no major postoperative complications took place. conclusion: tep is a demanding technique with serious learning curve. the use of a dilation balloon for insertion in the extraperitoneal area is not prerequisite. tep is an appropriate method both for first appearing and recurrent inguinal hernias. epiduralanesthesia instead of general anesthesia is no a contradiction for properly selected patients. aims: the aim of this study was to investigate the effects of preperitoneal carbon-dioxide (co ) insufflation during tapp (transabdominal preperitoneal) repair. materials and methods: male patients with inguinal hernia were include in our study. we obtain laparoscopic access at the umbilicus and introduce mm port. two mm working ports are placed lateral. diagnostic laparoscopy of the entire abdomen is necessary to rule out other pathology or contraindications for surgery. using aspiration needle we insuflate carbon-dioxide ( mmhg) preperitoneal at the level of anterior superior iliac spine while decrease abdominal gas pressure to mmhg. same procedure is made lateral to the umbilical artery. results: we found that preperitoneal carbon-dioxide (co ) insufflation during tapp facilitate the future parietalisation and even can reduce operating time in future improvements of the technique. there were no intraoperative complications related to this procedure. we did not found any potential risk of the technique when is use by trained surgeons. aims: laparoscopic inguinal hernia repairs (lihr) are performed more and more frequently because they offer some advantages; however, we cannot forget their specific complications. lihr are associated sometimes with peritoneal tears that can lead to bowel obstruction. we present two cases of bowel obstruction related to peritoneal defects post tapp procedure and review peritoneal closure, bowel obstruction and options to repair defects. a year-old male was scheduled for tapp due to bilateral relapse. two x tio mesh tm fixed with securestrapÒ, employed also for peritoneal flap closure, were employed. three days later he was readmitted with bowel obstruction with ct suggesting 'adhesions'. a year-old male had bilateral tapp in another centre. seven days later he presented with bowel obstruction. ct showed metallic tackers and suggested 'adhesions' results: first case: after four days of conservative treatment failure, a revisional laparoscopy showed ileum herniation through a peritoneal defect and firm adhesions to the mesh. bowel was labouriously separated and the peritoneal defect closed with two running sutures. he was discharged on the \ sup [ th \/sup [ postoperative day and three years later he is asymptomatic. second: after two days of conservative treatment failure, on laparoscopy, ileum was filmy adhered to polipropilene mesh through a big defect on flap closure. defect was closed with interrupted sutures. as tears persisted, an omental flap was created to cover the area. patient was discharged on the th day and continues asymptomatic three years later. conclusions: lihr bowel obstructions can be divided in adhesive disease and herniation. herniation can be early (through peritoneal defects) or late (trocar site). international guidelines recommends a thorough closure of peritoneal incision or bigger tears (grade b). the closure can be achieved with staples, tacks, running suture, or glue. these last two methods are more time-consuming but less painful. running suture seems to be the best, due to its low costs, tightness and low pain but sometimes can be technically difficult. low intra-abdominal pressures (= mmhg) facilitate suturing. when a herniation appears, careful bowel management is needed and running sutures are recommended. if tears persist, an omental flap can be useful. aims: application of a single port robotic platform to perform an entirely transanal tatme/ tata. methods: the following video demonstrates how a totally transanal proctosigmoidectomy is performed using a novel, single port (sp) robotic platform was used to carry out a totally transanal proctosigmoidectomy, single port robotic tatme/tata. a -year-old female patient with a clinical t n b rectal cancer at the cm level, status post neoadjuvant chemoradiotherapy ( cgy, xeloda) is presented. shown here is the open transanal dissection followed by docking of the sp robot, implementation of the single port instruments (fenestrated bipolar forceps, cadier, scissors, camera, clip applier) through a gelpoint path to complete a totally transanal proctosigmoidectomy including transanal tatme, ima/imv transection, splenic flexure release, and left colonic mobilization, loop ileostomy, and handsewn coloanal anastomosis. results: blood loss was cc. pathology demonstrated a moderately differentiated, rectal adenocarcinoma. the total mesorectal excision was complete (grade ), margins were negative, and all lymph nodes were negative for metastatic carcinoma. the patient was discharged on postoperative day after an uncomplicated hospital course. there was no postoperative morbidity or mortality. conclusions: application of the single port robot to transanal tatme/tata (sprtatme) is presented here. while much work remains to be done to validate the sp robot's safety, this first demonstration of a totally transanal tatme/tata establishes its feasibility and utility. this single port platform stands to greatly expand the application of natural orifice transluminal endoscopic surgery (notes). as shown, the sp robot offers more than sufficient visualization, technical control, and adequate reach to perform such an operation. we present an exciting new avenue by which to complete operations in an entirely transanal fashion, which are classically performed via a combined transanal and transabdominal approach. methods: this video shows the utilization of a new robotic platform to perform transanal endoluminal microsurgery, rtem. presented here is a year old woman with a recurrent rectal adenoma at the cm level, status post a previous tem resection in october . demonstrated is the utilization of the sp robot through a gelpoint path in order to perform a partial fullthickness and full-thickness resection. the robot is introduced through a mm in diameter cannula via a four-channel face-plate. the instruments' two-jointed mobility at the elbows and wrists as well as the novel navigation system are well demonstrated. the docking of the sp robot, utilization of the dissecting devices, and closure of the defect is shown. results: sprtem was performed with a blood loss of cc, and the patient was discharged on postoperative day . there was no postoperative morbidity, mortality, or moderate/severe pain. pathology showed tubular adenoma with low-grade dysplasia in a non-fragmented specimen with negative margins circumferentially. conclusion: initial experience using the sp robot for rtem is demonstrated here. the robot provides wonderful visualization and operative control to the surgeon. articulation of the robot's wrists and arms have the potential to facilitate technical aspects of the procedure. rtem stands as an exciting development in the field of transanal endoluminal surgery. introduction: the application of robotic approach in the esophageal surgical field is in its first phase. the microsuturing and microdissection capabilites of the robotic system can potentially overcome the traditional limitation of the laparoscopic surgery thus enhancing the indications of minimally invasive surgery. methods: we have performed a retrospective analysis of our prospectively maintained database that included patients who underwent robotic-assisted esophagectomy for malignant disease between and . results: ten out of sixteen patients had squamous cell carcinoma meanwhile six had adenocarcinoma. ten mckeown's and six ivor lewis were performed. the mean operative time was min ( - ) and the median blood loss was ml ( - ). no patients required conversion nor intraoperative transfusion. the morbidity rate was / ( . %) : a transitory laryngeal nerve paresis, a pneumotorax and pneumonia. the mean hospital stay was (range - ) days. an r resection rate of . % was achieved with a mean lymph node yield of ( - ). the -year disease free survival was . %, wheres the the -year overall survival was . %. conclusions: robotic assisted minimally invasive esophagectomy (ramie) is safe and feasible, it offers promising results while preserving a good oncology adequacy. this video shows our technique for the treatment of an esophageal diverticolum using a robotic left sided transthoracic approach, followed by a heller myotomy and dor fundoplication using a transabdominal approach. our case is a year old male, who suffered from severe dysphagia, halitosis and gastric reflux who on endoscopic and radiological investigations was found to have low grade and a cm wide esophageal diverticulum, cm from the lower esophageal sphincter. initially conservative management was attempted, however following poor compliance and the persistance of symptoms after year of therapy, surgical intervention was indicated. the operation was performed using the minimally invasive robotic system of the davinci siÒ, starting with the thorax time. the patient is positioned in left side decubitus. the camera-trocar is insert in the thorax via the fifth intercostal space the, two mm and one mm robotic trocars are added. the lung is liberated from pleural adhesions and the esophagus is then prepared exposing the diverticulum which is successfully removed with an endo-giaÒ. the esophageal muscle fibers, near the suture line is reinforced with separated vicryl stitches and the resected piece is extracted via endo-bag. a fr thoracic drainage tube is then placed and the trocar accesses repaired. the patient is the put in supine position with a °anti-trendelemburg angle. three robotic trocars (two mm and one mm) are placed and the robot docking is made from the patient left shoulder. the lesser omentum is divided to visualize and prepare the gastric-esophageal junction (gej) sparing the vagus nerve. the heller myotomy is then performed for cm over the gej and cm under it. the mucosal integrity is assured via laparoscopic and contemporary gastroscopic view. the gastric fundus is attached to the distal esophagus completing the dor fundoplication. post-operative care comprehends the removal of the thoracic drainage during the first post-operative day, the pain management and the progressive realimentation. the hospitalized period lasts day and the patient was dismissed without complications occurred. the uniportal video assisted lung lobectomies gained popularity all over the world during the last years. the technique is safely applied for peripheral pulmonary lesions, under cm, but more and more complex cases are being approached while the indications continue to evolve. our aim is to present the particular aspects of this technique in an -year-old female patient with a giant bullous lesion located in the lower lobe of the right lung. the preoperative work-up for this case is presented and commented. a multidisciplinary surgical team consisting of thoracic and pediatric surgeons was involved. a single . cm length incision in the fourth intercostal space was used for the access. due to the fact that the lesion involved almost the entire lobe and the margins were very close to the hilum, we have decided and performed a right lower lobectomy. dissection and stapling were quite difficult. all the anatomical structures had small dimensions, forcing us to perform an 'artery first approach' in a very narrow space. no complications during or after surgery were encountered. the patient was discharged after four days and she went to school on the sixth day. histopathological examination showed that the lesion was a type ccam (congenital cystic adenomatoid malformation). conclussion: the uniportal video assisted lung lobectomy was safety applied for a giant bullous lesion of the right lung. aim: dunbar syndrome, celiac trunk (ct) compression syndrome, caused by median arcuateligament is a rarely diagnosed disease because of its nonspecific symptoms, which cause adelay in the correct diagnosis. the aim of the study was to demonstrate the usefulness andadvantages of laparoscopic approach in the treatment of dunbar syndrome. methods: we performed laparoscopic release of ct in the department of general, minimallyinvasive and elderly surgery in olsztyn in . all of three patients suffered from severepain of abdominal cavity before the surgery. results: in two cases, there were a complete remission of the symptoms. in one case, there was animprovement. all patients reported relief of symptoms in the first days after the operation.there were no postoperative complications. conclusions: the laparoscopic treatment of dunbar seems to be safe and feasible procedure. thelaparoscopic surgery alone can often eliminate discomfort, while angioplasty and stentimplantation are no longer necessary. introduction: the advances in robotic surgery have permitted the application of such technology to various surgical fields, one of the last of these being hernia surgery. we present a case video of the treatment of a dual-hernia using a robotic retromuscular ventral hernia repair(rrvhr) using the davinci siÒ robotic system. the case report demonstrates the evolution of the trans-abdominal robotic umbilical prosthetic (tarup) in that it utilises a 'double docking' technique to allow the positioning of a large retromuscular mesh. methodology: our patient is a -year-old male who presented with chronic epigastric pain. the abdominal ct confirmed two abdominal wall hernias; an epigastric and supra-umbilical hernia with visceral contents and wall defect diameter of cm and . cm, respectively. using the minimally invasive robotic system of the davinci siÒ we adapted the well known retromuscular mesh technique. the operation was initially intraperitoneal with access to the retromuscular preperitoneale space using a right sided longitudinal incision.(as per standard tarup technique). we proceed with the dissection of the retro-muscular space until the left lateral edges of the rectus sheath, creating a preperitoneal space for the placement of a specifically modified ultrapro polypro-leneÒ x cm mesh. following this we repositioned the davinci siÒ in a symmetrical manner, with ports placed in the retromuscular space. the mesh is positioned and the peritneum subsequently closed with a v-lock sutureÒ. finally we opted for a negative pressure jackson-pratt drain, inserted preperitoneally. results: the patient was discharged on the nd post-operative day without complication follow up continued until months post operatively during which the patient remained asymptomatic, without signs for hernia recurrance . conclusion: the technique highlighted in our video demonstrates the utility of the robotic system in hernia repair. specifically the approach proved a success as it facilites the placement of the mesh totally extra-peritoneally with closure of the posteriore sheath without tension. the added advantages are that the port-sites are distant from the mesh thus reducing infective risk. additionally this technique allows the treatment of large peritoneal defects. surg endosc ( ) aim: to analyse the performance of a robotic fellow during a robotic total mesorectal excision (tme) at the end of the fellowship, and subsequently compare it with their mentor. methods: the fellow is exposed to robotic colorectal lists per week. during the fellowship, assessment of performance is recorded in a structured proforma covering aspects of autonomy, tissue handling and dissection. at the end of the fellowship, areview of cases performed by the fellow and the mentor was carried out in a blindly manner (video footage). results: robotic tme training was divided into modules in order of complexity and the trainee had to achieve sequential proficiency in each module, before progression. docking of davinci robotic system. inferior mesenteric artery exposure and ligation, development of medial to lateral plane and inferior mesenteric vein division. left colonic and splenic flexure mobilization. pancreas identification. rectal dissection (tme). qualitative assessments were recorded by the mentor; the fellow was 'able to perform with verbal help' most of the steps from early on. by the end of the fellowship, all steps were performed in a similar manner in terms of quality and oncological integrity when compared with the mentor. conclusions: at completion ofan advanced robotic colorectal fellowship, high quality trainees can perform every step of the tme dissection in a similar manner with the trainer, when assessed blindly, without compromising oncological integrity. aims: to find safe and simple method in robotic rectal low anterior resection with low tie arterial ligation and lymph node dissection around the root of inferior mesenteric artery. methods: we performed robotic rectal low anterior resection (rlar) by davinci si system in eight patients with rectal cancer. we applied low tie arterial ligation, just caudally to the origin of the left colic artery in all cases. during the procedure, we used tilepro function of davinci si system which enabled to display two other visual informations through external inputs under the normal -dimensional surgeon console view. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were displayed simultaneously under normal operative camera view in the surgeon console. results: left colic artery preservation was completely done in all cases. the mean time to find and expose the left colic artery from the first incision in sigmoid mesentery was min, which was drastically shorter than conventional method. this method needed lesser mobilization of inferior mesenteric artery (ima), and may be less invasive to autonomic nerve around the root of ima which is very important for ejaculation function. conclusion: robotic rectal low anterior resection with low tie arterial ligation was performed safely and in short time, using tilepro intra-operative navigation method. preoperative d-ct vessel branching simulation video and intra-operative real time ultra sound navigation view were very useful in the procedure. we present the method in video. nerve sparing tme and pelvic neuroanatomy for colorectal surgeons p. tejedor, f. sagias, j.s. khan aim: to describe the critical points in which the pelvic nerves can be damaged during a total mesorectal excision (tme) for rectal cancer and the benefits of robotic surgery for identifying these points. methods: there are critical points regarding pelvic neuroanatomy: superior hypogastric plexus (shp): located in front of l -s . the ganglionic sympathetic fibres form the right and left sympathetic trunk, travel along the anterior surface of the aorta and coalesce in the shp at the level of the inferior mesenteric artery (ima). superior hypogastric nerves: they take an anterolateral course into the pelvis. there is an avascular 'holy plane' around the rectum between these two nerves. inferior hypogastric plexus (ihp): lies over the posterolateral pelvis, almost parallel to the internal iliac arteries. this can be identified at the lower end of the rectum. neurovascular bundles(of walsh): in front of the denonvillier's fascia, at and o'clock position. they are responsible for erectile function. results: lack of knowledge or identification of key structures at these points can lead to increased risk of nerve damage and translate into poor functional outcomes. the ima is dissected up to the origin from aorta and here the shp can be seen. care is taken to avoid any damage to these structures. the tme plane is found at the back of ima as the inner most dissectible layer between mesorectum pelvic fascia. right and left superior hypogastric nerves are identified. dissection is carried out posteriorly, laterally and anteriorly. ihp is identified at the lower third of the rectum, when the dissection is about to reach the pelvic floor. care should be taken in not to go too far lateral and damage this plexus. in the anterior dissection, plane is carried in front of the denonvilliers' fascia. the neurovascular bundles can be seen at and o'clock position and the surgeon has to be careful to stay inside that plane in order to avoid damage. conclusions: the precise dissection in robotic surgery results in minimal tissue damage and better visualization and preservation of the pelvic nerves. aims: to describe and evaluate new contributions and eventual advantages of icg fluorescence to perform an icg guided bilateral pelvic lymph node dissection in a patient who underwent low-anterior-resection for rectal carcinoma. we also present the basic steps to avoid ileostomy during rectal surgery in which icg and ghost ileostomy play an important role. methods: a -year-old male patient was referred to our hospital due to abdominal pain and significant changes in usual bowel habits.colonoscopy showed a no obstructing cm middle rectal mass, which was reported as an adenocarcinoma.ct scan and mri revealed a mm polyp in the anterior rectal wall which was located cm from the anal verge. it was involving mucosa and sub-mucosa with muscularis propia invasion. no pathological lymphadenopathies or hepatic metastatic disease were found (stage t n ).a laparoscopic ultra-low-anterior resection plus icg lateral lymphadenectomy with total mesorectal excision was performed. a complete splenic flexure mobilization was performed to achieve a safe tension-free anastomosis. transection line of the proximal rectum was checked after icg intravenous injection. icg was injected around the tumor by inserting an anoscope, just before the surgery. after the dissection of the rectum, lateral lymphadenectomy was performed assisted by icg. an end-to-side anastomosis was made. and a vascular loop was passed around the terminal ileum to create a ghost ileostomy.the procedure lasted min. reactive protein c was monitored to identify an initial leak. the patient was discharge in postoperative day and no complication was detected. results: pathological exam reported a rectal adenocarcinoma. pelvic lymphadenectomy results were: negative nodes, negative nodes and negative nodes from right lymph node dissection, left lymph node dissection and rectosigmoid resection specimen respectively. no metastatic disease was found (stage t n m ). conclusions: in our experience, icg fluorescence imaging system offers important contributions to rectal surgery furthermore than evaluating vascular supply to the anastomosis. lymphatic mapping of the lateral lymph nodes and avoiding ileostomy could be a potential important use in the future. larger studies and more specific evaluations are needed to confirm its role in colorectal surgery and to find its limitations. background: robotic surgery for colorectal cancer is an emerging technique. potential benefits as compared to conventional laparoscopic surgery have been demonstrated. innovative robotic technologies have helped surgeons overcome many technical difficulties of conventional laparoscopic surgery such as hand-eye coordination, a two-dimensional view, and a restricted range of motion. robotic-assisted surgery was established as a new approach to minimally invasive surgery, overcoming these limitations. the following video shows a total robotic sigmoidectomy step by step on the basis of ourexperience. intervention: a -year-old male patient with no previous medical historyand a colon adenocarcinoma, cm from the anal verge, no distant metastases. it was decided to perform a robotic sigmoidectomy. target anatomywas located andwe proceededto the exposure of the mesenteric vessels from medial to lateral. a cautery wasused to open the peritoneum,up to the origin of the inferior mesenteric artery, and caudally past the sacral promontory.the vessels weretransected by ligasuretm. we performedthe complete release of the colon taking care to avoid injury to retroperitoneal structures. we usedligasuretm to section the mesocolon in order to prepare the transection of the proximal colon. indocyanine green was used to check the correct vascularization. an endogia tristapletm was used to divide the colon. subsequently, we sectioned the rectumand extracted the specimen through itwith no need to make any auxiliary incisions. we introduced the anvil of the suture device to perform the anastomosis. we sectionedand close the rectum with an endogia tristapletm. finally we opened the proximal colon to introduce the anvil,making a pursestring to fix it and create a side to end anastomosis. outcome: the surgery took min. the patient started oral intake h after surgery and left the hospital on the rd postoperative day. pathological examination ruled out a colon adenocarcinoma pt n . conclusion: total robotic sigmoidectomy is safe and feasible and can be a procedure of choice to achieve a good surgical qualityand avoid assistance incisions in patients with colon cancer. surg endosc ( ) with more and more data now advocating wait and watch policy for these patients which require close radiological and endoscopic follow-up but unfortunately around % of them have regrowth of tumour which will require surgical intervention. the use of robot for cancer resections is becoming more frequent especially in narrow spaces like in an obese male pelvis. the reason being better -dimensional views, more angulation of the instruments and exclusion of tremors, which in turn leads to better dissection and preservation of hypogastric nerves. in this video, we present a robotic low anterior resection for rectal re-growth in an obese -years old male patient. he was offered neoadjuvant chemoradiotherapy after discussion in mdt. he had an complete response with chemoradiotherapy and was decided to offer him watch and wait regime. unfortunately, he developed rectal re-growth in the first year of his follow up. imaging showed t lesion with no distant metastasis and was later confirmed on histology as well. after mdt discussion he was offered robotic low anterior resection. the video starts by showing the clinicopathological features of patient including his radiological and endoscopic images. robotic port sites are shown. the edited video starts with rectal dissection after ligation of inferior mesenteric artery and vein with emphasis on narrow pelvis and preservation of hypogastric nerves, seminal vesicles and intact presacral fascia. postoperative histology was ypt no and patient was discharged home after days with no postoperative complications. background: minimally invasive surgery for colon resection has improved patient outcome, however a minilaparotomy still is necessary to extract the specimen. this report describes a new approach that combine laparoscopic parellel overlap stapling left colectomy with natural orifice specimen extraction surgery, with the aim to minimize abdominal wall trauma. method: laparoscopic left colectomy for malignant diesease was performed using a standard five-port technique. after releasing the left colon via laparoscopy, divide the proximal and distal of specimen with -echelon, and put distal sigmoid colon and proximal transverse colon together. open sigmoid colon cm apart from distal margin, and incise transverse colon at proximal margin. take transverse colon and sigmoid colon side-to-side anastomosis via -echelon. incise posterior vaginal fornix to get into the abdominal cavity and extract specimen through vaginal. outcome parameters such as complications, conversions, operative time, postoperative recovery, and postoperative pain were prospectively recorded in a database. results: surgery was performed for patients with left-colonic carcinoma. no perioperative complications or conversions occurred. the median operating time was min. the median visual analogue scale score of postoperative pain was , and of patients needed analgesia on postoperative day . the median postoperative hospital stay was days. for malignancies, tissue margins were oncologically adequate, the averge number of harvested lymph nodes were . . the -week follow-up period was uneventful. conclusion: the described technique, a combination of laparoscopic parellel overlap stapling and natural orifice surgery, has the potential to avoid incision-related morbidity of the minilaparotomy in laparoscopic left colon resections. background: open surgical skills training has been well established over centuries, however, there are some significant differences in laparoscopic surgical skills training. it is an obvious advantage that the trainee and the trainer have the same view; however, some of the hurdles include the differences in tactile feedback, hand eye co-ordination, spatial awareness, depth perception and maximizing assistance. aim: we present a video highlighting some of the key challenges faced in laparoscopic colorectal surgical training, show-casing our systematic, structured approach. our approach: we have developed a structured approach starting with junior surgical trainees and progressing through to consultant level as per the levels below: level : attend courses/ workshops level : master camera work level : contra-lateral assisting level : intermediate level trainee-start operating with trainer scrubbed. the trainer is an additional member of the scrub team and stands on the same side as the trainee (does not replace any assistant) level : advanced level trainee-gradual progression from level . trainer un-scrubbed but standing next to the monitor throughout the procedure. level : trainer in theatre but out of sight of the trainee, with little interference level : progression to trainer-once proficiency is achieved at level / , the trainee is trained to become a trainer, for the junior and intermediate level trainees. within each level the complexity of the procedure increases as the trainee progresses through the level. junior trainees (years - of surgical training) are taken through levels - , intermediate (middle years of training) level or and advanced (last - years) up to levels . this way of training allows multiple members of the team to be trained simultaneously in every case. each operating list is preceded by team briefings where the role of every member of the team is clearly identified and followed by individual and collective feedback. conclusion: this training ladder proved very successful through the years. the feedback from trainees at all stages has been consistently positive. several trainees who have progressed to independent consultant practice, in the uk and abroad, are adopting this approach in their practice. introduction: despite the potential microsuturing capabilities of the robotic surgery, most of the esofago-jejunostomy after robotic total gastrectomy are still performed extracorporeal or through mechanical staplers. this can increase the cost of the procedure, the risk related to a improper functioning of the stapler. methods: we reviewed our prospectively maintained database analyzing patients from april to september , who underwent robotic total gastrectomy with hand-sewn esophagojejunostomy for gastric cancer. results: a total of patients were included in the study. the mean estimated blood loss was ml ( - ). the overall operative time was min ( - ). length of hospital stay was days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . no conversion was necessary nor anastomotic leakage occurred. the morbidity rate was / ( . %) and included a subhepatic abscess and wound infection trough pfannenstiel incision. a r resection rate was achieved in all cases. the mean of lymph node yield was ( - ). the -year disease free survival was %, the -year overall survival . %. the robotic-assisted hand-sewn esophago-jejunostomy is a safe and no time-consuming technique. it avoids the complication related to the stapler firing and it offers cosmetic benefit to the patient in terms of extraction site. introduction: colorectal endoscopic submucosal dissection (esd) is increasingly practiced for treatment of early colorectal neoplasia. however, colorectal esd is difficult to perform due to lack of retraction as well as instability especially over hepatic flexure. dilumen eip is an external flexible sheath introduced during colonoscopy to stabilize environment for esd. this video demonstrated the use of dilumen eip for performance of colonic esd at the ascending colon method and results: this is a years old lady who received screening colonoscopy and found a mm lateral spreading tumor (lst) type iia lesion at ascending colon distal to ileocecal valve. under general anesthesia, patient received colonic esd using dilumen eip. due to significant looping, the dilumen device was introduced with the techniques of double balloon enteroscopy. after identification of the lst, the balloon in the front would be deployed to the proximal to the lesion while both balloons would be insufflated and created a stable environment. the esd procedure started after submucosal injection with normal saline in mix with indigocarmine, epinephrine and hyaluronate. mucosal incision was performed over the anal side of the lesion, and after adequate submucosal dissection, clips were applied to attach the mucosal flap to the sleeve of proximal balloon and achieved retraction. the submucosa was adequately exposed for dissection using dual knife jet. this enhanced submucosal dissection especially at one area with significant fibrosis. after the procedure, complete closure of the mucosal defect was performed by clips and assisted by the front balloon. the pathology confirmed intramucosal adenocarcinoma with clear resection margins. discussion: the dilumen eip device stabilized the environment within the colon with the double balloon and provide adequate retraction for performance of colorectal esd. surgery, kobe city medical center general hospital, kobe, japan background: robotic surgery has been widely spread all over the world, but robotic gastrectomy is not common and difficult because of complex anatomy and wide-ranging operation fields. in addition, it had been performed only under a few high-volume centers for reasons of the limitation of national health insurance in japan, which means medical expenses not covered by insurance. the situation was changed from this april, so we started robotic gastrectomy to reduce complications more rather than laparoscopic gastrectomy. we report results and aim to present the methods in detail using da vinci si surgical system. methods: we place five trocars, one is umbilical endoscopy port, and other four ports are placed at the reverse trapezoid, almost fan-shaped. using the arm number , the organ can be lifted up so that sharp lymphadenectomy is able to be done by almost a scissor as the arm number while applying the countertraction by the arm number . in order to achieve a clear and bloodless lymphnode dissection while maintaining the oncological safety, we think not only the ultrasonic coagulating scissor but also the electrocautery of the scissor is very essential in robotic surgery. less postoperative complication such as pancreatic fistula or pancreatitis might be derived from robotic surgery because we can avoid pressing the pancreas during the suprapancreatic dissection of lymph nodes. the billroth i reconstruction can be performed using da vinci endowrist stapler under stable and inflexible surgical fields without needing help of surgical assistant. results: from october to december , patients with gastric cancer were operated robotic gastrectomy, included total gastrectomy. there was no conversion to open surgery and no conversion to other procedures derived from intraoperative complications, and the overall operation time is gradually decreasing from the th case. we are now on the way of learning curve shortening operation time, but robotic gastrectomy is no less safer and adequate than laparoscopic surgery. we will show our robotic procedures including lymphadenectomy around subpyloric and suprapancreatic area, and reconstruction with several important points in our video purpose: this report describes the benefits and drawbacks in the use of a novel articulating device (artisential), which has a multi-degree wrist freedom like the davinci endowrist, in performing complete single-port d lymph node dissection (lnd) in single-incision distal gastrectomy (sidg). methods: the artisential was used in performing sidg with d lnd for patients with advanced gastric cancer. all operations were performed by a single surgeon using a threedimensional camera and a passive scope holder in place of a scopist. the artisential was used mainly in the sb and suprapancreatic lnd, an area that is relatively far from the single port. in certain cases when the pancreas needed to be pushed down, such as obese male patients, the intraabdominal organ retractor was used to lift the tissue and the artisential to push the pancreas. operative results and short-term outcome were analyzed. results: twelve patients underwent the procedure without any intraoperative events, conversion to conventional laparoscopy, or surgery-related complications including postoperative pancreatic fistula. all patients underwent single port d lnd by complete exposure of the portal and splenic vein. mean operation time was . ± . mins. and mean number of retrieved lymph nodes was . ± . . the artisential was found to be useful in grasping the tissues behind the pancreas and the major arteries throughout most of the lnd. the articulating motion also allowed the narrow single-port field of view to be clearly seen without the instrument body obstructing the camera. conclusion: the use of artisential in sidg appears feasible and reproducible, and is mandatory in performing a complete d lnd in sidg. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states.the system technology utilizes an articulated multi-linked scope that can be steered along non-linear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation.the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. background: hepatobiliary procedures using a minimally invasive approachare demanding, especially in major hepatectomies. the use of da vinci surgical system allows to overcome some of the kinematics limitations of the direct manual laparoscopy maintaining the potential advantages of a minimally invasive approach . we herein present a case of left hepatectomy and local lymphadenectomy for hepatocellular carcinoma, carried out with the use of the da vinci xi. methodology: a -years old man with a long-lasting hbv chronic infection and ct scan and mri finding of a -cm solid neoplasia of the left hepatic lobe and gallbladder stones, was operated with the da vinci xi platform. the patient was placed in a supine position, with °anti-trendelenburg inclination. the trocars were positioned according with the intuitive indication for the upper quadrants surgery. results: the procedure was successfully completed in min.at first, an intraoperative us scan with the use of tile-pro technology was done to determinate the tumor extension. the hepatic parenchyma transaction and the local lymphadenectomy were performed with monopolar scissors and bipolar grasps. the left hepatic vein section was performed with an endoscopic vascular stapler. there were no surgical complications or need for conversion to laparoscopy or laparotomy. the post-operative course was uneventful and the patient was discharged days after surgery. conclusion: the da vinci xi can facilitate some technically demanding procedures and ultimately widen the range of application of minimally invasive surgery such as hepatic surgery. besides the well-known advantages provided by robotic surgery on d imaging, increased range of motion and augmented surgical dexterity, one of the most interesting and innovative features of robotic technology is the digitalization of the operative view; furthermore the tile-pro multiinput display allows the surgeon a d view of the operative field along with the ultrasound exam for a precise understanding of anatomy and vascularity and of tumor location. during the last few years, robotic surgery as well, as the latest innovation of minimally invasive procedures, takes its position in this particular field with the benefits of overcoming the limitations of conventional laparoscopy. our aim is to demonstrate the advantages of robotic surgery in procedures of hepatectomies, on occasion of a robotic hepatectomy performed by our team. methods: we present video fragments of a robotic left lateral hepatectomy procedure in an elderly female patient with a symptomatic gigantic haemangioma of the left hepatic lobe. we emphasize on the technical aspects and the advantages that the surgeon gains applying the robotic techniques in such procedures. results: the procedure was completed with minimal blood loss and the patient presented an uncomplicated post-operative course, with discharge on the third postoperative day, minimal need of analgesics and full recovery. conclusions: the excellent three-dimensional and high quality visualization that the robotic system offers, combined with the flexibility and the accuracy of the robotic instruments (especially on suturing), provide to the surgeon an important aid, in order to avoid serious complications, such as intraoperative bleeding and post-operative bile leaks. the restriction of the limitations of conventional laparoscopy is far more beneficial and promising for the evolution and the future of minimal invasive liver surgery. aims: the new da vinci xi surgical cart allows multi-quadrant and complex surgical interventions in a minimally invasive fashion. we present a case of robotic appleby left pancreatectomy using this platform and its specific operating bed. methods: a -years old woman with ct scan finding of a -mm hypo-vascular neoplasm of the pancreas body underwent surgery with the use of the new da vinci xi with four arms upper quadrants trocar' disposition. results: the procedure was successfully completed in min. the pancreatic body was mobilized in order to expose the portal-mesenteric axis. the gland was transected using a robotic endo-stapler as well as the splenic vein. after evaluating the patency of collateral circles with intra-operative ultrasound, the common hepatic artery and the celiac artery were transected. then we increased the right tilted position and the neoplasia was detached from the gastric body by a tangential gastric resection using the robotic endo-stapler. finally, the operation was accomplished with the transection of the posterior attachment of the spleen and the pancreatic tail. no conversion or intra-operative complications were recorded. the post-operative course was uneventful and the patient was discharged days after surgery. the da vinci xi with its specific tools helps in performing challenging procedures such as appleby operation for locally advanced pancreatic cancer. in our experience, the robotic endo-stapler permits the operating surgeon to directly control the transaction phase whereas the specific operating bed allows to perform minimally invasive multi-quadrant surgery and to obtain a better exposition of the operating field. results: the whipple procedure was successfully completed in min. thanks to the dvtm the patient's position changed during the intervention to improve the exposure, with the instruments left inside the abdomen and without undocking the robot. the dissection of the pancreatic head from the portal vein and the section of the retroportal lamina were performed with the use of the endowrist vessel sealer device. a personal modified end-to-side pancreatojejunostomy was carried out, with / prolene and gore-tex double layer suture. no intra-operative complications occurred and no conversions to laparoscopy or laparotomy were required. the postoperative course was uneventful. conclusions: the use of the new fully wristed vessel sealer extend makes easier difficult maneuvers such as the fine dissection of the pancreatic head from the portal vein and the section of the retroportal lamina, enabling an optimized approach for vessels sealing and cutting and tissue bundles. moreover, the dvtm allows patient's movements without undocking the system or removing instruments from the abdomen, enhancing the surgical workflow. background: necrotizing pancreatitis is a devastating illness which can develop in up to % of patients who suffer from pancreatitis. it carries great morbidity with an associated mortality rate between to %. many of these patients require drainage of fluid collections to treat sequela related to pain, per-os tolerance, and source control of sepsis if infected. the step-up approach to treatment of this disease has trended towards minimally invasive techniques, considering the morbidity of open debridement. as such, many centers have implemented the use of transgastric debridement via endoscopic cystogastrostomy. this technique, while effective in draining fluid and particulate necrotic tissue, has difficulty in resection of large necrotic tissue, due to instrument and anatomic limitations. current endoscopic accessories designed for polypectomy or foreign body extraction, for example, are not optimal for performing necrosectomy. to overcome this obstacle, additional access sites can be utilized to assist debridement. we describe the first laparoscopic assisted transgastric endoscopic necrosectomy through a percutaneous gastrostomy in a year old male with infected pancreatic necrosis secondary to biliary pancreatitis. aim: to investigate the feasibility of utilizing gastrostomy access to assist in debridement during endoscopic necrosectomy. methods: the patient previously underwent an open necrosectomy and gastrostomy tube placement for acute emphysematous pancreatitis. post-operatively, there was a persistent and enlarging cm infected walled-off necrosis (won). therefore, endoscopic cystogastrostomy was performed using a lumen-apposing metal stent. results: frank pus was evacuated. initial endoscopic necrosectomy was technically challenging due to the large volume of solid necrotic tissue. repeat endoscopic debridement utilized a surgical laparoscopic grasper via the gastrostomy site to aide solid debris extraction (video). this allowed for complete necrosectomy and resolution of the won. the patient did well and was discharged subsequently. conclusion: this is another emerging minimally invasive technique in the step-up approach for debridement and drainage of won. the use of the gastrostomy as a utility port for accessory instruments not only enhanced the technical aspects of the procedure but increased its efficacy as well. further experience is needed to validate the utility and reproducibility of this technique. objective: the presentation of the minimally invasive surgical approach for pancreatic necrosectomy guided by videoretroperitoneoscopy or var (video assisted retroperitoneoscopic), established in our center, as one of the option of the step-up approach treatment for acute necrotizing pancreatitis (anp) methods: the placement of the patient on the operating table should be in decubitus, with right lateral inclination, at - °on the horizontal surface. the pancreatic cell is approached using the drainage catheter previously placed by radiological control (ultrasound or ct) as a guide, which will allow access to the cavity with safety. an incision of - cm is made around the previously placed catheter, crossing the subcutaneous cellular tissue and muscular fascias, dissolving the musculature. it continues in a blunt dissection, until a loss of resistance is appreciated which generally coincides with the outflow of necrotic or purulent material. once the retroperitoneal cell is accessed, a mm trocar is placed and a pneumoretroperitoneum is performed. the -mm trocar allows the joint use of a mm and °optic and the surgical material that allows debridement and cleaning. the aspiration and hydrodissection of the necrotic material, and the extraction of the solid component of the necrosis are proceeded. once the collection is drained and the necrotic material removed, a wash and drain system is placed, like a -way foley type probe. conclusions: in conclusion, the var is an alternative surgical technique, valid and reproducible in the treatment of anp, which offers comparable results and even superior, in some series, to those of open surgery, with satisfactory results in terms of morbidity and postoperative mortality. aim: lung subsegmentectomy is suitable for small and deep, non-palpable lung nodules. since it is difficult to intraoperatively detect the arteries, veins and bronchi of the subsegment, as well as the intersubsegmental borders, complete video-assisted thoracic surgery (vats) for lung subsegmentectomy is challenging. we use preoperative three dimensional ct to detect the arteries, veins and bronchi of the subsegment before conducting complete vats subsegmentectomy, and perform intraoperative bronchoscopy to detect the bronchi and intersubsegmental borders. i would like to describe our experience of complete vats combined subsegmentectomy for a non-palpable lung nodule. methods and results: the patient was a -year-old woman. during health screening, a small groundglass opacity was observed in her right lung on chest ct. the nodule was mm in diameter and was located in s b (horizontal subsegment of the posterior segment) near s (the anterior segment). we preoperatively diagnosed the lesion as well-differentiated adenocarcinoma, and planned combined subsegmentectomy for s b and s a (lateral subsegment of the anterior segment) of the right upper pulmonary lobe. before the operation, the locations of vessels were confirmed by three-dimensional ct angiography. video-assisted thoracoscopic surgery was performed using four ports: two cm ports in the th intercostal space in the post-axillary line and in the angulus inferior scapulae line for the operator, a cm port in the th intercostal space in the mid-axillary line for the assistant, and a cm port for the camera in the th intercostal space in the mid-axillary line. the cm port was also used for removal of the resected specimen. intraoperative bronchoscopy was used for detecting the subsegmental bronchi. she was diagnosed with primary lung cancer (adenocarcinoma in situ, nonmucinous) postoperatively. the tumor was pathologically graded as tisn m . no tumor recurrence has been noted in follow-up of twenty two months. conclusions: the combination of preoperative three-dimensional ct angiography, intraoperative bronchoscopy and complete video-assisted thoracoscopic surgery can be used for performing lung combined subsegmentectomy. aims: minimally invasive surgery is increasingly widespread for the diagnosis and treatment of abdominal pathology. laparoscopy is a diagnostic resource for those cases in which mass biopsy is not approachable through image-guided puncture, and is often therapeutic in the same act. it avoids the morbidity and mortality associated with laparotomy, favoring the early treatment of malignant processes. methods: we present a case of a year old male who was incidentally diagnosed with an oval-shaped pelvic mass in the right lateral wall of the pelvis, adjacent to the vascular bundle of the right external iliac at its origin( centimeter), without sign of infiltration of surrounding structures. no other pathological findings on the abdominal computerized tomography and magnetic resonance imaging were found. due to its localization, it was not accessible to percutaneous biopsy. the first diagnostic impression was a benign tumor of the nerve sheath (schwannoma), without being able to rule out other diagnostic possibilities. to provide a definitive diagnosis the patient was subjected to an elective laparoscopic resection of the tumor. surgical procedure was performed using a millimiter and two millimeter, umbilicus for the optical system and operative on hypogastrium and left iliac fossa respectively. acleavage plan between the tumorand rightiliac vesselswas found. the exeresis of the masswas achieved, and it was extracted using an endo-bagÒ through the umbilical port site. a drain was put in the surgical bed. results: the patient had a short, uneventful post-operative course, being discharged on postoperative day . pathological examination revealed a lymphatic node with metastasis of poorly differentiated carcinoma, with suspected urothelial lineage. cystoscopy was performed with the finding of a centimeter lesion on the right ureteral orifice with calcifications on the surface. biopsies were taken, confirming the bladder origin of the tumor. conclusions: both diagnostic and therapeutic laparoscopy is useful on pelvic masses because of the direct vision into this narrow anatomical space, especially in obese patients, providinga detailed view that makes easier to isolate and spear the anatomical structures surrounding the tumor, minimizing the risk of tumor rupture and bleeding. surg endosc ( ) aim: indocyanine green (icg)-enhanced fluorescence has been introduced initially in laparoscopic surgery to provide detailed anatomical information during laparoscopic cholecystectomy and to evaluate vascular supply to garantee correct anastomotic perfussion in order to reduce the risk of anastomotic leak. the uses of icg are increasing, specially in hepatic and oncological surgery in order to identify centinel lymph node and lymphatic mapping.we propose the use of icg imaging during complex laparoscopic colorectal resection in cases presenting ureter obstruction, to prevent iatrogenic ureteral injury. methods: we present a case of a year old female previously diagnosed of pelvic endometriosis with severe pain and symptoms related with episodes of pseudo-occlusion .a colonoscopy was performed finding sigmoid cancer in an area of endometriosis in a narrow colon with difficulties to perform a complete colonoscopy that could be related to the process of pseudo-occlusion. the biopsy was informed as an adenocarcinoma.the ct-scan showed a dilatated left ureter in an area next to the sigmoid colon.we propose a preoperative strategy with a bilateral double j stent insertion, finding a ureter obstruction caused by the endometriosis.icg was injected through the ureteral catheter, guiding us during the surgery to avoid a iatrogenic ureteral injury. results: a laparoscopic left colectomy was performed. the icg allows us to follow the ureter during the surgery, disecting the colon properly from the area attached to the ureter. the prestenotic area of the ureter was marked dilatated up to two centimeters allowing the icg to identify it from the anatomic structures of the areas and guarateeing that there was not spill of icg out of the ureter avoiding a postoperative leak of urine. conclussions: when tumors, or another entities like endometriosis, produce a ureteral occlusion, icg could be injected through a j stent, allowing us to identify and to avoid an injury.icg fluorescence imaging is a safe, cheap, and effective tool to increase visualization during surgery, offering additional information of the anatomy in colorectal surgery. in this video we are going to show three cases of the robotic treatment of splenic artery aneurism and the evolution of the technology that we relied on for the preoperative planning and intraoperative navigation. our preoperative evaluation evolved from a tridimensional virtual reconstruction with augmented reality to patient specific anatomical d printed models, initially made of rigid materials and afterwards made of malleable materials, in order to reproduce hollow anatomical structures such as vessels, feasible to simulate the planned surgical plan. the choice of a robotic approach, in selected cases, allowed to restore the continuity of the splenic artery after the exclusion or excision of the aneurism, in order to preserve the spleen. aims: tumor-induced osteomalacia (tio) is a rare paraneoplastic syndrome in which patients presents bone pain, fractures and muscle weakness caused by the fibroblast growth factor (fgf- ), a phosphate and vitamin d-regulating hormone. in tio, fgf- is secreted by mesenchymal tumors that are usually benign but small and difficult to locate. when medical treatment is unsuccessful, surgical treatment is indicated and conclusive.this video shows our technique for tio surgical treatment guided by indocyanine green (icg) fluorescent angiography. methods: the patient is an -years-old woman with tio confirmed by blood sampling of fgf- , gallium- pet/ct scan and abdominal ct scan which identified a highly vascularized mm nodule in the mesentery along the ileo-colic vascular axis.the patient was scheduled for a diagnostic laparoscopy with tio removal.the patient was placed in supine position. three trocars were introduced in the left quadrants. identified the last ileal loop, following ileo-colic vessels, a small mesenteric bulge was found. the icg fluorescent angiography confirmed the localization of the ipervascularised nodule and helped to define its edges. the nodule was removed through monopolar energy and the hemostasis was optimized through bipolar energy. the specimen was extracted through an endobag using one of the trocar access. results: the postoperative course was uneventful and the patient was discharged on postoperative day . histopathological examination showed an extrasurrenalic paraganglioma. conclusion: tio are often difficult to locate for surgical removal. icg fluorescent angiography allows to facilitate tio localization and removal. the minimally invasive technique decreases perioperative morbidity and mortality. laparoscopic removal guided by icg angiography should be considered when tio needs to be removed and is difficult to locate. aim: this video shows our technique to perform laparoscopic resection of a voluminous left paraaortic paraganglioma. methods: the patient is a -years-old man with a recent medical history offever, lumbar pain and haematuria.abdomen ct scan, performed during admission at emergency department, revealed a x cm left paraaortic retroperitoneal mass with pseudo-aneurysm. after procedure of angiographic embolization (with spermatic artery sparing), the patient was scheduled for a laparoscopic resection of paraaortic tumor. the patient was placed in the right flank position. three trocars ( mm) in the abdominal midline and one trocar in left hypocondrium were placed. at initial examination of the abdominal cavity, voluminous left paraaortic mass arising in the contest of left mesocolon was found, dislocating posteriorly kidney vessels. the parietal peritoneum was divided and the paraaortic lesion was dissected on the aortic plane from medial to lateral and from down to up, preserving the inferior mesenteric vessels; the mobilization was carried on to splenic vein. the vessels, supplying the mass and arising directly from aorta, was isolated and taped with vascular clips. on the inferior margin of the lesion a large vessel, probably connected with previously embolized pseudoaneurysm, was dissected with vascular linear stapler. the mobilization was completed through difficult dissection from aortic plane and mesocolic posterior surface. the colonic perfusion was verified with fluorescence angiography. specimen was extracted through an endobag.a drain was left in pelvis. postoperative day .histopathological examination showed a morphological and immunoistochemical pattern for benign paraganglioma. conclusion: laparoscopic resection of paraaortic paragangliomas is feasible by skilled surgeon. the minimally invasive technique decreases perioperative morbidity and mortality. careful preoperative planning and surgeon's experience with vascular dissection and visceral mobilization are mandatory for a good outcome. aims: posterior retroperitoneal endoscopic approach has been considered for many years as a very complex and unsafe surgical technique. often attributed to a difficult location and visualization of retroperitoneal structures. in addition, surgeons were forced to work in a small and easily altered space due to discontinuous flow with constant changes of the retroperitoneal vision. lately this approach is emerging thanks to technological advances, mainly better visualization laparoscopic cameras and high definition screens, as well as continuous flow insufflators of co , maintaining stable and smoke-free cavity uninterruptedly. methods: it shows a management of a potentially serious complication and the reproducibility of the technique through the retroperitoneal approach. results: to operate with high pressure of neumoretroperitoneum allows to contain the hemorrhage and to value with relative serenity and security, the best surgical option to repair said injury being laborious due to the reduced workspace. conclusions: the posterior or retroperitoneal approach is feasible, safe and fast. although the possibility of injuring the vena cava in right adrenalectomy remains one of the most serious and feared complications. as shown in the video, posterior retroperitoneal endoscopic approach allows repair of vascular injury correctly and safely. methods: four patients undergoing adrenalectomy, two of them with right adrenal pathology and two left. minimally invasive access, endoscopic approach, is exposed in all of them. results: in the first two surgeries, right gland is shown. initially, transabdominal approach, which requires mobilization and separation of the liver to access the retroperitoneal space and subsequent proceed to adrenal extirpation. later, right retroperitoneal approach is observed, with a meticulous sealing of the adrenal vein prior to complete the dissection of the gland, despite the small cavity created by co . in the second part, both left adrenal approaches are exposed. transabdominal pathway is necessary to mobilize left colon and spleen to access a narrow space above the upper edge of the pancreas to locate adrenal gland. this is very different in posterior adrenal approach. conclusions: posterior or retroperitoneal approach is feasible and safe, allowing access to adrenal glands, located in retroperitoneal space, without across peritoneal cavity and its disadvantages. colon and small intestine mobilization is not necessary, with a lower rate of intestinal lesions and postoperative ileus. in the same way, liver or spleen mobilization is avoided. aims: when performing a laparoscopic adrenalectomy, especially in the setting of pheochromocytoma, one of the most important steps is to gain control of the adrenal vein early on in the procedure before great manipulation of the adrenal gland. we present the case of a year old female with episodic headaches and tachycardia and severe uncontrolled hypertension, found to have elevated plasma and urine metanephrines with ct scan localizing a . cm right sided adrenal nodule. the patient was prepared preoperatively with phenoxybenzamine until mildly orthostatic with dry mucous membranes and was taken for laparoscopic right adrenalectomy. methods: after positioning our patient in left lateral decubitus, ports were placed inferior to the costal margin. the right lobe of the liver was mobilized and retracted cephalad and the ivc was exposed. careful and meticulous dissection was carried up the ivc, however no main adrenal vein was encountered. the adrenal gland was then dissected circumferentially and was removed in an endoscopic retrieval bag. there was no difficulty in hemostasis and the patient was deemed to be hemostatic prior to withdrawal of the ports and extubation. results: our patient had no issues with hemodynamic stability and her blood pressure was within normal ranges during and following the case. her hemoglobin was stable postoperatively with . immediately post op and . on discharge. her pre-op hemoglobin was . . conclusions: our video demonstrates a right adrenal gland that was congenitally missing a main adrenal vein. it is very possible that small venous branches were taken with dissection however we believe this report to be important to note in the literature for surgeons performing adrenalectomy. surg endosc ( ) aims: adrenal cysts are the most frequently identified adrenal cysts, although they are a rare entity. typically they are presented by abdominal pain or palpable mass, but nowadays, cystic lesions of the adrenal gland are more often discovered incidentally by radiologic studies. adrenal cysts have an extensive differential diagnoses, which makes a difficult definitive diagnosis and a difficulty in later management. the management of an adrenal cyst can be summarized in three fundamental pillars: discard the functional status of the cyst, evaluation of eventual malignancy by images, and avoid possible complications (hemorrhage, infection), especially in large cysts . methods: clinical case: a -year-old male patient, with no history, studied for nonspecific pain in the right hypochondrium, without other accompanying symptoms. an abdominal ultrasound was performed, a cystic lesion in hcd without being able to identify the origin was seen. complementary explorations of interest are shown (ct), the biochemical study discards functionality of the lesion, negative serology for hydatidosis. the minimally invasive approach is the gold standard in the surgical treatment of adrenal pathology, so a laparoscopic approach is proposed for this patient. aims: endometriosis is a high incidence disease (approximately % of women) with a large impact on women's quality of life and fertility. endometriosis nodules surgical treatment is necessary every time there is evidence of active disease. the aim of this video is to present a minimally invasive technique for the resection of an endometriosis nodule from the abdominal wall. methods: a -years-old woman, with past history of endometriosis and a c-section, presents at the office with a palpable nodule at the rectus abdominis left lateral border, close to the umbilical scar. she had complaints of exuberant catamenial pain and magnetic resonance imaging (mri) showed a mm nodule compatible with endometriosis depot. this technique uses trocars ( ? mm) placed at the pfannenstiel scar. stepby-step as follows: (i) dissection of the pre-aponeurotic plane and isolation of the lesion (ii) lesion excision and its removal with sac (iii) closure of the aponeurotic defect braded suture. results: the post-operative period was uneventful and the patient was discharged home at post-operative day one. the aesthetic result was excellent and the patient was asymptomatic one month after the procedure. conclusion: endometriosis of the abdominal wall is related to previous c-section, is a rare event (incidence . - . %) and usually located in the subcutaneous fat underlying the scar. the presence of nodules in the depth of the muscle is much uncommon and particularly in this clinical case, the nodule was located cm cephalad from the previous pfannenstiel scar. this technique seems easy and reproducible in the authors' opinio. aims: general surgeons often face gynecological pathological findings, either along with other abdominal pathology, or as primitive cases that need laparoscopic expertise. with this particular presentation, our goal is to demonstrate the essential laparoscopic skills and the basic operative strategy that a general surgeon should be familiar with, in order to manage such cases. the presentation is made on occasion of a woman with multiple uterine fibromatosis of the pelvis, who was treated by our team. methods: we present video fragments of the laparoscopic excisional procedure for multiple uterine fibromyomatosis of the pelvis, highlighting the proper strategy in order to conclude the operation effectively and uneventfully, in a minimally invasive fashion. results: patients with multiple, large or other complex forms of uterine or pelvic fibromas can effectively be treated with a minimally invasive approach, with minimal blood loss, very fast recovery and minimal postoperative pain and complications. % of pregnancies require emergency surgery for a non obstetric indication, including acute appendicitis, cholecystitis, adnexal torsion, choledoco-lytiasis, hernias, intestinal obstructions, oncologic pathology or other less frequent indications. laparoscopic approach is the preffered surgical option for the patologies presented above. aims: to present the technical particularities and to analyze the outcomes of the emergency operations in pregnant women operated in hospital. method: a retrospective study including all the pregnant women operated in our hospital between - was performed. the preoperative workup and the surgical indication was discussed by a multidisciplinary medical team. the anesthesic and the obstetrical risk and their management was evaluated and specifically planned for each patient. the intraoperative and post-operative outcomes were recorded. results: patients with gestational age between weeks and weeks who underwent emergency laparoscopic procedures were included in the study. out of the cases we have performed appedectomies, cholecystectomies, adnexal torsions. with a min mean operating time, we had no major intraoperative complications; the technical challenges are presented and discussed. the hospital stay was , days ( - days). no major complications were associated with the laparoscopic approach in these cohort. one pre-term labour in a weeks gestational patient was post-operatively encountered. conclusion: laparoscopic surgery can be the first option for pregnant woman with non obstetrical surgical emergencies; challenges in diagnostic, management and surgical techniques of the multidisciplinary team are expected. the objective of this presentation is to demonstrate step by step the technique to the oncologic surgeon and gynaecologist in training, including some tips and pitfalls. this is a laparoscopic transperitoneal approach in a woman with advanced cervical cancer (figo ib ) that will be treated with exclusive radio-chemotherapy. the purpose of the laparoscopic lumbo-aortic lymph node staging is to define the irradiation field. in this indication false negative in pet ct ranges from to % (depending of the existence of pelvic fixation or not). the limits of this lymphadenectomy are: both ureters as the lateral limit of the dissection, iliac bifurcation as the caudal limit and renal veins as the cranial one. since the tumour is cervical and not ovarian, both ovarian veins are not resected. in the pathologic report, lymph nodes were examined free of cancer spread. the patient have had a radio-chemotherapy with restriction of the irradiation field on the pelvis. lymphocele is a frequent complication that only sometimes needs treatment ranging from dietary changing to percutaneous drainage. if conversion to laparotomy for bleeding this technique loose its benefice but this is a rare complication. this technique is feasible and safe but requires advanced laparoscopic skills. objectives: although extremely rare, isolated splenic metastases are being increasingly diagnosed due to the improvement of imaging, survival times, and surveillance of oncologic patients. this video alerts to the growing diagnostic dilemma with primary lesions of the spleen, particularly in patients with history of cancer, and reviews the laparoscopic splenectomy 'step-by-step'. case-report: -year-old male patient diagnosed with rectal cancer (g adenocarcinoma at cm of the anal verge) after a colonoscopy for rectal bleeding. thoracic and abdominal ctscan and pelvicmri, showed a ct n lesion, without distant metastases, except for a mm suspicious splenic lesion. cea- . ng/ml. after neoadjuvant therapy, a complete response was verified at the th week post-crt with a stable splenic lesion, and a 'watch-and-wait' program was initiated with no evidence of disease at the rd month. pet-ctscan did not show active metabolic features, despite an increase in the splenic lesion. in mdt, elective laparoscopic splenectomy was proposed and afterwards performed uneventfully. with the patient in semi-right lateral tilt, we approached the spleen inferiorly by dividing the splenocolic ligament. then we continued upwards, dividing the gastrosplenic ligament and exposing the splenic hilum, which was then carefully dissected, clipped and divided. finally the splenorenal ligament was divided and the spleen was extracted within an endobag, through a small pfannenstiel incision. pathologic report revealed a splenic lymphangioma. the patient is currently under a 'watch and wait' protocol surveillance with no signs of regrowth or relapse disease after year and months of follow-up. conclusion: one out of five colorectal carcinomas are metastatic at their presentation. isolated metastases to sites other than liver, lung or axial skeleton, are extremely rare, but can be found in the spleen. although the rare splenic secondary involvement is usually associated with breast, lung, melanoma, and gynecologic malignancies, if we consider solitary splenic metastases, colorectal and ovarian carcinomas are important sources. also, imaging including percutaneous biopsy, is frequently insufficient to clarify the nature of splenic lesions. for all these reasons, the decision-making process about this issue can be a true challenge, and will probably end up with laparoscopic splenectomy. therefore, surgeons must be familiarized with a standardized technique. sarcoidosis is a multisystem disease of unknown etiology characterized by the formation of noncaseating granulomas. sarcoidosis should be considered in the differential diagnosis of lymphoid disease. indications for diagnostic splenectomy includes a suspicion of a neoplasic process. the less invasive laparoscopic approach is the gold standard. case report: a -year-old female was referred to a general surgery department to complete a study to rule out lymphoid neoplasia. followed by hematology for cytopenias. biopsy of bone marrow and adenopathies were negative for lymphoid process. patient presented ct with multiple solid ( - mm) lesions in spleen, in thorax showed no pathological changes. laparoscopic splenectomy was performed. access with optical trocar, in mammary line. triangle -mm trocars after pneumo under vision. section with ligasure of gastroesplenic ligament with short vessels and phrenic-splenic ligament. identification and preservation of pancreatic tail. section of splenic vessels at hilar level (branches) with ligasure. lower pole release. release of posterior part with gerota and diaphragm. incision by aid helps in bag without fragmenting. review of hemostasis, extraction of trocars under direct vision. intraoperative findings: spleen with normal external appearance, not megalic. postoperative evolution: satisfactory. first hours without incidents and with analytical control without anemization. tolerance and mobilization starts without incidents. the histopathology report shows granulomas formed by epithelioid histiocytes with the presence of multinucleated giant cells of the foreign body type, in some perisinusoidal granulomas the giant cells with the presence of asteroid bodies in their interior. the material has been revised with the extension of special studies. conclusion epithelioid granulomatosis, non-necrotizing, which suggests sarcoidosis. the procedure lasted min. the hospital discharge was on the next postoperative day and no complication was registered. conclusion: splenectomy can be performed in a classic way, but at present the less invasive laparoscopic approach is the gold standard. indications for splenectomy include splenic tumours of unknown origin, suspicion of a neoplastic process, and splenomegaly. sarcoidosis should be considered in the differential diagnosis for lymphoid disease. postoperative pathological examination confirms the diagnosis. week-day surgery, university, sapienza, ospedale sant'andrea, rome, italy aims: we describe an interesting case of a female patient affected by a suspected echinococcus granulosus large cyst of the spleen. methods: a years old woman complained abdominal pain and a sense of gravity in the upper left abdominal quadrant. computed tomography scan(ct-scan) showed a centimetre (cm) cyst of the spleen with thickness of the wall and contrast enhancement uptake referred to an echinococcus granulosus cyst. the sierological blood test assessment, antigens and antybody markes, for echinococcus granulosus infection was negative. a laparoscopic procedure was planned. the patient was positioned on the right flank, four trocars were inserted along the left subcostal region of the abdomen: one millimetre (mm) trocar for camera, one mm for the assistant, and two of mm for instruments. a periombelical minilaparotomy was performed for the specimen extraction. results: post-operative course was uneventful. patient was discharged in third post-operative day. istopathological exam showed a simple epithelial cyst of the spleen. conclusions: laparoscopy is safe and feaseable in case of large cyst of spleen in condition of unclear nature of the cyst. laparoscopy permits to explore the abdominal cavity and to assess the cyst characteristics in a lack overlap between the radiological exam and blood test examination. surg endosc ( ) we describe laparoscopic splenectomy for recurrent splenic cyst after laparoscopic marsupialization and partial resection of splenic cyst. the patient was a -year-old woman with abdominal discomfort and with a -cm palpable mass in the left upper and inferior quadrant. she undergone years ago in another country a laparoscopic operation for splenic cyst. abdominal computer tomography revealed a cystic lesion of the spleen with concomitant huge splenomegaly. serology and oncological marker were negative. we performed laparoscopic splenectomy for the recurrent splenic cyst. the operation took min. histologic examination of the resected spleen revealed a chronic hematoma. the patient had no abdominal symptoms during months of follow-up. postoperative long term follow-up and examination by ultrasound or computed tomography is required after surgical treatment for splenic cyst to exclude the possibility of recurrence after spleen-preserving surgery. hand-assisted surgery is a recognized technique that combines the advantages of laparoscopic approach with the tactile feedback of the laparotomic one. it proved beneficial especially for the treatment of megaspleens due to lymphoma localization, thanks to safer handling of splenic vessels, major bleeding control and more effective detachment of superior splenic pole from the diaphragmatic dome. here we show an hand-assisted splenectomy for megaspleen reaching the omolateral anterosuperior iliac spine due to lymphoproliferative disease, in which the hand, inserted through a right subcostal minilaparotomy, was very useful during the dissecting manoeuvres, the splenic artery recognition and ligation and the isolation of the superior pole of the spleen from the gastric fundus and diaphragm. in any case of huge spleens, the specimen bagging is very difficult to perform in a pure laparoscopic way, not to mention the inexistence of capable endobag; besides, a minilaparotomy would be necessary for the spleen extraction. hand-assisted approach allow to overcome this not underestimable technical difficulty, reducing operative time with similar aesthetic and functional results to that of laparoscopic approach. aim: the evolution of technology and its application to the minimally invasive surgery of the thyroid gland offers new surgical techniques, like the transaxillary approach. this new procedure is still being implemented in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary endoscopic thyroidectomy and emphasize in the most relevant tips to take into account. also, current indications and limitations of this technique will be addressed. methods: a -year-old woman is referred for evaluation of a right thyroid nodule without any associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with well-defined edges and presence of peripheral vascularization. no other nodules are identified. fine needle aspiracion (fna) of the nodule describes a bethesda iii. after evaluation, a right transaxillary endoscopic thyroidectomy was performed. results: dissection begins in the subcutaneous plane above the pectoralis major muscle until identification of the sternocleidomastoid muscle. dissection continues towards the prethyroid muscles in order to perform a lateral approach of the thyroid gland. section of the upper pole allows better exposure of the recurrent laryngeal nerve (rln) which is being monitored intermittenly. identification and preservation of the parathyroid glands is the next step. surgery is completed with the section of the inferior pole of the thyroid along with the istmus. the postoperative period was uneventful and patient was discharged at h after surgery. final pathology revealed a cm nodule without malignancy. conclusion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients with benign pathology, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). further research is required to make a better assessment of the minimally invasive approaches in thyroid surgery. we present the video of a thoracoscopic esophageal leiomyomaenucleation. it has been widely demonstratedthe advantages of theminimally invasive approach in surgery. esophageal thoracoscopic surgery has been suggested as an alternative to open procedures, presenting less surgical trauma, lower risk of bleeding, less postoperative pain, lower wound infection and lower pulmonary morbidity, showing similar oncologic outcomes. although leiomyomas are the most commonof benign tumors of the esophagus, they are relatively rare, presenting an incidence of - per . autopsy series. in our case, the patient was diagnosed of leiomyoma located at the medium third of the esophagus. he referred a history of months of dysphagia for solid and liquids and retrosternal pain. the complementary studies were esophagoscopy, esophagography, ct and endoscopic ultrasonography. the patient was operated by a thoracoscopy approach using ports. it was completed the enucleation of the tumor following the closure of the muscular layer. methylene blue test confirmed no leaks. the patient was discharged on third day postoperative developing no incidences. pathology report: leiomyoma cm size, actin and desmin positive; s- , cd and cd negative. we want to demonstrate the advantages of a minimally invasive approach in this kind of pathology. aims: this video shows our technique to perform thoracoscopic enucleation of large esophageal leiomyoma. methods: the patient is a -years-old woman with a six months history of progressively worsening dysphagia. chest ct scan revealed a cm lesion of middle esophagus with extrinsic compression of mucosa and no increased fdg uptake on fdg-pet scan. barium swallow study showed a lateral deviation of thoracic esophagus due to extrinsic compression. endoscopic ultrasound confirmed the suspicion of esophageal leiomyoma. patient was scheduled for a thoracoscopic enucleation of esophageal tumor. she was placed in prone position and one-lung ventilation was employed. three trocars were placed in intercostals spaces on right hemithorax. azygos vein was identified and transected between vascular clips. esophagus was circumpherentially isolated from mediastinal structures. after myotomy, the lesion was dissected from submucosal-mucosal layer. since air leak test excluded injury of internal layer, muscular layer was closed with a continuous suture. the specimen was extracted through an endobag. a drain was left in place. results: the postoperative course was uneventful and the patient was discharged on postoperative day . final pathological examination confirmed esophageal leiomyoma. conclusion: thoracoscopic surgery in prone position allows removal of large esophageal tumor with several advantages. the minimally invasive technique decreases perioperative morbidity and mortality. introduction: spontaneous esophageal perforation is life threatening disease and requires emergent surgical treatment. recently, the efficacy of minimally-invasive surgery such as laparoscopic and thoracoscopic surgery for esophageal perforation has been reported. we report a novel technique of minimallyinvasive abdominal and left thoracic approach (malta) for spontaneous esophageal perforation. case presentation: -year-old male, who had been under hemodialysis due to iga nephropathy, complained of chest pain after vomiting several times. since the ct scan showed left hydropneumothorax and pneumomediastinum, and the gastrografin study demonstrated extravasation from left side of esophagus, we diagnosed him with the spontaneous esophageal perforation and planed emergent surgery. the patient was placed in the reverse trendelenburg position, and the legs were split, with the left side of the upper body lifted in order to perform thoracoscopy and laparoscopy simultaneously. first, we explored the thoracic cavity through a mm port in the left th intercostal space and added other ports. we identified the rupture site mm in size on the left wall of the lower esophagus and sutured the mucosa and the muscle layer with a running suture respectively. we covered the perforation section with pericardium fat and irrigated the cavity with physiological saline. then transferred to the abdominal cavity, no contamination was found in the abdominal cavity. a feeding tube was inserted into stomach through the round ligament of the liver and the operation was completed. the total operative time was min and the amount of intraoperative bleeding was ml including pleural effusion. postoperatively, the patient experienced left empyema pleurae but no other severe complications and was discharged on postoperative day . conclusion: we experienced a rare case of spontaneous esophageal perforation of a patient under hemodialysis. malta is an effective procedure for emergent esophageal operation because of great visual field of the chest and abdominal cavity without expanding contamination. introduction: digestive caustic injury is associated with high morbidity and mortality with stenosis in the long term. surgical treatment involves resection of the esophagus and reconstruction with the stomach, colon or jejunum. coloplasty provides several advantages but its vascularization is complex and involves anastomosis. classically, vascular assessment was achieved by palpation through laparotomy and color evaluation. indocyanine green (icg) allows a minimally invasive intraoperative angiography in real time. methods: a -year-old female with medical history of caustic ingestion and subsequent esophagogastric stenosis, carrier of feeding jejunostomy. . thoracoscopy (prone position): dissection of the esophagus from the hiatus to the upper thoracic inlet. . laparoscopy: patient in the supine position, placement of five trocars. total non-oncological gastrectomy, post-pyloric section of the duodenum and omentectomy were completed. mobilization of the righ, transverse and descending colon. measurement of the transverse colon with a tape (distance from the neck and the esophageal hiatus). individualization of the righ, middle (with its branches) and left colic arteries and placement of clamps at the right colic, right branch of the middle colic and left colic arteries. cc of icg were injected allowing for an assessment of the colon vascularization. section of the right branch of the middle colic artery. proximal section of the ascending and distal colon near the splenic angle, preserving the marginal arch. silk point to join the staple line of the descending colon and the pylorys. side-to-side mechanical antiperistaltic anastomosis between the distal endo of the coloplasty and the jejunum. finally an anastomosis between the ascending and descending side-to-side mechanical anastomosis using an assistance incision in the left flank was performed. . cervical dissection: extraction of the surgical especimen under laparoscopic control. vascular assessment with icg is performed before and after the side-to-side anastomosis is performed. results: there were no intraoperative complications. the patient was discharged on postoperative day . discussion: we describe the first case of total minimally invasive colonic interposition with icg assessment of the vascularization. this technique, although technically demanding, avoids the drawbacks of the open surgery and allows for a precise assessment of the vascularization of the graft. surg endosc ( ) introduction: large pedunculated fibrovascular polyps are uncommon, mostly benign, intraluminal massess, usually located in the upper esophageal tract. most frequent reported clinical manifestation is dysphagia, followed by regurgitation, chest pain and intestinal bleeding. ct scan, and mri are the key in the diagnostic work-up revealing a sausage-shaped intraluminal mass. endoscopy with ultrasonography and biopsy add important information for the diagnosis and pedicle location. surgical excision is deemed due to potentially life-threating complication related to airway obstruction. the most frequent polyp resection is performed through cervical esophagotomy or by direct esophagectomy. however, this approach is related to a high morbidity and mortality rate. in the last years, few excisions have been reported by a endoscopical approach with a lower post operative complication. material and methods: this video shows the surgical steps of a trans-oral endoscopic surgical resection of a giant ( cm) pedunculated polyp in a year old man. the procedure was performed under general anesthesia. a flexible endoscope probe was used and the distal end of the polyp was extracted through the oral cavity with a loop. the endo-gia stapler was used to cut the base of polyp and finally removed. the anatomo-pathological study confirmed the diagnostic of a fibrovascular polyp with no evidence of malignancy. results and conclusions: the patient had an uneventful recovery with no recurrency at years of follow up. this minimally invasive approach is a safe and feasible procedure to treat large esophageal fibrovascular polyps avoiding the complications related to more aggresive procedure. introduction: leiomyomas are the most common mesenchymal tumors affecting the esophagus and they usually grow in the mid to distal third of it. they tend to be asymptomatic, but sometimes they can grow to enormous size and produce dysphagia. case report: -year-old male asymptomatic patient was referred to our hospital due to an incidental finding. ct scan revealed a x mm rounded submucosal tumor on the dorsal side of the lower third of the esophagus. upper gastrointestinal endoscopy revealed a cystic lesion in the lower esophagus cm from the incisor teeth, with normal overlying mucosa. an endoscopic-ultrasound-guided fine-needle-aspiration of the mass was performed, which was reported as a likely leiomyoma.conservative treatment was performed, no growth was detected during eleven years of follow-up. but it became symptomatic, the patient complained of progressive dysphagia caused by compression so surgical resection was decided.laparoscopic enucleation of esophageal leiomyoma was performed. the tumor was reached by transhiatal dissection. a careful dissection of the mass was performed, preserving the vagal branches. an intraoperative endoscopy was performed to verify the integrity of esophageal mucosa and that the tumor was completely resected. the muscular layer was sutured after enucleation using absorbable suture material and the hiatus was closed with non-absorbable suture material. a dor fundoplication was also performed. a swallow test with a water-soluble contrast was obtained on postoperative day one. no pathological findings were found so the patient was asked to drink.histopathological exam revealed a tumor measuring mm mm mm consistent with leiomyoma.the procedure lasted min. the hospital discharge was on the third postoperative day and no complication was registered. conclusion: surgical excision is the mainstay of treatment and is recommended for symptomatic leiomyomas and those greater than cm. this case report demonstrates the technical feasibility, safety and minimal postoperative morbidity associated with minimal invasive esophageal surgery. introduction: total esophagectomy by means of minimally invasive surgery has proven to be a valid and effective alternative for performing this procedure. however, this procedure is not implemented in most centers. objective: demonstrate the technique of a total esophagectomy by endoscopic surgery for a benign esophageal stenosis. material and methods: clinical case: a -year-old female patient diagnosed with double esophageal peptic stenosis, treated on several occasions with endoscopic dilation by digestive, showing in the last endoscopy: severe esophagitis with stenosis impassable to cms. additional tests of interest are exposed. resolved: intervention: right thoracoscopy in prone position, dissection and complete mobilization of the thoracic esophagus, section of the azygos vein, pleural drainage. laparoscopic time, trocars, gastrolysis respecting the right gastroepiploic vessels, broad kocher until the cava is identified, vascular section of the vessels left gastric, full mobilization of the stomach, subxiphoid minilaparotomy, beginning of the cervical time with dissection of the cervical esophagus, section and fixation of this to a tube, externalization of the piece by abdominal route, creation of the gastric tubular with successive loads of gia, ascending posterior mediastinal plasty with manual esophago-tubular anastomosis, with placement of drainages and feeding jejunostomy. right operative with radiological control with gastrografin on the th day, discharge from hospital on the th day. asymptomatic one year after surgery, with radiological control without alterations. conclusions: the approach of esophageal peptic stenosis with minimally invasive surgery is safe and effective, adding the advantages inherent to this type of technique. (figs. , ) , showing a cystic lesion in the gastric submucosa with a well defined, medial and superior to the lesser curvature of the stomach with exophytic growth. it causes extrinsic compression of the cardia in the gastric body. within the differential diagnosis are gastric duplication cysts or gastrointestinal stromal tumor. a biopsy was taken, discarding the presence of neoplastic cells. finally, a study of digestive transit showed extrinsic compression at the cardial level, which causes difficulty in passing contrast. a laparoscopic approach was performed beginning with the dissection of the abdominal esophagus. the presence of a cystic lesion on the anterior face of the abdominal esophagus was identified (figs. , ) . we proceeded to the complete resection of cyst. the surgery was completed with °f undoplication anterior dor. the patient went home on the rd day without incident. results: duplication cysts are congenital malformations of the gastrointestinal tract contiguous with the esophagus, which can communicate with the esophageal lumen. most are diagnosed in childhood, but when it is diagnosed in adults, they used to be symptomatics. it is more frequent in men. although the pathogenic mechanism is unknown, it is caused by an anomaly during embryonic development. they are located in the thoracic esophagus, at the level of the lower and posterior mediastinum and, less frequently, in the abdominal esophagus, as in our case. they can give digestive symptoms(epigastralgia, vomiting) or respiratory symptoms. the diagnosis is made with eda, ct, and ecoendoscopy of choice, although it may be incidental. the treatment of choice in symptomatic patients is complete resection or cystic enucleation. in asymptomatic surgical is not defined, because it can cause complications, and, malignization due to degeneration is very infrequent. conclusion: the most of duplication cysts are diagnosed in childhood, although it's more frequent in adults to be symptomatic. surgical treatment can cure this disease. however, the choice between these becomes difficult in young patients, where the low incidence does not allow get series of long patients and decisions must be based on results achieved in adults. objetivos: to demonstrate the safety and efficacy of the laparoscopic approach in this infrequent pathology, pointing out the importance of having standardized the procedure to achieve better results. material and methods: case report: a -year-old man with progressive dysphagia until almost complete afagia, with clinical, endoscopic, radiological and manometric diagnosis,compatible with typical primary achalasia. chagas negative serology,we show the complementary studies of interest.dilatation is not performed, preoperative symptomatic treatment with calcium channel inhibitors. intervention: laparoscopic approach, trocars, aberrant left hepatic artery with signs of severe esophagitis,opening of the gastroesplenic-hepatic ligament, no retroesophageal window, dissection of the hiatus and inferior mediastinal, preservation and mobilization of the left hepatic artery and the anterior vagus,meticulous disection of the cardia,standardized myotomy: first proximal cms. with adequate simultaneous traction of both edges of the myotomy, then distal myotomy including - cm, including selectively the distal oblique fibers of les, tutorization with fouché and methylene blue to confirm good step and absence of leakage, dor-type funduplication, pts on each side, fixed to both pillars, hiatalmediastinal drainage. egd on the st day of normal po, dischargeat rd day, asymptomatic and with normal radiological control at year of age. conclusions: laparoscopic mh should be the first therapeutic option, in patients with primary achalasia, even in young patients. the length of myotomy, especially distal to ueg is one of the most important aspects of surgery, most authors (pellegrini) recommend that the myotomy extend - cm in the stomach, even up to cm below the ueg to achieve an effective disruption of the eei.the standardization of the procedure is fundamental to increase safety and effectiveness in these more complex cases. aims: the surgical treatment of giant hiatal hernias is a complex and demanding procedure, not only in terms of performing the operation in a minimally invasive abdominal fashion by avoiding thoracic approaches, but also concerning the management of large hiatal defects which contribute to high recurrence rates. our aim is to present our surgical technique for the reconstruction of such hiatal hernias, exploiting the benefits of the robotic approach and also to highlight the technical aspects of non-absorbable mesh placement in order to bridge effectively the hiatal defects. methods: we present video fragments from a procedures selected from a series of cases of robotic reconstruction of giant hiatal hernias performed by our team, in which a non-absorbable meshes were utilized to restore the hiatal gap. we emphasize on the clear benefits of robotic surgery in these cases and on the strategy of how to avoid high recurrence rates. results: all of our patients, who underwent reconstruction of giant hiatal hernias with this particular technique, experienced very good early post-operative results, very short hospital stay and no recurrence in a -month follow-up. conclusions: the robotic approach for the treatment of large hiatal hernias offers great advantages to both surgeons and patients, by eliminating the restrictions of conventional laparoscopic surgery, minimizing intra-operative incidents and post-operative complications. large hiatal defects are very effectively closed with the use of advanced suturing techniques and non-absorbable meshes in a tension-free bridging fashion. aims: mckeown esophagectomy is commonly used for invasive esophageal carcinoma. as the morbidity and mortality rates for esophagectomy are persistently high, minimally invasive esophagectomy in prone position is expected to reduce respiratory postoperative complications. there is still limited experience for the use of minimally invasive approaches in patients undergoing surgery after neoadjuvant chemoradiation and many concerns about the feasibility, safety, and oncological outcomes of these procedures are still present. methods: we present the case of a -year-old female with a middle third esophageal squamous cell carcinoma, who received neoadjuvant chemoradiation. she underwent laparoscopic and thoracoscopic (prone position) mckeown esophagectomy with hand-sewn esophagogastric anastomosis through a left lateral cervical incision. results: the operation was completed successfully, with no conversion to open surgery. the operative time was h with minimal blood loss and the patient was fed on day and discharged on day post-op. r resection was achieved and the number of total harvested lymph nodes was ( positive nodes, n ). conclusions: minimally invasive mckeown esophagectomy in patients with esophageal cancer and prior chemoradiation is feasible and safe procedure with acceptable oncological outcomes. results: preliminary results demonstrated that minimally invasive ivor-lewis esophagectomy procedure, provided of a better postoperative pain control and less respiratory complications. in order to standarise our procedure, the video shows how three different types of esophagogastric anastomosis are performed, depending on the patient characteristics, anatomical factors and safety and comfort for the surgeon: manual termino-terminal, mechanical termino-terminal and mechanical latero-lateral. conclusions: in our way to standardization, we are still looking for the best type of anastomosis, even though, we find out that, manually performed anastomosis are easier to performed, when the section in esophagus is lower, involving medium and inferior third. in the other hand, mechanical termino-terminal anastomosis seemed to be an ideal option for upper sections. more studies are needed in order to standardized one anastomosis, for all cases. because esophagectomy with radical lymphadenectomy is highly invasive, thoracoscopic esophagectomy (te) is attracting attention as a less invasive procedure. we first performed te with the left decubitus position in . in we developed a hybrid of the prone and left lateral decubitus positions for te, and a total of patients underwent te with a hybrid position . we introduced te with a hybrid position for the following three reasons: ( ) mobilization and lymphadenectomy around the middle and lower esophagus are easier in the prone position. thanks to artificial pneumothorax and the gravity, the middle and lower mediastinum are opened, and which give us good surgical field. ( ) lymphadenectomy along the left recurrent laryngeal nerve (rln) is more reliable and precise when performed in the left lateral decubitus position. we can dissect lymph node around the rln higher position in the upper mediastinum. ( ) unexpected events requiring conversion to thoracotomy (e.g., massive bleeding, injury of other organs, dense intrathoracic adhesion, resection of adjacent organs) are easier to deal with in the left lateral decubitus position. the patient is fixed on the operating table with the semi-prone position and we can easily change patient positions from the left lateral decubitus position to the prone position and vice versa using rotation system of the operation table. the upper mediastinal procedure including lymphadenectomy along the right and left rln is performed with the patient in the left lateral decubitus position, while the middle and lower mediastinal procedures are performed with the patient in the prone position with artificial pneumothorax ( mmhg). theabdominal procedures wereperformed by hand-assisted laparoscopic surgery (hals) and gastric tube reconstruction through aposterior mediastinal route was performed as a standard surgical procedure in our institution. the magnifying effect of thoracoscope enables us to perform more precise surgery and preserve nerve and vessels, and a hybrid position is thought to be feasible and effective methods. ivor lewis esophago-gastrectomy is a standard procedure for the treatment of distal esophageal cancer. among the years, the surgical community standardized the mininvasive abdominal phase. the thoracic phase is much more complex because usually all surgeons get in trouble in the phase of esophago-gastric anastomosis. in fact, is still very difficult and tricky to perform a mechanical circular anastomosis due to problems with the correct handling of the circular stapler through the minitoracotomy and is also difficult to place the anvyl in the proximal esophagus. the linear anastomosis (side-to-side) is a little bit easier but not so effective as the circular anastomosis in terms of leak rate. we think that robotic approach with its endowrist can allow us to overcome these limits and that a tailored double layer hand sewn esophago-gastric anastomosis could be the right choice. we treated patients with this approach and they were all uneventful in the post operative period except for a case of chylothorax we treated successfully with lipiodol injection in inguinal lymphnodes. we need more cases to analyze the technique in terms of leak rate and major complications but we think this is a promising and cost-effective procedure for robotic approach. aim: anastomotic leakage is one of the most dreaded complication after esophagectomy. indocyanine-green near-infrared angiography (nir-icga) intraoperative use has been recently introduced for visceral perfusion evaluation. in this video we present our technique for gastric conduit fashioning according to the nir-icga blood supply evaluation in a total minimallyinvasive ivor-lewis esophagectomy. methods: a years-old man affected by a siewert adenocarcinoma (ct , n , m ) underwent a preoperative neoadjuvant treatment according to cross protocol. at restaging ct-scan no more pathologic nodes were evident (ct , n , m ). the patient was submitted to a total minimally-invasive ivor-lewis esophagectomy. surgical procedure: after pneumoperitoneum induction and trocars insertion, the lesser omentum was opened and a lymphadenectomy at stations a, , , and performed. the esophagus was dissected at diaphragmatic hiatus with lymphadenectomy at station , and . the larger omentum was opened along the right gastroepiploic arcade, that was preserved, the short gastric vessels divided and gastric fundus mobilized. by evaluating the presence of an intense fluorescence at nir-icga, a tailored partial tubulization of the stomach was performed with multiple linear stapler firing. a right thoracoscopy was performed through trocars. the azygos vein ligated and divided. the mediastinal pleura was opened and the esophagus was dissected entirely with an en-bloc excision of nodes at stations , and . nodes at stations , , and were removed separately. the esophagus was sectioned above the azygos vein level and a purse-string fashioned. the cardia and the gastric tube were pulled up and a minithoracotomy performed. a new nir-icga was repeated to verify the good blood supply and tailor the site of anastomosis on a well perfused area. the stomach was opened and a circular stapler inserted. after the end-to-side esophago-gastric anastomosis fashioning, the tubulization was completed by linear stapler firing and the specimen removed. results: the post-operative course was uneventful and the pathologic examination revealed a cardial adenocarcinoma (ypt ,n , r ). conclusions: nir-icga is an interesting and easy-to use tool for surgeons. nevertheless in literature is still not clear which is the best parameter to measure the blood supply. large studied are needed. aim: uses and application of indocyanine green (icg) fluorescence in the field of surgery are growing exponentially. the safety and feasibility of its usage has been proven in several areas and various pathologies of surgery and surgeons are starting to incorporate it into their common practice. however, there are still several aspects to define regarding this technology. we present different uses of icg in the specific area of esophageal cancer. methods: we used icg fluorescence at different moments of a two-field minimally invasive esophagectomy. first of all, peritumoral injection of icg may offer a lymphatic mapping, both in the abdominal phase of the surgery and the thoracic one, improving lymph node dissection by allowing a more targeted and less morbid approach that includes all relevant nodal stations. at the moment of the gastric section, intravenous injection provides assessment of gastric conduit perfusion, therefore optimizing the construction of the graft to avoid the inclusion of poorly perfused areas that may increase the risk of leak of the anastomosis. besides that, the esophagogastric anastomosis can be tested in the thoracic phase of the operation in order to check an adequate perfusion and prevent further complications. results: we consider that icg fluorescence is a promising technology that could be easily introduced in the surgical routine of the esophageal surgeon as an instrument to assess the anastomosis perfusion. icg is also feasible in detecting lymph node drainage from the esophagus, although its technique of application needs to be defined. conclusions: icg fluorescence has opened a new world of possibilities in all the different surgical specialties. its use in the esophagectomy is safe, simple and feasible. in a near future, its application to esophageal cancer surgery could improve survival by predicting and preventing anastomotic leak and guiding in a tailored lymphadenectomy. further research is needed to demonstrate these promising applications. introduction: the oesophagectomy is currently still mandatory in the curative treatment of the malignant oesphagic pathology. this procedure is defined by important morbidity and mortality. the minimally invasive approach aims to reduce the complications without repercussion on the oncological outcomes, however it's not exempt from them being a demanding surgical technique like it is. aim: we present the video of three complications after a three-stage oesophagectomy (mckeown-like) with the thoracic stage via thoracoscopy and the minimally invasive surgical solution for both of them. methods: all three cases represent a three-stage oesophagectomy for malignant esophageal pathology. the first one was a -year-old male who suffered an intraoperative left main bronchus injury. the second case was a -year-old male with no intraoperative complications whatsoever. nonetheless, on the second postoperative day, milky drainage started to appear through the thoracic tube. the third and final case represents an intraoperative hemorrhage, which is the most common complication of this kind of surgery. results: the first case was diagnosed and treated intraoperatively with the use of an adhesive matrix. during the postoperative period the patient showed no further complications. the second case was a chylothorax, diagnosed on the second postoperative day. it was treated initially with conservative measures. due to bad evolution, he underwent surgery on the tenth postoperative day. we can see how we ligated the stump of the thoracic duct in the original surgery and then how we repaired the unexpected leak. after the second surgery, the patient was discharged on the sixth day. the last patient was also diagnosed and treated intraoperatively successfully, with no repercussion whatsoever in the postoperative time. conclusions: the minimally invasive surgery has many advantages in the upper gastrointestinal field. it is a demanding technique, so it is important to be able to treat the complications that may arise with this approach. surg endosc ( ) aims: the use of icg fluorescence is incrising in surgery, mainly as a test of vascular supply in colonic anastomoses. during the last years, other potential uses have been described, such as the identification of the sentinel lymph node and lymphatic mapping in oncological surgery. these advances could allow a better staging in order to decide the most appropriate treatment to each patient. gastric cancer is one of the fields where this could play a key role in the near future. we present a case of a patient who underwent a laparoscopic total gastrectomy with icg-guided d lymphadenectomy, where a personalized lymphatic mapping was performed. methods: a -year-old male patient underwent gastroscopy for gastric discomfort, and a gastric carcinoma was detected at the greater curvature of gastric body. endoscopic biopsy was informed as diffuse type gastric adenocarcinoma. the preoperative staging was completed with echoendoscopy and ct-scan (t bn m ). we decided to perform a laparoscopic total gastrectomy with icg-guided lymphadenectomy. the preoperative day, a gastroscopy was performed to inject . mg of icg in four submucosal areas around the tumor. results: intraoperatively, the lymphatic mapping marked by icg was checked, allowing the identification of the territory of drainage of the tumor to lymph nodes at the lesser curvature, the greater curvature and the splenic artery. a d lymphadenectomy and a total laparoscopic gastrectomy with roux-en-y reconstruction was performed. during the lymphadenectomy, we were able to observe marked lymph nodes in territory , and also observed that the paraaortic lymph node behind the celiac trunk did not become green and the lymphadenectomy at this area was not continued. the patient presented no postoperative complications, and was discharged on the seventh postoperative day. the histological results showed a diffuse type gastric adenocarcinoma pt n and isolated lymph nodes, being one of them possitive (corresponding to the adenopathy marked at the greater curvature). conclusions: lymphatic icg-mapping in gastric cancer is a potential revolutionary advance that could ensure a correct lymphadenectomy, avoiding lymph node understaging. it is necessary to continue carrying out studies that will allow developing protocols to define appropriate lymphadenectomy based on icg-mapping. introduction: petersen's hernia is one of severe postoperative complication after gastrectomy, which may result in massive resection of small intestine. it is considered an essential proscedure to close the petersen's defect for all cases after such reconstruction after gastrectomy as roux-en y method. we report a case of petersen's hernia after radical gastrectomy, which was repaired laparoscopically. patient: the patient was -year-old male, who underwent laparoscopic distal gastrectomy for gastric cancer (d lymph node dissection followed by roux-en-y reconstruction) two years ago. the closure of petersen's defect was not performed in the initial operation. he was aware of abdominal pain and visited emergency unit in our hospital. abdominal ct scan showed internal hernia of petersen's hernia. surgical procedure: in laparoscopic examination, dilation of small intestinal and mastoid ascites was observed. massive small intestine including y-limb entered into petersen's defect from left to right side. we carefully pulled through the small intestine and confirmed absence of ischemic change in the whole small intestine. then the petersen's defect was closed by continuous suturing with - non-absorbable barbed suture. results: the operation time was min and the estimated blood loss was ml. oral intake was started from the next day of the operation. there was no postoperative complication. the patient was discharged on the th postoperative day. conclusion: we could safely perform laparoscopic repair for petersen's hernia. regarding technical points in the procedure, it is important to judge the direction of the small intestine into the petersen's defect, to manage the dilated small intestine gently, and to close the petersen's defect by laparoscopic suturing. introduction: in this case we present a -year old male with a history of morbid obesity, sleep apnea and psychiatric affliction including alcohol and nicotine abuse. in he underwent a laparoscopic roux-en-y gastric bypass. the results were satisfactory, with no complications post-surgery, and a steady weight loss over time (pre: kg, post: kg). in november , he presented with complaints of dysphagia and weight loss ( kg in months). laryngoscopic examination by the otorhinolaryngologist was negative. he was referred to gastro-enterology for gastroscopy. biopsies showed a mildly differentiated adenocarcinoma of the gastro-esophageal junction with submucosal invasion. objective: after negative staging assessment, multiple treatment options were considered. the route of choice ended up being a laparoscopic radical gastrectomy with esophago-jejunostomy, with the objective to achieve optimal oncological results. methods: the procedure is demonstrated in this video. the gastric pouch as well as the remnant stomach, greater and lesser omentum were resected laparoscopically. due to the invasion of the carcinoma into the distal esophagus, a segment of the esophagus was resected as well. following anatomopathological examination on frozen section, the resection margins were reported malignancy free. results: postoperatively, there were no complications. ct scan with contrast showed no signs of leakage. anatomopathological examination confirmed the tumor to be a mildly invasive and poorly differentiated adenocarcinoma with local signet-ring cell differentiation (pt bn ). there was no need for adjuvant therapy. oral intake was sound. conclusion: adenocarcinomas of the gastric pouch are rarely seen following gastric bypass. this patient presented with complaints of dysphagia, and an adenocarcinoma was diagnosed. consequently, the patient had a total gastrectomy at our hospital. the surgery was performed laparoscopically, and was executed with success. to conclude, it is feasible to treat adenocarcinomas after gastric bypass laparoscopically via total gastrectomy and omentectomy. year old, female patient presented with upper abdominal discomfort and microcitic anemia. an ulcerative lesion was found on gastroscopy examination in body of the stomach (near the grate curvature). biopsy was done and pathology result showed poorly differentiated adenocarcinoma. chest computed tomography (ct) was without any significant findings. abdominal ct showed the lesion in stomach without enlargement of regional lymph nodes. her blood laboratory examinations were within normal limits, including serum cea. patient underwent laparoscopic total gastrectomy with modified d lymphadenectomy and roux-en-y esophagojejunostomy. total operating time (ort) was min. three days after operation, patient has developed none st elevated mi and respiratory failure. she was intubated. on day after operation she was extubated, on day patient started regular diet and was discharged home on day . final pathology result confirmed poorly differentiated adenocarcinoma of the stomach. this video shows our favourite technique for laparoscopic d subtotal gastrectomy. we usually perform the procedure with or trocars; after the coloepiploic detachment we perform the gastric transection first! this manoeuvre provides a perfect view for the lymphadenectomy. at the end of our dissection we transect the duodenum with seamguard reinforcement. before going on with the reconstructive phase, we prepare the roux en 'y' with double loop technique, usually without dividing the mesentery. then we remove the specimen through a periumbilical - cm minilaparotomy; we think it's important anyway to check margins and distance from the tumor before going on with the reconstructive phase. from the same minilaparotomy we retrieve the prepared limb for roux en y and we perform a side to side mechanical linear anastomosis outside.then we proceed with performing the anastomosis between the gastric pouch and the alimentary limb by laparoscopy. we like very much this technique for the increased exposition of tissues during lymphadenectomy. laparoscopic roux en 'y' d surg endosc ( ) a body-tc was performed in which axillary, mediastinal adenopathies and images suggestive of hepatic metastases were identified. the biopsy confirms a gastrointestinal stromal tumor. the case discussed in a multidisciplinary committee and the pet-ct study was completed, subcardial gist t n m was diagnosed. neoadyuvance was decided with imatinib for one month and surgery was performed using a laparoscopic approach. the approach was performed with trocars ( mm supraumbilical, two mm subcostal left and right, mm subxifoid and mm left flank). gastrectomy was performed with d lymphadenectomy following the oncological principles of subcardial tumors. the piece was removed in a bag by extending the mm port to mini-laparotomy. esophagogastric anastomosis was performed by hand assisted circular mechanical suture. methylene blue test was carried out. no nasogastric tube left, but drainage tutoring the esophagogastric anastomosis was left. results: the postoperative evolution was favorable. oral tolerance without incidents at fourth postoperative day. the patient was discharged without incidences on the seventh postoperative day. the pathological study of the piece was reported as subcardial gastrointestinal stromal tumor cm with respected surgical margins and lymph nodes free of malignancy, postoperative diagnosis of t n m . one month after surgery, the patient has adequate oral tolerance. she does not report gastroesophageal reflux and at months remains asymptomatic and with good evolution. conclusions: laparoscopic proximal gastrectomy is a technique that is not currently used but can be performed through a laparoscopic approach. it is a safe technique with good clinical and oncological results, especially in the early gastric cancer and gastrointestinal stromal tumors. however, long-term studies are necessary. laparoscopic gastrectomy is a perfectly safe option nowadays for the treatment of gastric cancer. every year the percentage of the laparoscopic approach is rising not only in the east but also in the west. we present a case of a year old female patient with a gastric tumor of the antrum-g adenocarcinoma with a ct n m staging. we perform a subtotal laparoscopic gastrectomy with a d lymphadenectomy and roux-en-y anastomosis. the patient begin clear liquids on the first post operative day and was discharged on the th. the final anatomopathological result of the specimen was a adenocarcinoma (g )-pt n . there were nodes resected all negative. the case was discussed in multidisciplinary team and was decided for clinical follow up with no further treatments. the patient was evaluated one month after surgery with no complaints and will continue the follow up. upper gi surgery, university of verona, verona, italy laparoscopic endoscopic cooperative surgery is an option in medium size submucosal cancers invading the muscular layer, mainly in border area were wedge resections are nor feasible.in this video we report a case of prepiloric gist treated with news technique (nonexposed endoscopic wall-inversion surgery).we think that this technique is feasible and safe and should be considered a valid option with a view to preserving the organ. aim: laparoscopic wedge resection or partial resection is a safe and feasible stomach preserving approach to gastric submucosal tumors (smt) such as gastrointestinal tumors (gist), and it has been widely performed recently. however, it should not be applied to the tumors at cardia in order to avoid stenosis or disruption of anti-reflux mechanism. we have introduced percutaneous endoscopic intragastric surgery (peigs) for smt at cardia since to preserve function of cardia. we will report the tips, techniques, and clinical result of our peigs. methods: from september to august , seven patients with smt at cardia underwent peigs in our hospital. we insert the mm port at umbilicus and investigate the abdominal cavity at first. then the incision is extended to . cm and lap-protector tm is equipped with the site to perform mini-laparotomy. using peroralendoscopy, a stomach was insufflatedand incision is made at an anterior wall of gastric body under direct vision. additional lap-protector tm is placed into the stomach so that the stomach is fixed on the abdominal wall. it enables us to keep direct access to gastric lumen and stable operative field. ez access tm is attatched on lap-protector tm , and intragastric operation is started. subsequently, two trocars are inserted using funada's gastropexy instrument. tumor is dissected by using energy devices, avoiding injury of capsule of the tumor. the defect of the gastric wall is closed by intragastric suturing. we should take particular care not to damage egj during the suturing, inserting peroral endoscopy in and out. results: the mean operation time was ( - ) min and the amount of intraoperative bleeding was . ( . - . ) ml. the maximum diameter of tumors was ( - ) mm. one case was low risk gist and otherwise were leiomyoma. the postoperative course was uneventful in all cases, without leakage or stenosis. total hospital stay was ( . - ) days. no patient had symptoms of esophagitis. conclusions: peigs for smt at gastric cardia is a feasible and safe approach, preserving function of cardia. our procedure achieves great stability and excellent visualization during the operation, which may have led to the fine surgical results. laparoscopic lymphadenectomyin gastric cancer is considered a feasible and safe procedure. data on the compliance of lymphadenectomy in the various lymph node stations is not yet well understood; moreover it is not clear if there are particular conditions relate to the patients impairing the oncological results. this video reports the use of the icg for the lymph node dissection of station number in a case of obese patient and a case of a cirrhotic patient. fist patient, m.a., was a year old man with distal cancer ct n and a bmi. second patient, t.d., was a year old man with distal cancer ct n and a alcoholic cirrhosis child b . in both cases, intraoperative endoscopy was performed to min before the onset of lymph node dissection. . mg of icg was injected into the submucosal layer in quadrants of the primary tumor. a laparoscopic subtotal gastrectomy was conduced with d lymphadenectomy. lymph note navigation were analyzed by novadaqÒ detector. using navigation system we removed the n basin. in both cases dissection were effective and pancreatic surface were easily detectable. number of lymph nodes retrieved was in the case of obese patient and in the case of cirrhotic patient. pathological tnm were pt n ( / n ?) in the first case and pt n ( / n ?) in the second. no n ? metastases were detected in n station for both cases. no pancreatic fistula was recorded. icg lymph node navigation system should be considered a valid support for the surgeon for completion of a correct lymphadenectomy in surgical challenging cases. aims: morgagni hernia is the rarest of congenital diaphragmatic hernias ( - %). its presentation is rare in adults and its finding is usually incidental. it was first described by giovanni b. morgagni in . it is located in the anterior region of the diaphragm. it is caused by a congenital defect in the fusion of the transverse septum of the diaphragm and the costal arches. the need for surgery depends on the presentation, it is recommended early repair before the development of complications. classically, the surgical approach was thoracotomy or laparotomy. currently, the tendency is to use a minimally invasive approach, which has shown good results, lower morbidity and faster recovery. the need to repair the defect with a mesh is controversial, recommended when it is not possible to close the defect without tension. the objective of this video is to demonstrate the safety and efficacy of the laparoscopic approach for the repair of this type of hernia, as well as the different types of mesh that can be used. aims: the treatment of the non-metastatic gastro intestinal stromal tumour (gist) is the surgical resection [ ] . the duodenal gastro-intestinal stromal tumour (gist) is a relatively rare clinical entity. from all the resected gist, only % are duodenal [ ] . the clinical presentation could vary from the most common acute gastro intestinal (gi) bleeding, chronic anaemia, but also acute abdomen caused by tumour rupture and intestinal obstruction. methods: a years old patient present at the emergency department of the chi poissy-st germain-en-laye (paris, france) with acute gastrointestinal bleeding. at the laboratory exams the haemoglobin was . g/ dl. the patient perform a computer tomography (ct) which shows two hyper vascularised lesion at the th duodenum; this lesions has an intra and extraluminare growing. the ct scan didn't show any other abdominal lesions. the patient were submitted to a minimally invasive surgical operation with the multiport laparoscopic technique. results: the resection of the th and th duodenum and of the first centimetre of jejunum was performed with a four trocar laparoscopic technique. a latero-lateral duodeno-jejunal mechanical anastomosis was performed. the operative time was min. the patient start with oral alimentation on the third post-operative day after a ct scan with oral contrast that was negative for anastomotic dehiscence and collections. the post-operative course was globally uneventful and the patient was discharged at fifth post-operative day without complications. the histological examination shows a low risk gist, cd positive and with a ki- inferior of % (classification tnm th edition pt aims: gastric volvulus are a rare condition that sometimes represent a diagnostic challenge for surgeons. here we present the video of a recent case of a gastric volvulus in our area that was treated with a minimal invasive approach. methods: we report the case of a -year-old woman who was admitted in the emergency room (er) with epigastric transfixing pain and impossibility to vomit that had started h prior to the admission. the physical exam showed good vital signs, and her abdomen was soft, with a tendency to tenderness with palpation in the epigastrium without guarding or rigidity. her lab tests were normal and the conventional radiology showed a double gastric bubble. we run an urgent computed tomography scan (ct scan) and a upper gastrointestinal (gi) endoscopy that showed a big type ii hiatal hernia that was complicated with a gastric volvulus. results: first, a nasogastric (ng) tube was placed for decompression of the stomach at the time when the endoscopy was made. the patient experienced a great improvement of the pain with that initial measure and remained stable. after almost a day of appropriate resuscitation, she underwent urgent surgery: we performed a hernia reduction, resection of the hernia sac, hiatal closure and a gastropexy and nissen fundoplication. the patient suffered no complications in the immediate postoperative time and was discharged after six days. conclusion: gastric volvulus are an uncommon emergency that we need to keep in mind. a simple abdomen x-ray can be very helpful, given that the double gastric bubble sign is a typical sign of this condition. it's always mandatory to perform an upper gi endoscopy in order to get to the diagnose and place a ng tube promptly. the surgery can be safely delayed in stable patients with no signs of ischemia, and a laparoscopic approach is associated with a shorter hospital stay and good long-term outcomes in this kind of patients. aims: during laparoscopic treatment of hiatal hernias the dissection can be complicated, but even more so the closure of the pillars, especially in giant hiatus hernias with a large defect. the use of prosthesis is controversial due to the lack of long-term studies and the possibility of secondary complications. the aim of this video is to demonstrate the safety of mesh hiatoplasty in hiatus hernia surgery. methods: we present the case of a -year-old woman with hypertension, hypothyroidism and right hemicolectomy for neoplasia years ago. the patient presented with malnutrition, with a weight loss of kg in the last months. a gastroscopy was performed in which a large hiatus hernia, that caused gastric volvulation, was shown. the upper gastrointestinal oral contrast study revealed esophageal tertiary waves and good passage to the gastric chamber, with an organoaxial volvulation of the stomach that was completely included in the thoracic cavity. results: through a five trocar laparoscopic approach, a large paraesophageal type iv hiatal hernia ( cm hiatal orifice) with complete herniation of the stomach and greater omentum to the mediastinum was observed. after reduction of the hernia content, complete dissection with partial resection of the sac was performed. an extended mediastinal dissection of the esophagus, with descent of the esophagogastric junction until achieving an abdominal esophagus of - cm, was carried out. after posterior and anterior phrenorrhaphy with nonabsorbable sutures, dislacement of the right pillar without diagragmatic opening was evidenced. it was decided to reinforce the hiatorraphy using a c shape composite mesh fixed with nonabsorbable sutures. the procedure was completed with a nissen-type fundoplication. postoperative course was uneventful and the patient remains without hernia recurrence months after the intervention. conclusion: prosthetic reinforcement in hiatal hernia repair can be a very useful resource in large hiatal defects in which a stress-free hiatus closure cannot be achieved. however, its use must be individualized according to the characteristics of the patient, the quality of the tissues involved and the result of simple hiatorraphy. aims: heller myotomy is an advanced laparoscopic surgical technique for the treatment of achalasia, a rare disease in which long time is needed to achieve the learning curve. surgical simulation, using animal models with an anatomy similar to humans, could help improving surgeon performance in a shorter time. the aim of our video is to show an ex-vivo and in-vivo animal model for heller myotomy training. methods: a cadaveric porcine model was developed using a tissue block including the esophagus and the stomach, without the diaphragm, mounted in a physical laparoscopic simulator. training procedures were also performed in an in vivo porcine model. experiments were carried out in the ' jesús usón' minimally invasive surgery centre in cáceres. results: the surgical technique is described step by step, first in the esophagus-stomach ex-vivo model and later in the live animal model. conclusion: surgical simulation using cadaveric an live animals offers a realistic representation, allows training in a safe environment, and can be very useful for advanced laparoscopic training in low incidence pathologies. introduction: esophagic perforation is one of the least frequent complications after laparoscopic nissen fundoplication, but it remains one of the most dreaded because of its morbidity and mortality rates and its technically difficult reparation. aims: to present how the authors dealt with an iatrogenic esophagus perforation post laparoscopic nissen fundoplication. methods: the authors report the clinical case of a -year-old woman who underwent a laparoscopic nissen fundoplication because of a symptomatic large hiatus hernia in a different hospital. the second postoperative day and after resuming oral intake, she presented respiratory and hemodynamic instability. she needed a chest tube that drained purulent content. the patient was referred to our hospital for clinical management. an urgent ct scan with oral contrast was performed without showing any leakage. results: in spite of the results, as the patient was unstable, she underwent an emergent diagnostic laparoscopy. it was found a small anterior esophagus perforation with right mediastinic collection. a running suture of the perforation was carried out and the nissen fundoplication was converted to a dör fundoplication. the operative time was min. she went to the intensive care unit for ten days. five days after the surgery, she was given methylene blue with no exteriorization through the drainages. as postoperative morbidity, she suffered from a right pneumonia and a thoracic collection that was treated by thoracic surgeons. the patient was finally discharged on the th postoperative day. she did well at home. she attended follow-ups without clinical reflux or any other particular condition. conclusions: esophagic perforation is a life-threatening complication that can be managed laparoscopically if it is detected soon after surgery and an expertise is available. surgical treatment of achalasia fails in - % of patients. the most frequent responsible cause is a previous incomplete myotomy, followed by fibrosis and aspects related with antireflux procedure. revisional surgery can represent a greater difficulty and a challenge. in this video we show revisional surgeries after heller's myotomy with dor fundoplication for achalasia. all cases presented a recurrence of the symptomatology and a revisional surgery was proposed. surgeries were characterized by the presence of a herniation of the previous fundoplication, fibrosis around the prior myotomy and/or the formation of a pseudodiverticulum. we show the steps followed and the aspects to consider during the dissection. these cases demonstrate that laparoscopic reoperation for achalasia is feasible, even after open surgery. aims: upside-down stomach (uds) is a rare type of large paraoesophageal hernia, characterized by migration of the entire or large parts of the stomach into the posterior mediastinum. uds is associated with severe complications like strangulation or volvulus development, possibly leading to acute gastric outlet obstruction and incarceration. surgical repair is the only curative treatment option and therefore recommended for these patients. standard procedure includes a hiatoplasty followed by an anti-reflux procedure. in a variety of studies, the use of mesh proved to be superior with respect to reduction of anatomical recurrences. methods: a -year old woman presented to us with reflux symptoms, dysphagia, dyspnea and tachyarrhythmias. she reported a weight loss of kg in the last months. ct scan and esophagogastroscopy showed a large paraoesophageal hernia, measuring approximately cm, with intrathoracic uds. results: we performed a laparoscopic hernia repair with dissection of the hernia sac from the posterior mediastinum, tension-free intrabdominal reposition of stomach and distal esophagus and hiatoplasty with biologic mesh (tutomesh tm ) augmentation. finally, a toupet fundoplication was performed to recreate the antireflux valve. in consequence of pronounced adhesions, a lesion of the muscularis of the distal esophagus occurred during surgery. the esophageal mucosa was unaffected. we treated the lesion laparoscopically with a simple interrupted suture (vicryl tm - ). an intraoperative patent blue v leak test did not identify any leaks. the recovery was uneventful. the patient was discharged on day after surgery and was free of symptoms in her follow-ups. conclusions: patients with large hiatal hernias and uds can be treated successfully and effectively with laparoscopic mesh repair. intraoperative complications can be handled laparoscopically in a safe manner. iatoplasty followed by nissen fundoplication represent the gold standard treatment of hiatal hernia; however it has been reported high hernia recurrence rate, especially in case of giant hiatal defects. in order to reduce recurrence rate, various techniques of hiatoplasty reinforcement have been implemented, such as prosthetic materials apposition. however, in literature have been reported various mesh complications such as esophageal and proximal stomach erosion and late esophageal perforation after ischemia, especially in case of synthetic non absorbable materials.in this video we are going to show the repair of a huge hiatal hernia by hiatoplasty and positioning of an absorbable biosynthetic 'bio a' mesh which is replaced by connective soft tissue over six months, therefore decresing complications and recurrence rate.as usual, we start with the mobilization of gastric fundus and isolation of diaphragmatic pillars by sectioning the aderences between them and herniated viscera. we proceed then with intrabdominal esophagus mobilization and higher mediastinal dissection in order to obtain an adequate esophageal lenght. after the exposition of the hiatus, we approximate the pillars with some non absorbable stitches and we reinforce the hiatus positioning a 'u' shaped bio a mesh over the esophagus, simply fixed to crura with single stitches. then we go on performing nissen fundoplication.the use of this prosthetic material appears to be cost-effective; first series in literature show very low complication rate and less recurrences of hiatal hernia than hiatoplasty without reinforcement. this video demonstrates the technique of laparoscopic identification and complete dissection of the sac of a totally intrathoracic stomach. identification of the sac is performed centrally by scoring the peritoneum overlying the arch of the diaphragmatic hiatus. the inferior edge is retracted and series of blunt dissection is undertaken. once the areolar tissue of the avascular plan is visualized; a raytec sponge is placed and advanced cephalad to expose the extra-saccular space. this raytec is kept in place to allow carbon dioxide to infiltrate and further delineate the anatomy. we then proceed with dissection at the left crus and right crus. complete reduction of the stomach can be achieved without grasping it. this can be performed by applying caudal retraction on the sac. this maneuver exposes the plane outside the sac. this space is divided into two compartments (right and left) separated by a septum which indicates the proximal extent of the sac. this plane is avascular and blunt dissection can easily free the hernia sac from the mediastinal structures and the pleura. this was followed by excision of the sac and hiatal repair which is reinforced with bioabsorbable mesh. the proximal short gastric vessels were then divided and a standard toupet fundoplication was performed. v -upper gi-reflux-achalasia introduction-objectives: the mixed giant hiatus hernias with paraesophageal component are hernias of difficult surgical correction, the laparoscopic approach of these implies a greater experience of the surgical team, given the complexity involved in its management, being the recurrence a complication that neither the use of meshes in this surgery has been able to avoid. in very high-risk patients, the gastric anterior pexy (boerema) may be an alternative to treat or alleviate the symptoms of these large hiatal hernias, although the frequency of recurrence with this technique is very high. material and methods. objective: the objective of the following case is to present a patient with type- hiatal hernia, barrett's esophagus without dysplasia and situs inversus. method: a laparoscopic partial fundoplication was performed on a -year-old male patient with a history of situs inversus totalis, who was seen in our general surgery service presenting a clinical history of retrosternal pain, heartburn and regurgitation. an endoscopy was performed, which reported hiatal hernia type and incompetence of lower esophageal sphincter, with squamocolumnar junction biopsies with report of barrett's esophagus without dysplasia. results: surgical time was programmed, for toupet type fundoplication; there were lax adhesions from omentum to wall, the lax hiatus and already known situs inversus. partial funcuplication was performed, with the usual technique adapted for structural anatomical abnormality, postoperative course without complications, oral initiation at the next day, drainage penrose type was set draining just a little serohematic liquid, diet was progressed and patient was discharged on the third postoperative day without complications. conclusion: situs inversus is the mirror image of situs solitus, which presents subdivision in situs inversus totalis, which is the most usual form, characterized by the mirror location of the intraabdominal and thoracic organs including the heart; in the case presented, the patient was referred with situs inversus and barrett's esophagus, performing laparoscopic fundoplication. the gold standard of surgical management is laparoscopic in benign esophageal pathology. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. in rare instances gerd is associatedwith type iv hiatal hernias in which the part of stomach and other organs migrate into mediastinum.nowadays this condition can cause problems for some surgeons.patient was a -year-old man. he was diagnosed with hiatal hernia in . the condition had beenhaving asymptomatic course until . patient takes omeprazole mg for years. he startedexperiencing chest pains when inhaled and dyspnoea in june . co-morbidities were: arterialhypertension, chronic obstructive pulmonary disease (copd) and obesity (body mass index was . kg/m ). the posterior mediastinum contained the part of stomach, large bowel and small bowelaccording to chest roentgenography. sizes of esophageal hiatus were mm. in our clinical centerwas performed laparoscopic removal of hernia, cruroraphy, mesh repair of the esophageal hiatus andnissen fundoplication in july. during the surgery stomach, the part of small intestine, greatomentum and transverse colon were relocated into abdominal cavity. after cruroraphy, repair of theesophageal hiatus with prolene mesh was performed. the patient was in intensive care during h.total enteral feeding was initiated on second day. patients had been discharged within days aftersurgery. patient was re-examined by a surgeon in september , there were no signs of a reccurence.this case report shows an efficiency and feasibility of the laparoscopic approach to the treatment gerdassotiated with a large defect in the phrenoesophageal membrane, allowing other organs, such as colon,great omentum and small intestine to enter the hernia sac. aims: the authors present a video with their standardized laparoscopic hiatal hernia repair and anti-reflux nissen procedure but using mm instruments and mm camera approach. methods: a years old female patient with a bmi presents a symptomatic gastro esophageal reflux disease (gerd) for years. manometry and ph-metry showed a lack of esophageal motor disorders and a severe acid pattern with a . demeester score. panendoscopy study showed a cm hiatal hernia and a los angeles-grade esophagitis. she decided not to go with ppi treatment anymore. a laparoscopic hiatal hernia repair and standardized nissen procedure is performed using mm instruments and a mm camera. case and technical details are shown in the video. results: the patient was discharged from hospital within a period of h with a rate in a eva acute pain visual scale. in a year follow-up, there has no been an anatomical or clinical recurrence. no chronic dysphagia, anatomical recurrence or abdominal wall complications have been reported with in this period of time. conclusions: depending on the patient characteristics, anatomical factors and surgeon mini invasive experience, hiatal hernia and anti-reflux mini invasive standardized repair using mm instruments, could be a safe and feasible option. more studies are needed in order to standardized this approach. background and aims: parastomal herniation a very frequent complication in stoma patients. in isolated parastomal hernias (type i or iii)* a laparoscopic sugarbaker repair with intraperitoneal mesh is our preferred technique. if a concomitant incisional hernia is present (type ii or iii)* we currently opt for a retromuscular mesh repair as described by pauli. we adopted a minimal invasive approach using the robotic platform. methods: we performed a robot-assisted laparoscopic pauli repair for a wide incisional henria needing component separation and a small parastomal hernia (type ii)*. a non-slit retromuscular mesh was placed after a bilateral tar (transversus abdominus release) and lateralization of the colon. results: the operation was performed robot-assisted laparoscopically with x trocars without the need to convert to an open procedure. the procedure lasted min. the patient was discharged from the hospital on post-operative day three. one month after the repair our patient presented with a parastomal skin infection for which she received surgical cleaning and wound dressing. ct scan three months postoperative shows good positioning of the mesh with a reinforced abdominal wall. conclusions: the modified sugarbaker repair in parastomal herniation is feasible following a pauli approach (retromuscular mesh positioning) completely laparoscopic, albeit robotically assisted. short-term follow up is promising. long-term postoperative follow-up in these patients is needed. methods: case presentation results: a -year-old caucasian female patient was admitted on emergency due to a progressive alteration of her physical condition and weigh loss, caused by intermitent and intense epigastric pain and vomisments. symptomes occurred several years prior to admittance, but used to be mild and rare. during the last months, the described episodes became more intense, lasted longer and occurred more frequently. percutaneous ultrasound raised the suspicion, while thoraco-abdominal ct revealed an enormous intrathoracic morgagni hernia and gastric volvulus inside the hernial sack. after a careful preoperative preparation (restoring of the nutritional and hydric inballances, amelioration of respiratory parameters), laproscopy confirmed the diagnosis; we gently reintegrated the herniated organs from the thoracic hernia into the abdominal cavity (small bowel, large omentum, transverse-doloco-colon, and thisted stomach); a laparoscopic exploration of the hernial cavity was followed by a thorough hemostasis. do to patient's frailty, we decided to leave the hernial sack in situ. surgical direct repair of the defect technique was done by using extracorporeally tied separate sutures through separate skin incisions. the postoperative outcome was completelly uneventful; patient was discharged on postoperative day . barium contrast at mounths followup showed a slight esophageal diskynesia, but normal gastroduodenal passage; due to aerocolia, the normal position of the transverse colon could be confirmed aswell; no signs of reccurrence where detected. conclusions: although very rare, a morgagni hernia should be suspected and included in the differential diagnosis of patinets with dispeptic syndrome and epigastric/thoracic symptomes. thoraco-abdominal ctscanning is the imagistic technique of choice. laparoscopic approach became the gold standard, being far mora addvantageous as compared to laparotomy or thoracoscopy. direct suture with extracorporeally separate sutures through separate skin incisions was chosen for being less time consuming; for the same resons, the hernial sack was left in situ, with no consequences so far. aims: the giant fibrovascular polyp is one of the rarest benign lesions of the oesophagus. the most common locations of origin are the upper oesophagus or crico-pharyngeal region. the lesion is more common in elderly population, particularly men. symptoms include dysphagia, odynophagia. management options include surgical resection or endoscopic removal with endoloop. we aim to demonstrate the optimal management of these rare lesions using an endoscopic approach. method: we demonstrated the management of -year old patient with a giant oesophageal polyp, excised by minimally invasive endoscopic resection. the patient was placed in supine position and tracheal intubation was performed under general anaesthetic before an endoscopic approach was taken. the oesophago-duodenoscopy visualised a cresenteric shaped lumen due to an intraluminal mass occupying two thirds of the oesophageal diameter. the procedure was a multidisciplinary approach with the upper gi surgical and gastroenterology consultants. the polyp stalk was located in the oesophagus at the level of t and an endoloop was manipulated to surround the polyp. the polyp was then separated from the stalk by cauterisation and resected from the patient. the stalk was then injected with adrenaline to prevent haemorrhage. results: the excised specimen was a cm polyp with stalk originating from the t -t level. histology confirmed diagnostic suspicions of a benign pedunculated fibrovascular polyp. the polyp was covered by non-keratinising squamous epithelium with a discoloured tip demonstrating ulceration. there was no evidence of dysplasia or neoplastic process. the video shows a case of laterally spreading tumour of the rectum with preoperative benign histology, paris classification -is g (granular type), ut n eus stage, kudo type iv, nice type . the neoplasm measured x cm, and extended from to cm from the anal verge, mainly located on the posterior wall. according to our local policy the indication was a transanal full-thickness excision. this was performed with the medrobotics flexÒ robotic system, used here for the first time outside the united states. the system technology utilizes an articulated multi-linked scope that can be steered along nonlinear, circuitous paths in a way that is not possible with traditional, straight scopes. the maneuverability of the scope is derived from its numerous mechanical linkages with concentric mechanisms. this enables surgeons to perform minimally-invasive procedures in places that were previously difficult, or impossible, to reach. with the flexÒ robotic system, surgeons can operate through a single access site and direct the scope to the surgical target. once positioned, the scope can become rigid, forming a stable surgical platform from which the surgeon can pass flexible surgical instruments. the system includes on-board d hd visualization. the flexÒ robotic system contains two working channels to accept a number of different surgical and interventional instruments including monopolar and bipolar electrodes, scissors and graspers for tissue manipulation. the video shows the introduction of the dedicated rectoscope, the connection of the flexible robot, and the way to operate the device performing a full-thickness excision, including suturing of the rectal defect by means of two running sutures by a v-lock / thread. while illustrating the technique the authors will comment pros and cons of the use of the device. surg endosc ( ) the video shows the use of a barbed suture of novel concept. this first prototype thread developed together with assut europe (roma, italy) is characterised by a bidirectional / barbed suture, of cm length overall, with two needles mm in diameter, and circumference. in order to fix the thread to the tissue, on the exact midline of the thread a small ( mm) ball of the same material of the thread is fused with heat. this button as the entire thread is made of polydioxanone, a monofilament material for long-term absorption, self-retaining. this small advancement offera a consistent advancement in endorectal surgery helping in making transverse wound closures as easy as never before. the first stitch is placed on the transverse midline of the rectal wall defect, this way approximating proximal and distal edges. the button keeps the thread under tension allowing the completion of a running suture towards one of the two lateral ends of the would. when the first end is reached the needle is cut and the other needle is grabbed in order to perform the other half of the running suture keeping the right tension on the thread, with no risk of lumen stenosis. the two lateral ends of the suture are self-blocked passing them through the last stitch. no need for clips, knotting or buttonholes to pass through. while illustrating the technique the authors will comment pros and cons of the use of the device. background: the usefulness of robotic surgery has been largely reported; however, there are not enough reports on gist's treatment. the aim of this study is to report a single center experience on gastric gist's robotic resection. gastrointestinal stromal tumor (gists) are the most common mesenchymal tumors (overall incidence - % of all gastrointestinal malignant tumor). they are most frequently located into the stomach. complete surgical resection still remains crucial for patients with gists. in cases of difficult localization of tumor (e.g. posterior wall, his angle) and bigger tumor (more than cm in diameter), there still exist apparent difficulty and limitation to conduct laparoscopic resection. robotic assistance provides wide movements of its arms and was recognized particularly useful in case of difficult tumor localization, especially for those positioned at the posterior side of the stomach wall or around the lesser curvature. methods: six consecutive patients were analyzed focusing on safety (conversion/complications rate; hospital stay), oncological (margin resection, recurrence rate) and feasibility (operative time, technical tip and tricks) profile of robotic approach. results: the mean operative time was ± min, the mean hospital stay ± days. conversion rate was nihil. no intra and post-operative (mean follow-up months) complications were registered. in all cases, the resections were classified as r . conclusions: our experience supports the usefulness of robotic system in case of gist located at anatomically difficult gastric portion, such as lesser curvature or fundus close to gej, confirming the excellent oncological outcomes ( % r ) and the encouraging safety profile ( %). regarding the operative time our data are similar or better as compared to those reported by the previous literatures and didn't differ by the most recent published data for laparoscopic gastric resection. the anatomical hand-sewn reconstruction performed by precise hand-sewn suture instead of the usage of mechanical staplers represents a real great advantages of robotic resection. in our series, no patients suffered from stenosis or leakage after operation. background: we describe the use of a different suture from those historically used in the elaboration of a widely spread surgical technique such as the nissen fundoplication, for the treatment of pathological gastroesophageal reflux or symptomatic hiatal hernias. in our unit we have implemented the use of / irreabsorbable barbed suture to close the diaphragmatic pillars and the °fundoplication with the same continues suture. aim: the objetive of the use of the irreabsorbable barbed suture in the nissen fundoplication is to shorten the surgical times, which would achieve benefits for the patient and the institution, increasing the number of ambulant patients and the number of patients to be operated the same surgical session. objective: the goal of the present study was to demonstrate that intraoperative icg fluorescent imaging is a safe technique that can be used in laparoscopy establishing the exact location of the lymphocele and reducing intraoperative risks. method: fifty milligrams of icg dissolved in ml of saline solution was injected via percutaneous drainage placed into the lymphocele to decompress transplanted kidneys weeks before a laparoscopic lymphocele marsupialization procedure. results: during the first exploratory laparoscopy, in the flank and right iliac fossa, near the renal grafts, fluorescence was identified in raised areas that were the internal side of the lymphocele lobes. the lymphocele wall was dissected and ml of serous fluid was aspirated after puncturing. a cm breach was then made in the cyst wall using the ultracision harmonic scalpel (ethicon us). afterwards, a pedicle of the omentum in the lymphocele core was interfered with and fixed by stitches. conclusions: laparoscopic surgery seems to be the preferred surgical option for the treatment of primary symptomatic lymphocele after kidney transplantation. intraoperative icg fluorescent imaging is a safe technique to establish the exact location of the lymphocele and reduces the risk of damaging urinary structures during surgery. conclusions: this method proved safe and risk-free, easily reproducible and without the need for a different toolkit than the one of a modern operating theatre. the preliminary analysis shows a strong correlation between the perfusional data so far obtained and the early outcome of the graft. thus opening the way to further analysis aimed to a future better management of post-operative immunosuppressant and support therapy. these results are quite encouraging, even if our study is at an initial stage. conclusions: results show a persistent dread across specialities regarding ai. rather than be seen as threat, ai should be embraced by clinicians as it will ease the ever-increasing daily workload faced. this will enable clinicians to focus their skills on patient centred activities, interventional procedures and development. despite current regulatory hurdles, ai implementation in medicine is unavoidable. this coming revolution presents a unique opportunity for endoscopist and radiologist to refocus their expertise in novel areas. project description: from february to july a three-armed proof of concept study was conducted at three hospitals, in three groups of patients; recurrent ventral hernia, aortic aneurysm, and healthy controls. patients were measured once at the outpatient clinic using an electronic nose based on three metal oxide sensors. measurement data were compressed to low-dimensional vectors using a tucker like algorithm, and used to train an artificial neural network (ann) to provide a classification between patients (? ) and healthy controls (- ). preliminary resultsa total of patients (hernia n = , aneurysm n = ) and controls were included in the study. based on receiver operating curve (roc) analysis, the ann could differentiate between recurrent hernia patients and controls with the following details: area under the curve (auc) . , sensitivity . , specificity . . aortic aneurysm patients and healthy controls could be differentiated with an auc of . , sensitivity of . , and specificity of . . the aeonoseÒ enose can reliably distinguish patients with weak collagen (recurrent hernia and aortic aneurysm patients) from healthy controls. validation of these results in a prospective cohort study is required before clinical application of the device. background: laparoscopic and endoscopic cooperative surgery (lecs) has been performed gastric submucosal tumor (smt) or duodenal tumor. although minimally invasive surgery using thoracoscopy has been the usual approach for esophageal smt, the treatment method combined thoracoscopy and endoscopy has not been established. in addition, submucosal endoscopic tumor resection (set) for esophageal smt was reported using the technique of submucosal tunnel. aim and project description: we planned to resect large esophageal smt located in the upper or middle thoracic esophagus by a combined endoscopic and thoracoscopic approach. because set is only recommended for tumors up to mm in size owing to the limited submucosal space available and the left thoracic approach is restricted by the aortic arch and the trachea. preliminary results (case presentation): a -year-old woman was diagnosed with a benign schwannoma of length mm originated from either the submucosal or the muscular layer of the middle thoracic esophagus by endoscopic ultrasonography, enhanced computed tomography, and ultrasound-guided fine-needle aspiration biopsy. since the tumor had increased in the years and she had a symptom of dysphagia, we planned to resect it by a combined endoscopic and thoracoscopic approach. on endoscopy in the supine position, a submucosal tunnel was created mm proximal to the cranial edge of the tumor, and its only oral end was dissected from the mucosal and muscular layers. this was followed by the resection of the entire tumor by left-sided thoracoscopic procedure in the prone position. endoscopic mucosal closure was achieved by using clips. no postoperative complications were observed. large benign esophageal tumors can be safely excised with minimally invasive surgery by using a combination of endoscopy and thoracoscopy. background: haemorrhage remains a major cause of morbidity and death in all surgical specialties. in laparoscopic surgery. relatively small amounts of blood can obscure the view of the operative field and increase the risk of injury to surrounding structures. excessive bleeding often leads to longer hospital stays, increased healthcare service utilisation, and higher healthcare costs, among other negative consequences. aim: the aim of this study was to analyse the feasibility of purastatÒ, a new synthetic haemostatic device, made of self-assembling peptides in laparoscopic colorectal surgery. project description: this was a prospective observational non-randomised study. consecutive patients undergoing laparoscopic colorectal surgery were enrolled. informed consent was obtained from all patients inclusion criteria was the need employ a secondary method of haemostasis when traditional methods such as conventional pressure or utilization of energy devices to control the bleeding were either insufficient or not recommended/ appropriate due to proximity to ureter, pelvic/sacral veins and other important structure. preliminary results: twenty patients were enrolled ( males ( %) and ( %) females). mean age was years (± , years). all patients were undergoing elective laparoscopic colorectal cancer surgery ( right hemicolectomy, sigmoid colectomy, anterior resection). we utilised mls of purastatÒ in all patients. the mean area of application was , cm (± . cm ) with the amount of purastatÒ needed per centimetre being . mls. the mean time to apply the product was secs (± s), whereas the mean time to achieve haemostasis was , secs (± . s). there were no post operative complications in this cohort of patients. mean operative time overall was min (± . min). none of the patients experienced delayed post-operative bleeding and the mean hospital stay was days (± . ). in conclusion, according to the purpose of this preliminary study, we have demonstrated that purastatÒ can be easily used in laparoscopic surgery and it is a safe, effective haemostatic agent. this is a feasibility study and additional controlled studies would be useful in the future. during last three years the laparoscopic method of surgery in the presence of common bile duct stones was carried out. after performance of intraoperative cholangiography and visualization of stones in the common bile duct laparoscopic, choledochotomy and bile duct stones extraction was undertaken in patients, using flexible choledochoscopy control. in all patients with gallbladder stones was then performed laparoscopic cholecystectomy. results: laparoendoscopic intervention on common bile duct was successfully performed in patients ( . %) and the operation was completed by common bile duct drainage by kehr. in patients due to technical difficulties conversion to open surgery was carried out. postoperative morbidity in the form of bile leakage were diagnosed in patients ( . %). in three cases they stopped spontaneously in - days after the operation. patients were operated on repeatedly and additional suturing on choledocholithiasis was carried out. postoperative mortality was . %. the death of the patient of years was caused by acute cardiovascular failure. institute for image guided surgery, ihu-strasbourg, strasbourg, france; hepato-digestif, nouvel hôpital civil, strasbourg, france; image guided surgery, nouvel hôpital civil, strasbourg, france; gastroenterology, chu-besancon, besancon, france; gastroenterology, chu-lyon, lyon, france; gastroenterology, clinic de trocadero, paris, france background: eus is difficult to learn and has a steep learning curve. therapeutic eus (teus) is even more so. simulators, ex-vivo models and phantoms are the most common current teaching modalities but are felt by many to be unsatisfactory for high-level training. aim: we designed a training curriculum for teus that uses high-fidelity animal models and present a validation study performed by teus experts. project description: different simulated pathologies were created in each of acute pigs. teus experts performed therapeutic procedures in two or more animals over two days. each intervention was evaluated simultaneously using a structured survey by an non-expert observer. data included demographics and procedure details as well as likert-scale evaluation of the quality, realism and education utility of the simulations. global evaluation of the experience was captured from the experts as written comments. all data was consurrently registered and subsequently analysed by two blinded surgical educators. methodology: three types of models were created using surgical access: -tumors (injection of types of hydrogel), -retro-gastric collections ( - cm long intestinal loops filled with oatmeal, oil-water and gel), -obstructions (bile duct and ureteral ligations days prior to experience). gastric, pancreatic and liver tumor models were used for fna and fnb practice. retrogastric fluid collections and choledochal/ureteral obstructions were used for cyst gastrostomy, hepaticogastrostomy, gallbladder drainage and kidney drainage. results: experts age: - , median intervention time min , total of interventions evaluated, overall quality of experience: ( %) ranked - (excellent), ( %) from - (good), ( %) from - (poor), / procedures were successfully completed. models were rated good to excellent quality ( - ) in ( %), poor quality in ( %). for % ( ) of the interventions the model was considered not good enough to be repeated (solid retrogastric tumor and peripheral hepatic lesion). conclusion: high-fidelity live animal models with simulated pathologies are considered to be excellent training tools by experts and may provide a better learning experience for teus. surg endosc ( ) in our short videos, we present scenarios in which this technology helped: to distinguish a significantly dilated cystic duct from the cbd, to identify an anterior cystic artery in the contest of acute inflammation, to identify a luksha duct, to exclude a leak after endoloops positioning on cystic duct. intra-operative augmented visualisation of biliary anatomy with indocyanine green cholangiography is an essential technology tool with the potential to extend the hour window of safety for emergency cholecystectomies, with significant logistics benefits. introduction: endoscopic resection of subcardial polyps has its limitations; especially when it is necessary to dry out the entire gastric wall. uniportal intragastric surgery is a good alternative for the exeresis of subcardial premalignant lesions with gastric preservation. patient and method: we present a video with two cases. technique: we perform a laparoscopy to explore the entire abdominal cavity, then we open a . cm hole in the great curvature; a cm incision is made in left hypochondrium and the uniportal device is placed inside de stomach. we inject serum in the submucosa with a endoscopic needle. when submucosa is completely separated from muscular lay; a submucosal exeresis can be made; but when there is not a complete separation after injection, then we perform a entire wall resection with a cm circular margin. if a complete wall resection is made, then we close the defect with a barbed suture. results: case : years old male with a , cm subcardial polyp, preoperative biopsy was informed as severe dysplasia. in the laparoscopy we saw an unknown lesion in the great curvature that looked like a gist. we placed the uniportal device intragastric and proceed to the submucosal serum injection. as the submucosal lay was completely separated from muscular, then we performed a submucosal exeresis. we close the gastrotomy and made the resection of the great curvature lesion with endostapler. the pathological analysis confirmed the severe dysplasia in subcardial lesion and gist in the great curvature lesion. patient was discharged with no complications after days. case : years old male with a cm subcardial polyp. preoperative ecoendoscopy suggested a muscular layer infiltration but only severe dysplasia was found in the previous biospsy. laparoscopy did not found more lesions, and uniportal intragastric device was placed. a complete wall resection was made, and the defect was closed with manual barbed suture. pathologyst confirmed severe dysplasia and unaffected margin. patient was discharged with no complications after days. conclusion: uniportal intragastric surgery is feasible and safe and may be useful for subcardial premalignant lesions when endoscopic resection is very difficult or not feasible. introduction: the role of icg in bariatric surgery is still unclear. knowing the lack of perfusion in the gastric pouch could be of great interest in revisional surgeries; but the question remains: is the current icg technology reliable enough to make intraoperative decisions in bariatric surgery? methods: we have carried out a check of tissue perfusion with icg fluorescence in several cases of primary and revisional bariatric surgery. a solution of , mg/kg was injected intravenously and icg fluorescence was performed. we looked for the correct staining of the entire gastric pouch and the intestinal loop trying to rule out areas of tissue ischemia. results: the cases in which the test was performed showed a minimal delay of - s between the intestinal loop stain and the pouch. a correct staining was observed in all but one case shown in the video. is the case of a years-old male patient who was operated years earlier in another center; at that time he underwent a sleeve gastrectomy. we evaluated the patient for persistent gastroesofhageal reflux of years of evolution with esophagitis. we offered revisional surgery to perform gastric bypass and hiatal closure. fundus was dilated so a funduplasty was performed instead of using endostappler in the vertical side of the pouch. manual anastomosis gastric bypass was performed. when the icg test was performed, a corner of the pouch does not stain green (an area of , cm) . so the decision was to resect that part and redo the anastomosis or wait and see. it was decided not to resect and the patient was discharged two days later with no complications and good outcome with a months follow up. conclusion: icg fluorescence may be useful in bariatric surgery in the future but more evidence is needed of its usefulness in making intraoperative decisions. background: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging of lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results for detection of aberrant lymphatic drainage outside of the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems in order to identify the lymphatic nodes. aim: the objective of the experimental study is to investigate the possibility to detect the sentinel lymph nodes after the injection of different solutions with indocyanine green in the subserosal colonic layer in the pig. project description: twelve female large white pigs were operated with laparosocpic approach and spies optic filter (karl storz, germany). indocyanine green was injected in the subserosa of the colonic wall ( ml at points). lymphatic flow was observed at - - - - and min, searching for the migration of the icg by the lymphatic channels and its introduction in the sentinel nodes. preliminary results. the identification of the sentinel nodes is very difficult with the solution of icg-sterile water. with this technique we can see the lympjatic channels but not the lymphatic nodes. the adition of % human albumin as a transporter of the icg is very helpful for the correct identifiaction of the lymphatic channels at - - min and the correct visualization of the lymphatic nodes at min after the bowel injection. addition of other transporters like dextran solutions may be helpful too but the time to the correct visualization is longer. there was significant difference among the three groups as regards postoperative les and postoperative ph metery.the incidence of persistent dysphagia was significantly higher in the group i. postoperative gerd symptoms were significantly higher in group iii ( . %. p \ . ). recurrent achalasia was significantly higher in group i ( patients . %, patients in group ii ( . %) and nil in group iii (p \ . ). in conclusion: longer myotomy on the gastric side ([ . cm) ensures complete division of the les with better outcomes in term of resolution of dysphagia but may be associated with higher postoperative gerd. therefore, a myotomy length of . to . cm on the gastric side provides a balance between relieve of dysphagia and development of postoperative gerd. c/t scan of his abdomen revealed a large groin hernia with signs of small bowel obstruction and collapsed distal bowel. emergency theatre was organised for this patient with anaesthetic assessment prior to his surgery. initial plan of local exploration with possibility of small resection was changed once he was under full anaesthetic with muscle relaxation. his abdominal girth provided an opportunity to utilise laparoscopic intervention as an initial approach. laparoscopy with degree lens revealed moderately distended loops of small bowel and a large omental mass along with a loop of small bowel incarcerated in right direct inguinal hernia site. background: robotically assisted surgery is a rapidly developing modality of minimally invasive surgery with proven advantages in the management of cancer. despite its increasing prevalence, there is still an ongoing debate regarding its future role in colorectal surgery. while the prospective randomised multi-centre studies provide research evidence for its potential efficacy, an assessment of its effectiveness and realistic outcomes in everyday clinical practice can add an important perspective to this discussion. the international robotic colorectal registry will allow to compile and pool the international robotic colorectal experience. aims: the aim of the international robotic colorectal registry is to monitor the safety and outcomes, as well as the quality of specimen of robotically assisted colorectal surgery for malignant and benign diseases of the colon and rectum. the primary endpoint is a composite oncological failure. the secondary endpoints include anastomotic leak, resection margin involvement, conversion rate, operative time, post-operative -day morbidity and mortality, long term oncological outcomes, quality of life, functional outcomes and cost-effectiveness. project description: the international robotic colorectal registry is a multicentre web-based, online secure database. the registry has been awarded an ethical approval by a relevant national committee. all surgeons performing robotic or robotically-assisted surgery are invited to participate. the data collected includes patient demographics, cancer characteristics, operative details, histology of the specimen, wound healing, post-operative therapy, readmission, quality of life and functional (bowel, urinary and sexual) outcomes. all the sensitive patient information is encrypted before its introduction into the database. preliminary results: so far, twenty robotic colorectal centres have joined the international robotic colorectal registry. the preliminary results will be published once patients have been enrolled. univariate and multivariate analyses will be performed to identify possible risk factors for poor outcome.the possibility to record open, laparoscopic or other minimally invasive colorectal procedures will facilitate comparison of the outcomes of the robotically assisted surgery and other modalities. the registry will also allow each surgeon enrolled to monitor their skill progression and outcomes over the time. results will be published in surgical literature and presented internationally. background: gastric outlet obstruction (goo) due to benign strictures is an uncommon surgical entity today. this paucity relates to the decrease in its aetiological factors in the modern era as well as to advances in both prevention and medical as well as endoscopic treatments of such condition. the most common of causes relating to peptic ulcer disease, has been subdued for decades with quality acid control medications. on the other hand advances in gastroscopic dilatations skimmed even more the frequency of these cases from arriving to surgical intervention. aim: this presentation gives an update on the standing of this pathology and its surgical management today. it will also shed a light on our early experience in this condition at the royal hospital of muscat in oman. project description: a case series of all patients with goo, who were surgically managed between and results: there were a total of patients, males and females. the cause of obstruction was peptic ulcer disease in , corrosive injury in , iatrogenic perforation in and idiopathic hypertrophic stenosis in . emergency presentation was seen in . management included jaboulay pylorpolasty in , resection in (distal gastrectomy in , total gastrectomy in ) or a bypass (gastrojejunostomy) in . in of the above, the procedure was done by laparoscopy. post operatively, temporary gastric paresis delayed recovery in , however all symptoms resolved in . there were no recurrences at minimum of years of follow up. in spite of advances in medications and gastroscopy interventions, we still seem to identify this condition within our population. although infrequent, they demand awareness from surgeons since they could be managed successfully, especially laparoscopically, with minimal morbidities and early recovery. the introduction of advanced laparoscopy to the unit's setup in recent years, made such option feasible with satisfactory and durable outcomes. background: gists of the upper gi are found mainly in the stomach ( - % of cases) and small intestine ( %). duodenal gists however, comprise a smaller subset with a frequency of to %. the optimal surgical procedure for duodenal gist is still evolving. since wide margins and extensive lymphadenectomy are not required, restrain from more radical resections in this area would be a valid option. aim: this is a video case report of a patient with a gist involving the third part of the duodenum treated by laparoscopic lateral duodenectomy and end-side roux-en-y duodenojejunostomy case report: years lady presented with recurrent mid abdominal postprandial pain with anorexia, nausea and occasional vomiting an ultrasound showed well defined hypoechoic mass of . . cm at the right para-aortic region . ct scan defined the mass as retroperitoneal, intimately related to the pancreas uncinate process and the third part of duodenum with no clear cleavage line between them. an mri endorsed the diagnosis of gist of the duodenum. she was operated upon through a laparoscopic lateral duodenectomy including the gist at the third part of the duodenum. a frozen section confirmed the clear margins . reconstruction was done by a roux-en-y duodenojejunostomy with the alimentary limb taken cm from the dj flexure. she had an uneventful post operative recovery and was discharged well. the histology confirmed a low grade gist tumour hence no further treatment was needed. at follow up six months later, she was doing well and gaining weight. conclusion: complex anatomy of the pancreatico-duodenal area makes conserving the duodenum for tumours rather than a major resection a challenging option. in our case however, with the disease in the third part being of a moderate size, a lateral duodenal wall resection including the mass was possible rather than a segmental resection. this procedure could be an ideal choice for benign, moderate sized tumours in the third and fourth part of the duodenum. background: during laparoscopic colectomy, laparoscopic lymph node dissection and extracorporeal intestinal anastomosis is commonly performed. an umbilical incision of - cm and wide-range mobilization of the intestinal tract is required for extracorporeal anastomosis. previously, we introduced intracorporeal overlap anastomosis in june as a minimally invasive treatment. here, we report its short-term outcomes. aim: we retrospectively compared the surgical outcomes of cases of extracorporeal anastomosis and cases of intracorporeal anastomosis, all of which were performed between june to may . procedures: after lymph node dissection and sufficient mobilization of the intestinal tract, the proximal and distal intestines were resected perpendicularly to the intestinal tract with a -mm linear stapler. the anastomosis was performed after the specimen was extracted from an umbilical incision. the opposite sides of the mesenteric margin cm from the staple line of the one intestinal tract, and cm from the staple line of the other intestinal tract, were marked, and then the respective intestinal tract was positioned to join the opposite mesenteric sides together. an insertion hole was made in the intestinal tract at the marked site. side-to-side anastomosis with a linear stapler was performed, and then the insertion hole was closed with a linear stapler after several temporary sutures. preliminary results: in the extracorporeal anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. furthermore, there were three intraoperative cases of bleeding ( . %), and two postoperative cases of lymphorrhea ( . %) that occurred. however, in the intracorporeal overlap anastomosis group, the mean operation time, blood loss, and post-operative days were min, ml, and . days, respectively. additionally, there were no cases of intraoperative complications, and only one postoperative case of lymphorrhea ( . %). conclusion: intracorporeal overlap anastomosis in laparoscopic colectomy is safe and feasible, and can be used as a minimally invasive treatment. nowadays d-printing it's not a new technology any more but with an exponential developing. there are beliefs that in % of everything that will be produced will be d-printed. in medical field this technology knows the same exponential developing. first used in orthopedics and maxilo-facial surgery now d-printind is used in many other fields for different reasons, like preoperative training models, surgical special instruments, in medical education, etc.. liver surgery is in continuous developing and this is the reason why we need experimental liver model for training and testing. a best liver experimental model should heave liver consistency, to be flexible, to heave the same ultrasound feedback, to be cheap and easy to be reproduced. this is why we developed a liver experimental model made of gelatin by a simple recipe, using a dprinted mold, created after a human liver ct-scan. first was made the segmentation of the liver. after segmentation we create the d virtual liver model and the negative image of the liver, which was used for creation of the pieces of the liver mold, with connections between them and a hole on the top to pour the gelatin solution. ( ) ethical concerns on learning and training with real patients and substitutives such as animals, and ( ) reconciling time devoted to learning with clinical practice, considering the european work time directives. simulation in medical education is and has been the preferred route to address both pedagogical needs. virtual simulation has proven to be a valid tool for training; however, current systems restrict usage to tasks and modules offered, without possibility of personalization. we present the minimally invasive surgery simulator scenario editor (mis-sim) an environment where users can create, edit and run virtual reality tasks designed for medical training. the environment features an editor allowing users to develop learning tasks, defining its learning objectives and task goals in an easy way. a first proof of concept has been implemented for surgical training and training activities (demostrators and short courses) have been carried out in three european sites: spain, the netherlands and hungary. during training activities, different exercises have been created and uploaded to the contents' database. trained technical skills include handeye and bimanual coordination, instrument handling and pulling. preliminary results with users have shown mis-sim training potential, although some functionalities should be made easier. personalization has been highlighted as the key added value of mis-sim with respect to the current competitions in the market: the ability for target users to use virtual reality based learning tools while remaining in complete control of the learning process. mis-sim aspires to break the barrier between vr and medical education by empowering users to create their own tasks. with mis-sim teachers/course creators and learners (healthcare professionals & future healthcare professionals) will benefit from an innovative tool to ( ) create personalised medical learning contents tailored to preferred learning styles, allowing the creation of individualize learning paths; ( ) improve the efficiency of training by focusing on the training needs of the learners and ( ) share and sell vr-based didactic contents. c. tiu , p. sánchez-gonzález , m. chmarra , d. gutiérrez , c. guzmán-garcía , l. sánchez-peralta , g. wéber , f. sánchez-margallo , b. pagador , j. dankelman aims: currently surgical training is largely based on the improvement of technology enhanced learning solutions. the progress of engineering and the diversification of training facilities outside the operating theater results in an even greater contribution of technology in the future. the main reasons to encourage these changes are increased efficiency of simulators and directly increased patient's safety. the goal assumed by the easier project is to develop multi-skill, online platforms for minimally invasive surgical (mis) procedures-based on common pedagogical principles with reference value in a multinational space. the platform will allow the connection of external assets (such as simulators) to centralize all training data from residents. this work presents the milestones of the project during its first year of life. methods: the consortium's activity started with a knowledge elicitation process organizing brainstorms and workshops including experts in mis and interventional techniques, from spain, romania and hungary. this experience led to the formulation of a questionnaire that was implemented online and sent via email to surgeons and residents from the participating countries. results: accumulated experience was used to define the pedagogical needs of the platform. the pedagogical needs form the starting point for defining the technical requirements and specifications. based on them, the design of the platform has been achieved, including its architecture and communication protocol between external assets design, facilitated by the use of state of art educational standards. discussions and conclusions: next steps include the implementation of the easier platform, as well as the definition of cases studies selected by the clinical partners in spain, romania and hungary to solve applications of the platform dedicated to cholecystectomy, lumbar puncture and arthroscopy. a pedagogical model, built by the experience of the consortium, is being used to guide instructional design of the course. finally, results will be validated in a multi-center validation study.the easier project will create a training platform with reference value for european surgery. the structure of the consortium, based on the confluence between collectives with clinical, technological and pedagogical experience, will generate a complex learning tool in surgery embodying technology-based training systems with clinical experience. background: the treatment of groin hernia is an important part of our daily surgical activity. aim: we proposed to evaluate outcomes of the laparoscopic trans abdominal pre peritoneal treatment (tapp) of the groin hernia. project description : one hundred and fifty patients who underwent a tapp for a groin hernia were included in a retrospective study between january and november . results: the gender ratio was . the average age was , years. twenty percent of patients had a history of abdominal surgery. the operative indication was a unilateral hernia in % of cases, associated with an umbilical hernia in % of cases, a recurrent groin hernia in % of cases and a bilateral inguinal hernia in % of cases. the conversion rate was . %. the hernias were classified according to the ehs classification in l type in % of cases, l in % of cases, l in % of cases, m %, l r in % of cases and f r in % of cases. a contralateral inguinal hernia was discovered in % of patients. a polypropylene mesh x cm was fixed by a stapling in % of cases and by a suture in % of the cases. the average operation time was min. the hospital stay average was , day. an antalgic treatment was prescribed in % of patients. the average time to return to normal physical activity was days. a postoperative seroma was noted in % of patients. no cases of mesh suppuration were noted. chronic pain was noted in two patients. no recurrence was noted with an average follow-up of months. conclusion: laparoscopic treatment of the groin hernia by tapp had good results concerning the postoperative pain, early recovery of physical activity and aesthetic damage. a larger setback is needed in our study to evaluate the recidivism rate. background: surgeon's training in ultrasound is viewed and understood differently in different parts of the world. if in united states the need for surgeons' training was accepted and taken over by the american college of surgeons, in europe the practice is completely different from one country to another, from one city to another, from one department to another within the same premises hospital. in some european countries, surgeons currently use ultrasound for diagnosis-especially in urgency, for follow-up, intraoperative, or as guidance for many surgical gestures. during this time, access to ultrasound of other surgical specialties-gynecology, urology, ophthalmology-is considered natural. material and method: once the decision to initiate an ultrasound course for surgeons was taken, a team of experts with technical or clinical expertise in ultrasound was organized. at the initiative of the technology commission, the courses were to be organized at the eaes congresses or others communication events endorsed by eaes. starting from the importance of each ultrasound application in surgery, it was decided to develop different modules to solve different training needs. at this time, the course offered at seville covers the capitols like abdominal ultrasound, guided punctures and trauma. a module dedicated to intraoperative ultrasound is under construction and will be available in november . the course has a skill abilities dominant character, two thirds of it being thought of as a hands-on application on stationary, classical ultrasounds with large screens and also on small size wireless actual devices. results: after his debut in frankfurt in , the course was resumed in london and in bucharest, twice. in this process, new modules and better teamwork skills have been developed. the participants' satisfaction quizzes, coming from all continents, were really encouraging. for the intraoperative ultrasound module the team approach is unique. students will have the opportunity to practice on live animals both laparoscopic and open abdominal procedures conclusions: the ultrasound for surgeons course initiated by eaeas was received with interest. the team will seek to inspect the real needs of training surgeons in this field and will complement and diversify the current platform surg endosc ( ) preliminary results: a total of procedures ( - , ± . ) were carried out in patients. the indications included acute ( leaks following esophageal resection, rupture of the strictured anastomosis following pneumatic dilatation) and chronic conditions (esophagopleurobronchial and gastropleurobronchial fistulas following the resection of esophageal diverticulum and sleeve gastrectomy). the initiation of the therapy was in , and day in case of acute conditions, and after years of the duration of the unsuccessful therapy in chronic cases. the successful closure was observed in patients, patient passed from mods and ards. in case, the initiation of evac was provided as a combined surgical and endoscopic intervention (ct proven distant intraabdominal abscesses). in chronic cases, was discontinued due to the haemophagocytic syndrome of unknown etiology, in the second one, success in reduction of the lesion and symptomatology with long term duration was observed following just applications of evac, despite minimal remanent leakage. the success is to our experience linked to early initiation of the therapy and presumes complex intensive care. the future investigation should specify the timing including preemptive use of evac and the combination of evac with other endoscopic, interventional and surgical therapeutic modalities. the aim of this feasible study is to investigate complete robotic esophagectomy with total mediastinal lymph node dissection (retm) only by the robotic arms. methods: the patient is placed hemi-prone position with one lung ventilation by blocking balloon tube under general anesthesia. the robotic trocar for st arm of da vinci xi surgical system is placed in the th intercostal space (ics) on the scapular line, the trocar of nd arm is placed in the th ics on the posterior axillary line, rd arm trocar is placed in the th ics on the middle axillary line, th robotic arm trocar is placed rd ics on the middle axillary line, and assistant trocar only for taking in and out of gauze is placed in the th ics on the middle axillary line. on the upper mediastinal lymph node dissection, robotic camera exchanges from nd to rd robotic arm to close and identify anatomical structures. esophagectomy with lymph node dissection starts from middle and lower mediastinum to upper mediastinum including along bilateral recurrent laryngeal nerves. all procedure perform under close-up view along the robotic enhanced anatomy to preserve organ functions. background: complete stenosis of the duodenal lumen secondary to a surgical suture in the treatment of a duodenal ulcer is a rare complication. the usual surgical resolution corresponds to a gastrojejunostomy associated or not, to an antrectomy with vagotomy, as a treatment for the peptic disease. the endoscopic resolution of this complication requires the use of complex maneuvers and specific therapeutic instruments. aim: to describe the endoscopic resolution of iatrogenic occlusion after raffia of perforated duodenal ulcer. description: a -year-old man was admitted to the emergency service for four days of pain and abdominal distension associated with abundant retention vomiting. performed ten days ago of a perforated duodenal ulcer, in which manual raffia was performed in two planes and drainage. abdomen and pelvis ct showed great distension and diffuse thickening of the gastric wall. the endoscopy showed abundant gastric retention content, pylorus, and bulb edema, and complete closure of the duodenal lumen, secondary to suture material; it was possible to count three suture threads. with a tipped papillotome and electrocautery, all the suture threads were sectioned, identifying a filiform opening through which a hydrophilic guide is inserted under fluoroscopy until it is sure to overcome the stenosis; we dilated the trajectory with a dilator of mm in diameter and cm in length. with a contrast medium, we observed an adequate trajectory and installed a partially covered duodenal metal stent (hanaro stent) of cm in length by mm in diameter in order to sustain the dilation. he was sent to home with inhibitors of the proton pump. after two weeks, the stent was removed, without complications. he was controlled two weeks after withdrawal, and, with pharmacotherapy, the patient was asymptomatic, making a normal life. conclusion: in this case, the result was positive. satisfactory results can prevent major surgery, which reduces the risk and possible complications. a new range of non-invasive medical tools with a remarkable improvement on the existing market. a manual laparoscopy, with the important novelty of having a bending head with a high degree of movement. this head can get multiple spatial positions to work in surgery and is very easy to use, with the same scissors thimble who controls it, so its performance and learning is very simple and intuitive. the tools can be easily reusable and they can be cleaned and sterilized by ordinary methods, very ergonomic and lightweight . the generic type models we initially have developed are focused in general surgery, but gradually we will develop new applications and different heads for specific medical conditions such as arthroscopy, laryngoscopy, otolaryngology, ophthalmology, orthopedics . its operation is simple and functional, simply moving the scissors thimbles, where they have a dual role, combining the head tilt and the action of opening and closing of this is achieved.the design of this tool allows us to work with some degrees of unparalleled freedom from the existing tools. our instruments replicate the movements of the robot with a simple handheld mechanical instrument, our philosophy is to position our instruments in between a long empty field between the surgeon and the robot. the tip of our instruments are providing the surgeons with angulations impossible to reach with the traditional instruments unless applying huge movements from the hands of the surgeon. we consider that this devices will have a very fast acceptance from the market as this robotic type movements can be managed by the surgeon through the traditional mm trocars, without the need to change to a new surgical technique, just with the traditional method and a very brief training. background: diverticula of the middle thoracic esophagus are infrequent, its etiology may be secondary to traction or pulsion mechanisms. when the etiology is a mechanism of pulsion, they are associated with esophageal motor disorders and its prevalence is estimated between . % and . % of the population . they are rarely symptomatic and the diagnostic is usually incidental. the most common symptoms are episodes of food impaction, chest pain or bronchoaspiration.diverticulectomy and esophageal myomectomy by minimally invasive approach is the treatment of choice in those with large size or associated symptoms. aim: to describe a clinical case of esophageal diverticulum solved by minimally invasive surgery approach. clinical case description: a -year-old patient with a history of epilepsy and hypothyroidism consulted for atypical chest pain and dysphagia to liquids and solids. a study with esophagogastroduodenoscopy was performed: cm from the dental arch, a large wide-mouth diverticulum was identified. we complete the study with an esophagram with barium: voluminous diverticulum in the right lateral face of the middle esophageal and a thoracic ct scan showing esophageal diverticulum located in the carina, from . x . cm to cm from the esophagogastric junction. due to the suspicion of associated motor disorder, high resolution manometry was performed showing a significant motor disorder with alteration of peristalsis and exit obstruction with incomplete relaxation of the inferior sphincter and superior hypertonic sphincter. preliminary results: the patient underwent surgery: diverticulectomy and complete esophageal myotomy by thoracoscopy minimall invasive approach. the patient evolved favorably and was discharged after days with a previus control esophagram without pathological findings. currently, it remains asymptomatic months after surgery. , rectus muscles are re-approximated from xiphoid to pubis using laparoscopic running self-locking, pds sutures to restore anatomy and physiologic function of the abdominal wall. unlike the standard access to the abdominal cavity executed with lateral access, the lap-t technique is performed through sopra-pubic aesthetic approaches, using one mm and one mm bariatric ( cm) instruments laterally, and one mm camera in the middle.the entire procedure is performed in gas-less laparoscopy, with laryngeal mask and intra-peritoneal liquid anesthesia. the repair is consolidated placing an intra-peritoneal semi-absorbable mesh. preliminary results: in all cases abdominal functioning was successfully restored; no higher pain related to the continuous laparoscopic suturing has been reported compared to bridge ipom laparoscopic repair, while allowing for a more physiologic outcome and a stronger repair. the use of miniaturized instruments allowed for minimal tissue trauma and accurate surgical gestures; the tiny trocar sites did not require skin suturing and might reduce the risk of trocar hernias. no intra operative bleeding, no seroma formation, chronic pain, nor mesh infection have been recorded. % follow up at months, % at months with no recurrences observed. the lap-t technique allowed for a sound and anatomic reconstruction, reduced trauma, faster recovery and more satisfactory aesthetic results. surg endosc ( ) background: anastomotic leakage is a serious complication, associated with significant morbidity and mortality. one possible cause of anastomotic leakage is insufficient vascular supply. markers of sufficient perfusion include pink color of the bowel wall, visible peristalsis, palpable pulsations and bleeding from the marginal arteries. these signs are subjective and may be misinterpreted even by experienced surgeons. aim: the assessment of bowel perfusion with the use of indocyanine-green fluorescence angiography might be helpful in decreasing the number of anastomotic leaks. project description: we report a case report of a middle-aged patient without significant medical history who was treated by transanal total mesorectal excision (tatme) for rectal carcinoma. the patient underwent neoadjuvant treatment with radiochemotherapy. during the surgical procedure, indocyanine-green fluorescence angiography showed adequate perfusion of the bowel. the postoperative phase was uneventful and the patient was discharged home on the th postoperative day. preliminary results: indocyanine-green fluorescence angiography is a safe, cost-effective and feasible tool for assessment of tissue perfusion during colorectal resections. background: to properly learn how to perform a laparoscopic suture, along with safe tissue handling, applying an appropriate magnitude of the force on the tissue is essential. for this reason, it is fundamental to investigate and validate if training with real-time visual force feedback improves the suturing performance of laparoscopic novice surgeons. capturing all of the forces applied in laparoscopic in surgery in an unobtrusive way has been difficult in the past. sensor has supplied a novel force-sensing film (forcefilm) that can detect all of the forces applied with laparoscopic instruments without changing the surgical workflow or operation of the instruments. aim: to evaluate the effect of visual force feedback on surgical performance, applied force and surgeon's ergonomics during training of laparoscopic suturing using the sensor technology (sensor medical laboratories ltd.). methods: twenty novice laparoscopic surgeons participate in this study. they perform a laparoscopic suture on an ex vivo stomach tissue from a pig. participants are assigned, in a random fashion, to either group that receives visual force feedback (a) or the control group (b) without visual force feedback. five training trials (t -t ) are carried out in order to assess the learning curve. in addition, an evaluation pretest (t ) and posttest (t ), without visual force feedback but recording the force applied, will be performed after the training trials. the applied force on the tissue and visual force feedback of each instrument are provided by means of the sensor technology. it accurately measures the forces exerted on the tissue from the instrument tip and wirelessly communicates the force information to the surgeon via visual force-feedback. during each trial, several parameters are evaluated such as execution time, applied force, surgical performance, and mental and physical workload. preliminary results: laparoscopic training using visual force feedback leads to an improvement of suturing skills with a reduction of the applied force and therefore providing a potentially positive effect on patient outcomes and surgeon's ergonomics. background: hiatal hernia is a frequent disorder, characterized by a protrusion of any abdominal structure other than the esophagus into the thoracic cavity through a widening of the diaphragmatic hiatus. current anatomic classification is mainly based on the location of the gastroesophageal junction and the presence of a true hernial sac, differentiating sliding from paraesophageal hernias. there is no solid evidence to support an association between gastric carcinogenesis and peh. however, chronic reflux is considered as one of the strongest risk factors of developing adenocarcinoma of the esophagus and proximal stomach. aim: herein, we report a case of an -year-old caucasian female with dysphagia, regurgitation and heartburn accompanied by an iron deficiency anemia, a remarkable total body weight loss and recurrent lower respiratory tract infections due to microaspirations. subsequent work-up with ct, upper endoscopy and barium esophagram confirmed the presence of synchronous distal gastric adenocarcinoma and a giant paraesophageal hernia with complete intrathoracic stomach. after mdt discussion and keeping in mind the patient's age and comorbidity, a d laparoscopic distal gastrectomy with a synchronous hernia reduction with posterior cruropexy was scheduled. project description: the patient was placed in a supine position, five thoracoscopic ports were introduced, and a diagnostic laparoscopy of the abdominal cavity was performed. the stomach was identified through the dilated hiatus into the left thorax. the hernia sac was dissected away from mediastinal structures, then excised to untwist the stomach. after reduction of the stomach to abdominal cavity, a total d ? gastrectomy with a roux-en-y reconstruction was performed. maintenance of optimal vision during minimally invasive surgery is crucial to maintaining operative awareness, efficiency and safety. hampered vision is commonly caused by laparoscopic lens fogging (llf) and lens condensation which has prompted the development of various antifogging fluids and warming devices. numerous tricks have been proposed to overcome this issue, such as heating the scope into a sterile thermos flask filled with hot water, or using one of the commercially available antifogging solutions. however, whether one method is superior to another remains elusive. as most surgeons know, none of these tips are totally efficient, as they don't treat the cause: the temperature difference. taking into account this need, we have developed ehs (endoscopy heater system), a thermoadjustable system by microcontroller, which is implemented in the manufacturing process of the rigid endoscope focused on laparoscopy. the technology enables the self-modulation of the temperature of the endoscope within the different conditions during the surgery, avoiding the % of laparoscopic fogging. with the adoption of ehs surgeons get a clear field of vision avoiding continues repetitions of extraction and insertion of the endoscope in the body during the intervention. in this way the risks of the patients are reduced with a more efficient and shorter duration procedure. ehs also represents an alternative that meets sustainability criteria by reducing energy costs and eliminating much of the waste currently generated by this procedure. therefore, this innovation will disrupt the laparoscopic device market by enhancing safety and effectiveness without introducing new components that could complicate surgical procedures. case report: we presented the case of a year old women with chronic coloenteric fistula. conservative treatment was unsuccessful. the orifice was then closed with two subsequent clips, and the patient recovered well. to our knowledge, this is the first successful case of coloenteric fistula treatment with ovesco discussion: ovesco system is a technique that enables the closure of gastrointestinal defects (perforation sites, leaks, fistulas) . after the system application, the patient can be treated at home as was the case with our patient. a successful closure of the leak or fistula is possible when no extraluminal abscess is present. in our case, we had a cavity (previous sinus or abscess) that drained into the small bowel, thereby forming the coloenteric fistula. this allowed us to succeed with a fistula closure, as the cavity could drain into the small bowel conclusions: looking through the reports, one notes that the success rate of the otsc system procedure for insufficiency of anastomosis or colorectal fistula was - %, but only nine successful reports of chronic colorectal fistula were found). a % success rate is reported if the clip is placed within a week of occurrence of the leak . on considering the financial side, clips could reduce costs and time of hospitalization and avoid patients having to undergo a surgical repair . the major advantage of ovesco clips seems to be their ability to grasp more tissue compared to the standard clips and their strong grip on the wound margins because of their sharpened teeth. the drawback of the clips in fistula sealing is their incomplete grasp when the tissue is fibrotic. most authors agree that ovesco is not very appropriate for fistulas larger than - mm. inguinal hernia repair is one of the most performed interventions in minimally invasive surgery. in this opportunity we report a new technique through the use of innovative devices such as the robotic clamp and magnetic deviceswith this technique and thanks to the magnetized devices and the robotic clamp we have demonstrated to reduce the surgical time between - min as well as to optimize the ergonomics of the surgeon.we explain the technique with a demostrative video and exposition of the devices that are necessary for make it.with this new technique we get a greater capacity of mabiobra for dissection of the peritoneum and later a greater facility for the suture of the same in the repair of the inguinal hernia. a motorized and computerized laparoscopic tool that can be customized to the specific surgeon and procedure a. szold aim: surgeons have different levels of skill and use instruments for different tasks, but laparoscopic instrument are commonly simple mechanical instruments that allow limited degrees of motion, and the same instruments are used regardless of the surgeon or the task. robotized articulating instruments so far have added degrees of freedom, but perform in a standard way for all users and procedures. technology: human xtensions has developed a \ u[hand-held \/u [ motorized smart laparoscopic instrument, that was recently introduced in human procedures. the device has several features that enable to customize it to the user and procedure. the degrees of freedom can be reduced from to , the scale of rotation motion has options that can control both speed and range of rotation, a feature especially useful for the variable types of suturing tasks. results: the variable features were tested in different procedures requiring suturing and grasping. the combination of all optional settings made the instrument customizable to the different skill levels of the surgeons. as such, it enabled to control the complexity of the device and take the surgeon through the learning curve until full control of all features was achieved. in addition, the combination of different controls was used for performing specific tasks requiring different levels of maneuverability. in september , the results of a bomss survey regarding the routine use of pre-operative bariatric surgery were published. they found that % of units surveyed considered routine preoperative ogd completely unnecessary. as part of newly launching bariatric services in a single isolated centre we protocoled that all bariatric patients had to undergo pre-operative ogd, including a clo test, and reviewed if the ogd findings had influenced our surgical choice of operation and any necessary treatment before surgery. all patients embarking on the bariatric programme since its launch in jan to sept were included and had an ogd. the results of these ogds and all the clo tests were reviewed. these ogds were all performed by a single consultant to minimise any potential subjective differences. of the patients, ( %) tested clo positive of which had normal findings on ogd. patients had a hiatus hernia, gastritis, oesophagitis, gastritis and oesophagitis, had other findings e.g. ulcers, polyps' or nodules. the positive clo patients underwent eradication of h pylori. studies have shown that this is a treatable and preventable cause of gastritis/ gastric cancers and potential surgical complications causing prolonged hospital stay in % of patients. knowing about the presence of a hiatus hernia prior to surgery also contributed to the surgical planning, including allowing time for the concurrent correction of the hiatus hernia in the operation. all patients with demonstrable oesophagitis ( %) had their operative choice changed to roux en y gastric bypass thus aiming to prevent post-operative reflux which would have been exacerbated had they undergone a sleeve gastrectomy instead. carrying out a pre-operative ogd had a significant impact in operative choice and additional treatment before surgery and therefore should be advised in all patients. general surgery department, ahievran university, kirsehir, turkey the majority of fatalities worldwide in people under the age of years are caused by trauma . blunt mechanisms account for . to . %of injuries [ ] [ ] [ ] [ ] ,with the abdomen being affected in . to . % of all traumatic injuries. this case contribute to the literature: a patient with sleeve gastrectomy has distorted anatomy at duodenogastric junction, if has bat,her/his small bowel perforation(sbp) will occur on more distal segment. this a unique case before unpublished. years old female who had sleeve gastrectomy years ago presented to emergency department sustained blunt abdominal trauma (bat). when she arrived pyschical exam (pe) revealed an abdominal guarding, tenderness, normal vital signs but those increased h later. wbc values also increased h later. hb was normal.fast showed in cm thickness fluid early in douglas pauch (dp), mm in supravesical, mm diameter in dp h later. abdominal ct: mm diameter fluid in interloop, dp free air at th h from the accident. a diagnostic laparoscopy(dl) was done with diagnossed acute abdomen.there were a sbr-located cm from treitz, intraperioneal fibrin deposits and fluid-repaired with a primary suture.the patient discharged on days without any event. repeat ct scans are recommended for patients with initial suspected bowel injury. we could not do this; cause ct exam could taken in rush hours only but we did repeatly pe that peritoneal iritation signs increased,resulted a dl,surgical therapy. according to the literature; dl may be a good treatment option in these patients, to reduce morbidity or mortality, time to surgery has been emphasized. long interval between presentation and surgery was found to be associated with complications. very few reports of isolated jejunal transection following blunt abdominal trauma have been published in literature. the literature mentioned; the patients with sbp are hemodinamically stable on arrival to the hospital like our case are, a rupture of the jejunum was seen just distal to the duodenal-jejunal flexure but there were a perforation cm below treitz ligament and caused me to think the patient had sleeve gastrectomy and some brid around gastric and duodenal proximal jejunal part of intestines and also caused a new descended treitz ligament. normally external forces across to spine produce a blast effect on small bowel between treitz and ileocolic ligament. introduction: sasi bypass is a novel metabolic/bariatric surgery operation based on minigastric bypass and santoro's operation.it can be offered for patients with weight regain after sleeve gastrectomy. sleeve gastrectomy (sg)is a commonly performed bariatric procedure.weight regain following sg is a significant issue.yet,the understanding of this phenomenon is still unclear.rates of regain ranged from . % at years to . % at years.sasi bypass was an option for some candidates having sg done years back and failed to achieve the required weight loss or having weight regain.in sasi bypass, resleeve gastrectomy of the dilated gastric pouch is done followed by a side to side gastro-ileal anastomosis. the aim of this study is to report the clinical results and the outcomes of sasi bypass as a therapeutic option for patients with weight regain after sg methods: we conducted a retrospective study for morbidly obese patients having history of sg done more than years back and failed to achieve and/or to maintain the required bmi. exclusion criteria:patients with recent history of laparotomy(less than months). procedure was done at sidra hospital in kuwait from november to november . using ports, resleeve gastrectomy was performed over fr bougie tube starting cm above the pylorus then gastro-ileal anastomosis (side to side)was performed cm above the pyloric ring to an ileal loop counted cm from the ileocaecal valve. data was collected from the patients including:weight loss progress,laboratory full results. discussion and results: during the study period: morbidly obese patients with a mean bmi of ± kg/m were evaluated. -%ewl(excess weight loss)reached % at one year. -diabetes was cured in the known diabetic patients (type )within months,and the one known type diabetic patient had better control and less insulin daily doses(results were guided by glycated haemoglobin results every months). follow up laboratory results were normal in % of patients (all were kept on regular vitamins and proteins supplementation)-one patient had postoperative leak(day )from the anastomotic line that was treated conservatively. conclusion: sasi bypass is a promising operation that offers a good weight loss for morbidly obese patients having weight regain after sg conclusions: our study demonstrates a good agreement between the degree of liver steatosis and monocytes fat accumulation as well as between plin levels in liver and circulating monocytes. this suggests that ectopic fat deposition is a generalized feature of insulin resistance in obesity. sg reverses monocyte fat accumulation and restores insulin signalling, which correlates well with insulin sensitivity. moreover, circulating mmp levels significantly dropped after sg suggesting that the state of generalized inflammation characterizing obesity normalizes. her stomach stapled,a foreign tube like body was seen on cut surface of the stomach.the foreign body seen in dissected stomach wall is the tube is in placed a gastric banding insuflation tube.a laparatomy was made and the tube is extracted her stomach sutured primarily,nasogastric decompression,peritoneal drainage was made.her peritoneum was drained.she has a septic condition, leave in icu for a long period.her general status being well and discharged from hospital in days.we learn after the operation not before;she had a gastric adjustable banding and extraction the gastric band, but the tube of gastric band is not removed. alkhaffaf et al present a case of fistulation of the lagb tubing into the jejunum a review of the published data to identify the salient learning points with this and similar rare complications fistulation from lagb tubing is a rare complication that tends to follow removal of an infected port. the clinical presentation is nonspecific, rendering the preoperative diagnosis difficult. the tube and band can be removed laparoscopically, with closure of the small bowel fistula site. securing the tubing to the abdominal wall fascia after intentional detachment from the port might reduce the incidence of this complication.katherine j et al report a late and rare complication of a small bowel obstruction in a -year-old woman from an lagb placed for years. although not a common complication, one that could easily see the safety record of lagb patients tarnished if this small subgroup of patients is not acted upon promptly by emergency departments' unfamiliar lagb surgery. in our case we already made esophagogastroduodenoscopy before operation, ofcourse take past medical history from the patient.the patient hide past operation (gastric banding and removing band and port but leaving insuflation tube). ). there was no difference in the two groups regarding follow-up rate. basic demographics were the same, and other long-term results were similar between the groups. regression models for both post-op complications and failure as defined by baros score did not show that gender is a risk factor. discussion and conclusions: in our study, revisional sleeve surgery were similar. we did not see any significant difference in post-op complications, success of the operation as defined by baros, or subjective feeling of the patients. we do believe that gender-specific outcomes should be taken into consideration in optimizing patient selection and preoperative patient counseling, and that in the case of a sleeve post a band gender is not a risk factor for complication or failure of the procedure. objective: the internal hernia is a rare but a potentially fatal complication of laparoscopic roux-en-y gastric bypass (lrygb). the aims of this study are: ( ) to determine the impact of mesenteric defects closure on the incidence of internal hernia after lrygb; ( ) to determine the symptoms, characteristics and management of internal hernias after lrygb. the median interval between lrygb and reoperation was months in group a and months in group b. the median percentage of excess weight loss (%ewl) was % vs %, respectively (p = . ). the median percentage of total weight loss (%twl) was % vs %, respectively (p = ns). patients, % ( in group a), were admitted to the emergency room with acute abdomen pain. a ct scan was performed in patients, %, and showed signs of occlusion in all cases. the most common symptoms were abdominal pain and vomiting. the surgery was performed using laparoscopy in patients, %, and using laparotomy or conversion in patients, %. conclusions: the closure of mesenteric defects during lrygb is recommended because it is associated with a significant reduction in the incidence of internal hernia. our study intends to analyze the long term results of sleeve gastrectomies performed by d laparoscopic approach. materials and methods: a prospective cohort study was conducted to perform gastric sleeve for morbid abesity. all surgeries were performed by the same surgeon over a period of two years. the operating surgeon is a senior most laparoscopic surgeon with vast experience in laparoscopic surgery. during two years period, cases were operated using d laparoscopy system. scientific calculation was done using spss release . windows software. results: patients, female ( %) and male ( %), with median age of . ± . ( - ) the excess weight lost (ewl) was % in the first year, % in the second, % in the third, % in the fourth, % for the fith year, % for the sixth and % for the seventh. postoperative complications were stenosis of the sleeve always located in the incisura and treated with endoscopic dilatation except one that required conversion to oagb. three leakages, all of them reoperated with drainage and introducing prosthesis by endoscopy in the same act. we have never had a postoperative bleeding of the sleeve. conclusions: d gastric sleeve laparoscopy is a safe and feasible technique for morbid obesity and related pathologies. the ewl is correct in long time. complications are rare but is necessary to have a good level of suspicious in order to a rapid solution. the worst complication is the leak of the sleeve. the oversewn of the gastric section is a good technique to avoid this complication. surg endosc ( ) aims: leak is one of the common complications of laparoscopic sleeve gastrectomy that result prolongation of hospital stay, morbidity and even mortality. methods: i report new approach for the treatment of leaks presented to me post laparoscopic sleeve gastrectomy with laparoscopic roux en y bypass to the leak site at the level of gastroesophageal area. this new approach is possible and feasible, and avoids stenting due to high failure rate, prolonged hospitalization and saves life of patients. results: all leaks healed days from surgery due to well vascularized small intestinal patch, except for leaks that healed after weeks of conservative treatment. aims: analyse the effect of one anastomosis (mini) gastric bypass (mgb/oagb) in the treatment of gastro-esophageal reflux in patients previously submitted to laparoscopic sleeve gastrectomy (sg). methods: a retrospective analysis was performed on the data of patients who underwent mgb/ oagb after a previous sg at policlinico san marco, italy, from january to june . a total of patients, female and males ( % f/ % m) underwent mgb/oagb after sg, due to the development of significant gastro-esophageal reflux disease (gerd), refractory to proton pump inhibitors (ppi), detected with the gerd questionnaire (gerd-q) and esophagogastroduodenoscopy (egds). in three patients ( %) a weight regain was also observed (mean bmi . kg/m , range . kg/m - . kg/m ). mean patients age was . ( - years old). before sg none of the patients had declared symptoms of gerd or was subjected to a therapy with ppi, preoperative egds did not show signs of esophagitis. mean bmi of the patients who developed gerd without weight regain was . kg/m ( kg/m - . kg/m ) at the time of surgery, with a medium ewl% of % ( . - . %). patients were treated unsuccessfully with ppi for at least six months before programming revisional surgery. mean gerd-q score was . results: after mgb/oagb, with a mean follow up of months ( - months), mean bmi was . kg/m and gerd-q score was . however, five patients out of ( %) developed an anastomotic ulcer or a grade c esophagitis. we did not observe any post-operative immediate complication nor any death. conclusion: mgb/oagb is a simple, effective and safe surgical procedure for patients who underwent a previous sg and who developed gerd, with satisfactory results in the short and medium post-operative time, even if there is still concern regarding the complications linked to biliary reflux. v.s. kyosev aims: laparoscopic adjustable gastric band (lagb) was one of the common techniques in bariatric surgery worldwide. the advantages included the possibility of regulation, ease of placement, acceptable weight loss and low rate of perioperative complications. a late complication of lagb is penetration of the gatric band through the gastric wall and migration into the lumen of the stomach. hereby, we present three cases of gastric band migration following lagb. methods: from to we observed cases of gastric band migration in between and years after lagb placement. the patients were hospitalized in surgical department complaining of sudden sharp epigastric pain, nausea and vomiting, with symptoms onset in the last few days. all patients underwent abdominal ultrasound examination, x-ray investigation of the abdomen with oral contrast administration, fibrogastroscopy. in cases the imaging studies revealed gastric band migration into the stomach's lumen and in case-obstruction of the jejunum by the gastric band. all patients underwent laparoscopic surgery. results: two of the patients underwent gastrotomy, extraction of the gastric band and roux-and y-gatric bypass. the patient with jejunal obstruction underwent laparoscopic enterotomy, extraction of the gastric band and cholecystectomy due to concomitant cholecystitis. two of the patients had no additional perioperative complications and were discharged at the th postoperative day. one patient developed fever, left pleural effusion and partial insufficiency of the gastrointestinal anastomosis in the early postoperative period without the need of surgical treatment. the patient was discharged on th postoperative day. all patients were prescribed a diet and monthly blood test of ion balance. conclusions: lagb was one of the most common treatment methods due to the epidemic spread of morbid obesity in western countries. detailed knowledge on possible lagb complications is essential for the treatment of these patients. the diagnosis of lagb complications is often delayed due to its relative rarity and nonspecific clinical manifestations, but in most of the cases it requires emergency surgery for management of life-threatening conditions. results: there was conversion in patients (short mesentery of the small intestine). such postoperative complication like anastomotic leak in patient ( . %) and staple line bleeding in patient ( . %), which was managed laparoscopically. compensation for type diabetes was achieved in ( %) patients, improvement was recorded in ( . %), dyslipidemia in ( . %) and ( . %) patients, arterial hypertension in ( . %) and ( %) patients respectively, what led to metabolic syndrome resolution in ( %) patients. the liquid is allowed to take after day. average postoperative hospitalization- . ± . days. %ewl for months %. conclusions: laparoscopic mini gastric bypass is an effective method of surgical correction of body weight and metabolic disorders in patients with morbid obesity and allows to receive an adequate and stable correction of arterial hypertension, lipid and carbohydrate metabolism, which are components of a metabolic syndrome. introduction: over time laparoscopic sleeve gastrectomy lsg has become the most popular bariatric operation worldwide. a critical step during lsg is ensuring sleeve-size consistency. gastrisail device (gastric positioning system) is a three in one surgical device replacing the standard bougie used in lsg for the application of suction, decompression and to serve as a sizing guide for gastric sleeve creation. the aim of this study is to evaluate the possible merits of gastrisail device in lsg over the standard laparoscopic sleeve gastrectomy. methods: a prospective study of patients randomly divided into two groups: group a composed of twenty patients who undergo lsg with the use of gastrisail and group b composed of twenty patients who undergo lsg with the standard bougie without the use of gastrisail comparing both according to operative time, consistent sleeve formation, delineation and visualization, intraoperative and post-operative complication rates, the lenght of hospital stay,gastric pouch design and percentage of excess weight loss (%ewl). results: regarding intraoperative time, the mean time was . ± . and . ± . for group a and b respectively,while no patients in group b had consistent sleeve formation, patients ( %) had consistent sleeve formation. delineation and visualization were accomplished in % of group a patients, was not accomplished at all in group b patients. the alignment of the stomach was reached in patients in group a but no patients at all in group b, the mean of hospital stay was . ± . and . ± . for group a and b respectively, the smaller tube design illustrated by gastrograffin x-ray at rd post-operative day was accomplished in patients ( %) and patients ( %) in group a and b respectively. there was no significant difference in %ewl in both groups. conclusion: the use of gastrisail device is superior to the standard lsg in consistent sleeve formation, visualization, delineation and good alignment and accomplishment of a small tube design while no significant difference in %ewl. bariatric surgery has spread all over the world. since japan has few patients with morbid obesity compared with western countries, it has been implemented only in limited facilities. however, bariatric surgery in japan is rapidly spreading recently, and many facilities are about to install bariatric surgery. effects of bariatric surgery are known to last for a long time, but some cases require reoperation which is called revision surgery due to late complications or rebound. because of thick subcutaneous and visceral fat, open surgeries are not even always a good solution to make surgery easier in morbid obese patients and all procedures must be completed laparoscopically. therefore, especially in revision surgery, the incidence of complications tends to be increased. as the number of bariatric cases to be increased in japan, cases requiring revision surgery is likely to increase. in revision surgery, it is necessary to select the procedure according to patient condition, and it is necessary to familiar well with those procedures. we will present cases that underwent revision surgery in our department and show the clinical outcome. we have done four revision surgeries after sleeve gastrectomy so far. operative indications are mid-gastric stenosis and rebounding disease. for stenosis cases, we performed roux-en y gastric bypass with distal stomach resection, and for rebounding cases, we performed re-sleeve gastrectomy with duodenal-jejunal bypass. average interval from initial operation to revision surgery is months in rebounding cases, and months in stenosis cases. duration of operation was min in average, and mean estimated blood loss was ml. no postoperative mortality was observed. in rebounding cases, excess bmi loss at year after surgery was . % in average, and both cases achieve diabetes remission at year. one cases of mid-gastric stenosis required a nutritional support with formula diet temporally. in particular after sleeve gastrectomy, revising to roux-en y gastric bypass, re-sleeve gastrectomy, and adding the duodenal-jejunal bypass will be the main techniques. along with an increase of bariatric surgery in japan, it is necessary to acquire sufficient knowledge and skills to carry out revision surgery. methods: we present the case of a year old woman who underwent lsg after lagb removal and lgcp. the patient underwent preoperative endoscopy and barium swallow, with no sign of stomach perforation or erosion. we emphasize that the patient, had undergone three operations of gastric band placement, gastric band removal and gastric plication before sleeve gastrectomy. however, a successful lsg was achieved. results: no severe postoperative complications were mentioned. conclusion: weight loss in the first year was % of the excess weight.sleeve gastrectomy after gastric band removal and gastric plication, for morbid obesity seems to be safe and efficient, especially in casesof absence of gastric erosion. surg endosc ( ) department of surgery, patients were observed with serious septic complications many years after gastric banding operation. we detected a female dominance ( female, male) in patients with a mean age of . years. the leading symptoms were: dysphagia, upper abdominal tenderness and pain, spontaneous fistula formation, fever, masked septic signs, bowel and urinary obstruction. patients underwent video-endoscopy, chest and abdominal ct (computed tomography), fistulography and cystoscopy. results: in still morbid obese patients, laparoscopic procedures were performed with a conversion rate of %: atypic gastric and cardia resection in cases, gastric suture in cases, small bowel resection and suture in - cases. in one case, fistulectomy, abscess evacuation and combined urinary bladder suture and drainage were carried out. the duration of the surgeries were over h with minimal blood loss (\ ml). the foreign bodies were completely removed in every case. intraoperative complication was not occurred. early physiotherapy were promoted, oral feeding were gradually built up from the th postoperativ day depending on the type of the operation. early postoperative complications included recurrent fistula formation (n = ) and wound infection (n = ). all the fistulas were closed after conservative treatment. average hospital stay was days, regular check-ups were held on the rd, th and th months of follow up. conclusion: gastric banding is the most common, routine and safe technique for the treatment of morbid obesity. the development of late, severe septic complications draws attention to the crucial importance of follow up. the surgical management of these patients is recommended in specialized centers in regard to difficult operative conditions and atypic treatment options. aims: single anastomosis duodeno-ileal bypass with sleeve gastrectomy (sadi-s) has been proposed as an alternative to biliopancreatic diversion with duodenal switch (bpd-ds) in order to maintain the outcome of the original procedure simplifying the technical complexity and to avoid potential complications. moreover, it potentially represents the more natural second step bariatric procedure after sleeve gastrectomy (sg). we aimed to report the initial experience with sadi-s of our high volume bariatric center. methods: retrospective analysis of patients who underwent bariatric procedure between july and november was conducted. the primary aim was the evaluation of the safety of sadi-s, defined as the rate of postoperative complications. the secondary endpoint was the bariatric efficacy of the procedure, defined as percentage excess weight loss (%ewl). results: among patients who underwent bariatric procedures at our institution ( . %) patients were scheduled for sadi-s. all patients had multiple comorbidities. initial indication for sadi-s was failed sg in patients (median pre-sg bmi . kg/m \ sup [ \/sup [ ; median months after initial operation respectively) and primary procedure in patients (median pre-operative bmi . kg/m ). the surgical procedure was accomplished with robotic-assisted approach in cases (median operative time min) and with laparoscopic trocars standard approach in the remaining cases (median operative time min). the duodeno-ileal anastomosis was fashioned using a double layer hand-sewn running sutures. no patients showed early post-operative complications, the median postoperative stay was days. at a mean follow up of months the median %ewl was . . to date no patients experienced surgical. one patient develop wernicke encephalopathy months after surgery, but he was non-compliant to multivitamin supplementation. conclusions: at least in a high volume bariatric center sadi-s, both as second step after sg and as primary surgical option, seems to be a safe and effective bariatric metabolic procedure based on solid physiopathologic principles. on the other hand, longer follow-up is necessary to support the use of this procedure as a better alternative to bpd-ds. m.r. elkeleny , a. abo khozima git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery department, faculty of medicine,alexandria university, alexandria, egypt four bariatric cases . female patient with intragastric balloon, minor leak from the balloon leading to ballon migration to the jejunum;hance, small bowel obstruction occured. emergency diagnostic laparoscopy was done, enterotomy and extraction of the balloon, direct repair of enterotomy and balloon extraction through mm port site. . male patient presented after days of lsg with small bowel obstruction due to entrapment of small bowel loop through one of the port sites;therefor, emergency laparoscopy was done with reduction of the herniated segment and closure of the port site. . female patient presented with stricture of the ogj after re-sleeve gastrectomy managed by balloon dilatation which recur after weeks .she was managed by expandable metallic stent for weeks with good response and the stent was removed. . -year-old male patient presented with sever peripheral neuropathy following months after sleeve gastrectomy, and the patient was getting worse; thus, he used wheel chair. he has been making good progress on vitamin b complex injections. the aim of our study was to compare histopathological findings of gastric specimens to preoperative clinical symptoms and to conclude about the need for ugi endoscopy as a routine prior to surgery. methods: the last two years, morbid obese patients were selected to undergo laparoscopic sleeve gastrectomy (lsg) in our institution. for the needs of our study, all of them had ugi endoscopy and were reviewed for upper gi symptoms. histopathological reports obtained according to our protocol, after surgery. results: gastric histology from specimens revealed: no findings in / patients ( . %), gastritis in / patients ( . %) and focuses of incomplete intestinal metaplasia without dysplasia in / patients( . %). finally, two minor leiomyomas with low cellular proliferation rate were fully excised in a patient's specimen. there was no inconsistency between preoperative symptoms and gastric histology, while leiomyomas found were no reported to ugi endoscopy due to size. conclusions: some of the patients with clinical features of food intolerance, gastroesophageal reflux disease, and peptic ulcer disease had finally findings in histopathology of their stomachs. history of helicobacter pylori infection implements a raised incidence of mucosa pathology as well. because only one case revealed carrying significant pathology (leiomyomas), we consider that is safe to proceed with surgery in an otherwise asymptomatic patient based on his previous medical records and blood tests. aims: splenic abscess following laparoscopic sleeve gastrectomy (lsg) is a rarely seen complication. the aim of our study was to present a case of splenic abscess in a morbid obese patient who underwent lsg. as the main concern in these cases is leakage from the staple line, we present our diagnostic and treatment approach. methods: a -year-old, female morbid obese patient (bmi . kg/m ), without any predisposing risk factors, underwent elective lsg in our department. following an uneventful course, she was discharged at the nd postoperative day. however, at the th postoperative day, she readmitted to our unit with high temperature of . o c, left upper quadrant tenderness and leukocytosis. contrast computed tomography (ct) revealed an abscess at the upper pole of the spleen , cm in maximum diameter, without leakage from the staple line. results: the patient was treated with broad-spectrum antibiotics and radiological percutaneous drainage of the abscess. although there was a partial clinical improvement, a week later, a new ct scan revealed the continuous presence of the abscess. despite the stable general condition of the patient a laparoscopic splenectomy was performed and a gradual recovery was followed. the presence of splenic abscess without splenic trauma or leakage from the gastric staple line, is an extremely rare complication and only a few cases have been previously reported. the cause has not yet clarified, but the proposed mechanism involves infarction of the spleen, due to vascular compromise and subsequent infection. most of the reported splenic abscesses were diagnosed during the late postoperative period. in our report we present a case of early onset, hence highlighting the need of clinical awareness for early diagnosis and treatment. introduction: obese surgical patients with obstructive sleep apnea (osa) have a higher risk of peri-and postoperative desaturations and subsequent morbidity and mortality. currently, the best perioperative management of patients without known osa remains unclear. although routine osa screening has been advocated, sleep studies are costly and time consuming. we hypothesized that bariatric patients can be safely monitored on a surgical ward by continuous postoperative pulse oximetry without preoperative screening for osa. objectives: to evaluate outcomes of continuous postoperative pulse oximetry without preoperative osa-screening, and to compare the results to outcomes of patients with osa and continuous positive airway pressure (cpap) treatment. methods: all patients who underwent bariatric surgery between and were included in this single-center retrospective cohort study. all patients were postoperatively monitored with continuous pulse oximetry on the surgical ward. patients with less than two documented saturation measurements were excluded. patient files were reviewed for osa diagnosis, cpap usage and perioperative details. primary outcomes were -day complication rates, intensive care unit admissions due to cardiopulmonary causes and postoperative desaturations of spo \ %. secondary outcomes were icu admissions following all causes, length of stay and rates of reoperation and readmission. results: in total, patients were included. patients ( %) were preoperatively diagnosed with osa, ( . %) were cpap users. complications occurred in . % of patients without osa and in . % with osa(p = . ). desaturations were documented in . % and % (p \ . ), respectively. in both groups, patient was admitted postoperatively to the icu for cardiopulmonary causes that could be related to osa (p = . ). both recovered without further complications. icu admissions, regardless of cause, occurred in . % of patients without osa and in . % with osa(p \ . ). no significant difference between groups was observed in complications based on clavien dindo classification, length of stay, reoperation-and readmissions-rates. conclusions: these findings suggest that continuous postoperative pulse oximetry without preoperative osa-screening is a safe perioperative management strategy for bariatric surgical patients. future studies are needed to assess cost-effectiveness of pulse oximetry vs. routine preoperative osa-screening in a prospective clinical setting. background: the pathology of colon is one of the most pressing and socially significant problems of modern health care, because it leads to reduction of the working population employed in manufacturing, in some cases to disability and reduced quality of life. mini invasive surgery of the colon has a great advantage: speed recovery, shorter hospital stay and better cosmetic results, the quickest return of patients to work. as a result, mini invasive endovideosurgery is firmly established in clinical practice of coloproctology. objective: the choice of optimal surgical method for treatment of colostasis, achievement of favorable outcomes of treatment. introduction: laparoscopic roux-en-y gastric bypass (rygb) is one of the most important bariatric surgical procedures performed worldwide and it can produce an important loss of weight with reversal of metabolic disorders like diabetes and dyslipidemia. even though it has good results, some complications occur after gastric bypass. a rare but serious complication of rygb is the so-called postprandial hyperinsulinemic hypoglycemia. its prevalence has been estimated less than % of cases and its pathophysiology remains unclear. methods: the aim is to present a case series of reversal surgery in patients with severe hiperinsulinemic hypoglycemia after rygbp in the hospital general universitari de la vall d'hebron. unit of endocrine-metabolic and bariatric surgery (eac-bs center of excellence for bariatric and metabolic surgery by ifso). it is a retrospective analysis of a prospective database same surgical team. we present in this study, the main features of those patients. results: between and , patients underwent a laparoscopic reversal procedure to normal anatomy and age mean was year ( years to years). mean preoperative body mass index (bmi) was . kg/m (range - . kg/m ) and were women. all patients presented hypoglycemia symptoms years after and the longest was years after the procedure. the first step of the standard approach was a laparoscopic reversal to normal anatomy with resection of the alimentary rygb limb in cases. a concomitant sleeve-like gastrectomy (sg) was added. four patients presented postoperative complications: gastrogastric anastomosis leak ( ) introduction: laparoscopic sleeve gastrectomy is the most performed bariatric procedure, but complications might interfere with patient's long-term evolution based on its compliance and tolerance, surgical attitude and unpredictable evolution. materials: we present the case of a female obese patient, with type ii diabetes mellitus and blood hypertension, with multiple, sequential bariatric minimally-invasive interventions: sleeve gastrectomy in complicated by postoperative acute gastric dilation and mediogastric stenosis, reoperated for viscerolysis and cholecystectomy, with endoscopic gastric dilations, initially converted to functional one anastomosis gastric bypass ( cm limb), with a non-adjustable gastric ring positioned instead of stapled division. the last operation was complicated months after by persistent biliary gastro-esophageal reflux, chronic abdominal pain, and gas bloat syndrome. in the patient underwent conversion to laparoscopic r-en-y gastric bypass, with gastro-enteral anastomosis resection, band removal and viscerolysis. results: conversion to r-en-y was complicated by biliary leakage post-viscerolysis, treated with laparoscopic approach in the \ sup [ th \/sup [ po day. after multiple surgical and endoscopic interventions, the patient presents short-term favorable outcomes, with no reflux or abdominal pain, with further weight loss and diabetes improvement. conclusion: bariatric surgery has unpredictable evolution in same cases, and conversion to r-en-y seems to be the best solution. lgcp is widely used in developing countries due to its lower cost and good results. material and methods: we performed in our department lgcp for morbid obesity. excess weight loss (%ewl) was % at month after surgery and % at one year. in cases revision surgery was needed for different complications and in cases for inadequate weight loss or weight regain after month follow up. in cases we performed sleeve gastrectomy (in cases after taking down the plication) and in cases we performed a re-plication in one row. results: the rate of revision surgery was % overall and % for inadequate weight loss (excess weight loss \ %) or weight regain. major complications occurred only in one patient (leak with abscess) but it was solved by laparoscopy. minor complications as vomiting and nausea appeared in patients ( %) and were solved with medication. after one year follow up %ewl in these cases was %. conclusions: revision surgery after lgcp is possible. a new plication or sg was the option in our series with good results. further studies are needed to evaluate the use of lgcp in the armamentarium of bariatric surgery. background: roux-en-y gastric bypass (rygb) is one of the most commonly performed bariatric procedures around the world.however, rygb it sometimes carries the risk of rarebut serious long-term complications such as malnutrition and liver failure. we report a case of laparoscopic reversal of rygb. methods: in march , a laparoscopic rygb was performed for a -year-old female without comorbidities and with a bmi of kg/m . all laboratory test results at the preoperative evaluation were within the normal range. abdominal ultrasound revealed moderate hepatic steatosis and oral endoscopy a hiatal hernia with grade b esophagitis. one year later, patient experienced an important weight loss of kg (from to kg) with a bmi of kg/m . however, patient presented general weakness, abdominal pain, ascitis lower extremitiy edema, anemia, progressive caloric and protein malnutrition, vitamin (a, d), mineral (copper) and folic acid deficiencies, nonalcoholic steatohepatitis (nash) and liver function was progressive worsening. results: a laparoscopic reversal of gastric bypass was performed. the operation was successfully performed via laparoscopy. operating time was min. postoperative was uneventful and patient discharge home at day . hepatic biopsy revealed nash with steatohepatitis of % (fibrosis f - / ). eight months after reversal of gastric bypass, patient has improved her clinical situation (no asthenia), maintains of weight ( kg) and has improved her nutritional status and liver function parameters. conclusion: laparoscopic reversal of rygb is technically feasible and might be performed safely after thorough preoperative evaluation in carefully selected patients with malnutrition and liver failure. conclusion: laparoscopic sleeve gastrectomy it's a safety obesity procedure before major abdominal hernia repair. it's a minimally invasively technique with an absence of anastomoses. these factors prevent fewer complications, without using the small bowel, and skin problems and allow resolution of obesity-associated co-morbidities. body weight loss after surgery may be an opportunity to repair the severe loss of domain incisional hernia. bibliography borbély, y., zerkowski, j., altmeier, j., eschenburg, a., kröll, d. and nett, p. general surgery, benhazi medical center, benghazi, libia, general surgery, royal bahrain hospital, manama, bahrain obesity is a worldwide epidemic with an increasing incidence trends and as a consequence obesity related health problems become priority to healthcare authorities in all the countries. laparoscopic gastric plication is an emergent restrictive procedure which claimed to be low cost because they do not need staplers and carries less complications as compared to laparoscopic sleeve gastrectomy. we present here a years female who was operated for morbid obesity four months back where she underwent laparoscopic gastric plication with no immediate post operative complication and her wight loss was adequate. two days before presentation to our emergency department she started to complains of sever attacks or upper abdominal pain and vomiting.clinical examination was unremarkable apart of abdominal tenderness in left upper abdomen. all blood routine were normal and all inflammatory markers were within normal range.ct abdomen showed large cystic lesion around the greater gastric curvature containing fluid and raised possibility of collection. patient was admitted to hospital, in despite of medical treatment her pain persists and necessitate immediate laparoscopic exploration. gastro-gastric hernia at the greater curvature through loosen ethibond suture that was used to plicate the stomach in the previous surgery. we released the suture to liberate the strangulated stomach which is not gangrenous. re-plication was not possible because of the extensive gastric wall edema and as preoperative discussion with the patient she refused conversion to sleeve gastrectomy no intervention was done. post surgery patient was free of symptoms and tolerating oral diet and discharged home on third post operative day with no complications. gastro-gastric herniation could progress to gastric wall gangrene which will result in high morbidity and even mortality. high index of suspicion is required to diagnose the condition . preoperative patient counseling is important to explore the surgical options if deemed necessary to convert to another bariatric procedure. k. chouillard, a. d'alessandro, l. chahine background: bariatric surgery is the best available, long-term treatment for morbid obesity. currently, laparoscopic sleeve gastrectomy (sg) is the most commonly performed bariatric procedure in france. despite its safety and efficacy, long-term complications of sg are not rare including gastro-esophageal reflux disease (gerfd), twisting, stenosis, insufficient weight loss, and weight regain. the goal of this study was to analyze the pattern and short-term results of surgical revision in patients with sg. methods: revisional bariatric surgery, regardless of its motivation, was always a multidisciplinary decision after clinical, biological, endoscopic, and radiological assessment. patients who had revisional surgery after sg were retrospectively identified and subsequently divided in subgroups according to preoperative body mass index ( we aim to present the management and the particular aspects of the surgical technique in a gastrobronchial fistula after gastric sleeve . the mean time between intervention and diagnosis is . - . months. methods: between and , laparoscopic gastric sleeve resections were performed in our bariatric center. we had one case of gastrobronchial fistula associated with an inferior lobe abscess of the left lung, diagnosed months after the gastric sleeve. the patient was subject for medical treatment for h, than a laparoscopic intervention was performed in order to drain the lung abscess and the gastric fistula and to place a feeding jejunostomy. . months after this intervention ( . months after gastric sleeve) a laparoscopic roux-en-y fistulojejunostomy was performed. the evolution was monitorized with blood tests, upper gi contrast series and ct scans. results: the surgical drainage of the lung abscess, along with the antibiotherapy, controlled the infection and allowed the lung cavity to reduce in size, and thus the drainage tubes introduced in the thorax through the diaphragmatic orifice were retracted progressively. also, the feeding jejunostomy allowed a proper nutrition for the patient with a good recovery. however, . months after the drainage intervention, the gastric fistula was not healed, and a decision to interrupt the communication with the lung cavity was made, by creating a laparoscopic fistulojejunostomy. after this, the evolution was favorable, with the healing of the lung cavity, oral feeding was permitted and the jejunostomy was suppressed. conclusions: the treatment of the gastrobronchial fistula is complex (medical, endoscopic or surgical), phased and long lasting until healing. surgery was our initial choice for treatment due to the existence of the lung abscess, which needed to be drained. key words: gastrobronchial fistula, lung abscess, laparoscopy, fistulojejunostomy s.i. filip, i. hutopila, c. copaescu introduction: leakage remains one of the most dreadful complications in metabolic surgery. the main cause of leakage is poor tissue oxygenation due to inadequate vascular perfusion. the study of intraoperative tissue perfusion in real time due to icg enhanced fluorescence could provide valuable information for the surgeon in order to prevent postoperative fistula. aim: to present our experience in using icg enhanced fluorescence in laparoscopic bariatric surgery material and method: in cases of gastric sleeve, cases of gastric bypass and in cases of revisional surgery or redo cases we used intraoperative icg mediated fluorescence to assure the optimal vascularization of the involved tissues. in our video we present intraoperative aspects before and after using icg in different cases. results: in all cases of primary gastric sleeve and gastric bypass with intraoperative use of icg we did not encounter inadequate perfusion. in one case of redo gastric bypass after failed vertical banded gastroplasty for morbid obesity despite intraoperative laparoscopic normal aspect of the gastro-jejunal anastomosis, icg mediated fluorescence allowed to identify an unexpected ischemic anastomosis and we could prevent consecutive postoperative leakage. discussion: presented cases are discussed and result with referral to literature is made. conclusion: intraoperative use of icg is a valuable tool in assessing the perfusion of the tissues and provide essential information for the surgeon in order to avoid postoperative leakage. , including patients, hemostasis with clips has been performed in all cases. however, among these cases nine patients required reoperation for early postoperative bleeding. in five cases a bleeding source from the stapled line was identified while in cases no identifiable source was found. during the second period ( to present) patients were submitted to bariatric surgery and hemostasis was performed by over sewing with a running suture. among these cases reoperation for postoperative bleeding was needed in cases ( . %), but no bleeding from the staple line being encountered ( %). the difference has statistical significance. no significant complications related to the use of this type of reinforcement were encountered. conclusions: over sewing the gastric stapled line in bariatric surgery is superior to hemostatic clip application in preventing the postoperative bleeding from the stapled line postoperative bleeding. a protocol of active search of the bleeders during the bariatric procedure should be implemented and respected in all the cases. gastroenterological surgery, saitama medical university international medical center, hidaka-shi, saitama, japan intestinal endometriosis is a rare disease which is associated with about to % of patients with endometriosis, and it is favorable to the rectum and sigmoid colon. here we report cases (shown in the table) underwent laparoscopic resection for intestinal endometriosis. there were no postoperative complications in all cases, and all patient was discharged on - \ sup [ th \/sup [ postoperative day. before the operation, of patients were diagnosing intestinal endometriosis, and it was difficult to preoperatively diagnose. among them, the symptoms at the time of menstruation were clear was one case. in case of submucosal tumor, preoperative diagnosis seems difficult. additional image examination at menstruation may be useful for diagnosis. d dissection was performed for case , , because malignant disease could not be denied as a preoperative diagnosis. of them were strongly doubted endometriosis in surgical findings. in intestinal endometriosis surgery, pelvic adhesions and fibrosis are often advanced. in the sigmoidectomy, the average operation time was min and the blood loss was ml. in the rectal resection, the average operation time was min and the blood loss was ml. in case and , pelvic adhesion was severe, residual rectum could not be straightened, and side to side anastomosis was performed. in intestinal endometriosis surgery, intestinal anastomosis method should be considered flexibly. conclusion: laparoscopic surgery for intestinal endometriosis was safe, but technically difficult because of fibrosis and adhesion. it is important to accurately diagnose from clinical symptoms and image also intraoperative findings. anastomotic method should be decided according to the case. aim: the aim of the study was to identify and highlight some of the complications one can encounter in bariatric surgery-specific-sleeve gastrectomy and discuss the therapeutic options one has at his disposal. methods: the study was retrospective. we identified a number of patients which had a sleeve gastrectomy done in our clinic for a year period. of these had important surgical complications encountered during the surgery or in postoperative care. results: the group included patients, with an average bmi of [ kg / m . average hospital stay was days, with an average of . days which increased to days when fistulas were encountered. the most frequent surgical complications were bleeding from the gastric suture ( ) and gastric fistula ( cases). other complications encountered were wound hematoma. surgery was required in of the cases of bleeding and of the fistula cases required reintervention. one case was resolved with endoscopic stenting. conclusions: laparoscopic gastrectomy is considered a safe procedure with good results for the patient. although complications are rare they pose a series of technical difficulties for the surgeon due to the weight of the patient and frequent comorbidities which come with obesity. a thorough understanding of the symptoms and good follow-up ensures the best results. aims: to achieve additional weight loss or to resolve band-related problems, a laparoscopic adjustable gastric banding (lagb) can be converted to a laparoscopic roux-en-y gastric bypass (rygb). there is limited data on the feasibility and safety of routinely performing a single-step conversion. we assessed the efficacy of this revisional approach in a large cohort of patients operated in a high-volume bariatric institution. to the best of our knowledge this series represents the largest single-center study on conversion from lagb to rygb methods: between october and december , a total of patients who underwent lagb removal with rygb were identified from a prospectively collected database. in all cases, a single-stage conversion procedure was planned. the feasibility of this approach and peri-operative outcomes of these patients were evaluated and analyzed. results: a single-step approach was successfully achieved in ( . %) of the patients. during the study period, there was a significant increase in performing the conversion from lagb to rygb single-staged. no mortality or anastomotic leakage was observed in both groups. only patients ( . %) had a -d complication: most commonly hemorrhage (n? = ? / ), with no significant difference between the groups. conclusion: converting a lagb to rygb can be performed with a very low morbidity and zero-mortality in a high-volume revisional bariatric center. with increasing experience and full standardization of the conversion, the vast majority of operations can be performed as a single-stage procedure. only a migrated band remains a formal contraindication for a one-step approach. surg endosc ( ) . six months after surgery the mean hrql score, was ( - ) in patients underwent to lsg and . ( - ) in patients underwent lgb. twelve months after surgery the mean postoperative questionnaire score was ( - ) in patients who underwent lsg. at ph-manometry the mean percentage time of acid reflux in orthostatism was . (range - . ) and in clinostatism . (range . - . ). the mean demeester score at the distal electrode was . ( . - . ). conclusions: in asymptomatic patients, complete gerd evaluation before bariatric surgery allows better selection of surgical procedure, to reduce the postoperative occurrence of severe or de novo gerd. postoperative gerd evaluation provides useful data regarding the impact of lsg on gastroesophageal reflux. a larger patient sample size is required. aims: vertical calibrated gastrectomy (usually know as gastric sleeve) as unique technique gives better results than the roux y bypass in terms of improvement of anthropometric measures, reduces comorbidities and has a lower rate of postsurgical complications, with an improvement of quality of life. material and methods: an observational, longitudinal, retrospective and comparative study with patients, aged - years,during a period of years. everyone must comply with the protocol of the unit. demographics of the population and the anthropometric data will be measured in the presurgical consultation, the month and the year after the surgery: weight, height, bmi, weight loss percentage,bmi percentage and percentage of excess weight lost. we took data on the cardiovascular risk by the framingham score. the quality of life is measured by baros scale. mayor comorbidities are hypertension, diabetes, dyslipidemia. complications will be measured in absolute frequencies. for de statistical study, we apply type t student or chi square being statistically significant p equal to or less than . . results: there was not statistically significant difference between the techniques of surgery month (p = , ), but they were evident to the year of the same (p . ). not gender or age differences were apparent. mayor complications did not appear in gastrectomy (no leaks), highlighting the number of bleeds with this surgical technique. the bypass there were two leaks. there was no statistically significant difference in cardiovascular risk (p = , ) between the two techniques. there was a more significant decrease in number of comorbidities in gastrectomy against the bypass, with a total disappearance of patients with dyslipidemia. there were no statistically significant differences in baros score, although it was higher in gastrectomy. conclusions:-the vertical gastrectomy as unique technique can be considered superior in the short term, as well as safe, according to the aec quality parameter. we think it will be necessary to continue their studies into the medium-long term. aims: analyze the impact of different bariatric surgeries technics in carbohydrate metabolism and pancreatic beta cell population of none obese adult wistar rats. methods: we used twenty healthy not obese adult wistar rats divided in five groups randomly assigned. each with n = . the control groups were divided into fasting control (f) and sham (surgical control). the surgical groups were separated into vertical gastrectomy (gs), % resection of the middle small bowel (ri ) and gastric bypass (gb). in each group was assessment: beta cell mass modifications, pancreatic islets histomorphometry, proliferation, apoptosis and neogenesis in beta-cell pancreatic population; intraperitoneal glucose test tolerance, body weight and food intake. statistical analysis as evaluated using mann whitney test. results: the malabsorptive and restrictive group have a significantly smaller increase weight than the control groups. the intraperitoneal tolerance glucose test reports incremental glucose area under curve (auc) was significantly higher in the malabsorptive group and lower in the restrictive group compare to the control groups during the second (p \ . ) and third (p \ . ) month of the study. the beta-cell mass was significantly higher in the ri group compared with control groups respectively. there was a significantly increased number of beta-cell per pancreatic insulin positive area in gs and gb. proliferation was significantly increased in ri and gb group, and significantly decreased in sg compared. there was no significantly difference during apoptosis assessment among surgical and control groups. in neogenesis differences between groups were assessed qualitatively by the presence pdx - expression, being higher in rygb. the endocrine pancreas in our model is altered by the anatomical and functional conditions arising from surgical techniques. carbohydrate metabolism conditions are affected by temporary adaptive processes due to surgical alternatives. there is a hyperplasia and hypertrophy of the beta cells in surgeries with a malabsorptive component, as well as greater neogenesis. these results could explain part of the existing relationship between the enteropancreatic axis and the existing incretins. m. buza, c. copaescu introduction: nowadays, we have high volumes of obese patients for whom surgery is the answer, but unfortunately the psychological evaluation has no standard recomandation in preoperative evaluation of bariatric patients. it is argued that surgery success, in addition to the operation itself, relies on behavioral changes and that one of the goals of the preoperative assessment is to prepare the patient for the postoperative period, aiming to optimize surgical results. aim: although no formal standard exists in the literature, there is growing recognition of the important elements to be addressed and the appropriate means for collecting the necessary data to determine psychological readiness for these procedures. methods: information regarding the components of the clinical interview and the specific measures used for psychological testing are discussed. given the limited data on predicting success after surgery, determining psychological contraindications for surgery is addressed. additionally, the multiple functions served by the psychologist during this assessment procedure are highlighted along with the value of this procedure in the patients' preparation for surgery as well as the postoperative follow-up. in our center of excelence for bariatric and metabolic surgery (coe) we introduced since a mandatory pre-and postoperative psychological evaluation for all patients addressing the metabolic program. results: psychological evaluation of patients before bariatric surgery is a critical step, not only to identify contraindications for surgery, but also-and more so-to better understand their motivation, readiness, behavioral challenges, and emotional factors that may impact their coping and adjustment through surgery and the associated lifestyle changes. postoperative follow-up is necessary. the psychological evaluation of the patient undergoing bariatric surgery is an invaluable piece of the larger pre-and post-surgical assessment, aiming better results in the short and long term after bariatric surgery. introduction: a mesenteric cyst is defined as a benign abdominal tumors that is located in the mesentery of the gastrointestinal tract, identified in * of , hospital admissions. mesenteric chylous cysts are rare pathologic entities that often present with unspecific symptoms. the preoperative diagnosis requires all the common abdominal imaging techniques. usually the correct diagnosis may be made only at the operation stage or during the histological examination. all mesenteric cyst should be resected in order to avoid their complications, complete surgical resection is recommended and curative in the majority of cases with a low risk of local recurrence. the laparoscopic approach is the gold standard in the treatment of intraabdominal mesenteric chylous cyst. laparoscopic resection provides less pain, shorter hospital stay, and early recovery for the patient. case report: we report a case of -year-old saudi woman who presented to our clinic complaining of upper abdominal pain and mass in the epigastrium for one week, no history of nausea, vomiting, or recent changes in bowel habits. her medical and family histories were clear and she had never had any abdominal interventions. abdominal palpation revealed a smooth-surfaced mass palpable in the left upper quadrant, ultrasonography and with computed tomography of the abdomen revealed an approximately mm unilocular cyst closely related to the mesentery in the left side of upper abdomen not related to the pancreas .the cyst was excised by laparoscopy complete surgical excision to avoid recurrence within healthy borders, it is contained milky white fluid. the histopathological findings were chronic inflamed mesenteric cyst. a review of the literature considering this rare entity was also performed to evaluate our treatment strategy. conclusion: mesenteric chylous cysts represent a diagnostic challenge and they should be considered when a physician encounters an intraabdominal mass. usually the correct diagnosis may be made only at the operation stage or during the histological examination. the treatment of choice is the complete surgical excision that can be safely performed by laparoscopy. surg endosc ( ) background: diverticulum of appendix is relatively rare, and appendiceal diverticulitis was reported to have a higher risk of perforation than appendicitis. in the us and europe, because of the high risk of perforation, preventive appendectomy is recommended to appendiceal diverticulosis, even if the patient has no abdominal pain. methods: we retrospectively reviewed the records of post-operative patients, who were diagnosed appendicitis or appendiceal diverticulitis on the pathological findings in our institution from january to october . all patients were performed computed tomography (ct) before operation. patients underwent laparoscopic surgery, including appendectomy, cecal resection, ileocecal resection and right hemicolectomy, while patients underwent open surgery. total of cases of appendiceal diverticulitis were analyzed in our study. result: patients had abdominal pain before surgery. patients were diagnosed appendiceal diverticulitis by preoperative ct. all patients underwent laparoscopic surgery ( appendectomy, cecal resection, and ileocecal resection). on the pathological findings, perforation of appendix was found in patients and the pseudo type of diverticula with no muscle layer was found in all patients. patients with appendicitis were treated surgically during the same period. among them, a perforation of appendix was found in cases. the perforation rate was . %. on the other hand, the perforation rate of appendiceal diverticulitis was . % in our study. conclusion: the perforation rate of appendiceal diverticulitis was higher than of appendicitis in our study. for the examination of the treatment strategy, including preventive appendectomy, the accumulation of more cases will be expected. case presentation: a -year-old man was referred to our hospital with right lower quadrant abdominal pain for days. his fever was . °c. his white blood cell count was , / ll, and c-reactive protein level was . mg/dl. ct revealed multiple diverticula of cecum and appendix. micro-abscess and free air were found around appendix. we diagnosed this case as appendiceal diverticulitis and laparoscopic appendectomy was performed. a perforation was found in resected appendix. microscopic study revealed a pseudo-diverticulum. the inflammation of appendix was stronger in serous membrane side than in mucosa side. this finding accorded with appendiceal diverticulitis. introduction: in order to reduce the abdominal trauma and the length of scar incisions (also during laparoscopic surgery) many approaches during the last decade has been proposed, such as single access laparoscopic surgery (sals). the aim of our paper was to update the data of our previous paper with a greater cohort of patients and a longer follow-up, also showing the single access laparoscopic left colectomy (salc) technique in particular with inferior mesenteric artery preservation imap (valdoni's technique). materials and methods: we made a retrospective analysis from october and october of all patients who underwent a sals approach for colorectal disease in the department of general and mininvasive surgery of san camillo hospital of trento. statistical analysis was performed using ibm spss statistics . continuous data were expressed as mean ± standard deviation (sd). categorical data were expressed as absolute number and percentage. the results are presented as -tailed values with statistical significance if p values \ . results: from october until october , salc for colorectal surgery were performed in our unit. of this , were for left colectomy. in cases we performed an imap. the salc with imap were performed only in case of benign disease. the mean operative time was . ± . . only one intraoperative complication were recorded, that was a splenic capsule tear, resolved with apposition of fibrillar haemostats. according to clavien dindo classification there were in particular grade ii complications, a bleeding solved with blood transfusion and one pancreatitis solved with medical therapy; grade iiia complications that was anastomotic bleeding solved endoscopically (the two complications raised in patients with imap) and iiib complications due to anastomotic leakage which needed reoperation. the mean length of incision was . ± . cm. logistic regression did not show any correlation between imap and any complications. conclusion: in conclusion, salc is a safe but very challenging technique which need a longer learning curve than the conventional laparoscopic one. in laparoscopic colectomy, also, imap seems to be safe and effective without correlation with post-operative complications also if performed in single access laparoscopic approach. aims: to describe an infrequent anatomical variation that can give rise to diagnostic and therapeutic difficulties. methods: patient with ivermark syndrome (situs ambiguus and polysplenia) with acute appendicitis and bibliographic review results: a -year-old male who consulted for flank and right hypochondrium pain of h of evolution, associated with nausea without vomiting, no fever noir other symptoms. to the physical examination good general condition. painful to palpation selectively on the flank and right hypochondrium, with involuntary defense and positive decompression at this level. the signs of rovsing and psoas were negatives. in the analytical performed leukocytes of , with neutrophils in % and rpc (reactive protein c) in mg/l. abdominal ct (computed tomograph): cecum and the ilio-cecal valve were visualized at the subhepatic level with tubular structure on the side and seemed to correspond to the cecal appendix which is increased in size ( mm), with findings suggestive of acute appendicitis. sigma and descending colon located in right hemiabdomen. second per duodenal portion located anterior to the superior mesenteric artery. superior mesenteric vein located to the left of the superior mesenteric artery, rotating around it, (radiological signs compatible with intestinal malrotation). no free fluid collections nor pneumoperitoneum. laparoscopic appendectomy on phlegmonous acute appendicitis without incidents. correct post-operative course, being discharged at h; the pathological anatomy was reported as acute appendicitis in phlegmonous. conclusions: ivermark syndrome is a genetic alteration with a multifactorial inheritance pattern, characterized by an alteration in the situation of the mesenteric vessels, which leads to abnormal rotation of the intestine during the embryonic period and alteration of the situation of different intra-abdominal organs, without a specific pattern that is pathognomonic, is associated with congenital heart anomalies between and %. reaching adulthood only between and % of them. a case of acute appendicitis is presented in a patient with this anomaly, which can lead to diagnostic and therapeutic difficulties due to the anatomical variations involved. abdominal tomography is the image method that provides the best performance for the diagnosis of acute pathologies in this type of patients. background: the clinical manifestations which occur in relation to decompression during scuba diving are variable. mild symptoms have often been reported in gastrointestinal tract. this is one of the severe cases with gastrointestinal barotrauma. ischemic colitis caused by air embolism very rare, therefore it is to be reported and discussed. case presentation: a -year-old man visited our emergency room with diffuse abdominal pain and bloody diarrhea days ago. the patient was a skilled diver who took seafood through diving for years. two days before presenting, the patient had severe abdominal pain just after diving for h at a depth of meters. he was immediately transferred to a local hospital for hyperbaric oxygen therapy, but there was no improvement with the symptom. abdomen ct angiography showed terminal ileal, ascending, sigmoid colonic and rectal decreased enhancement with wall thickening. sigmoidoscopy showed diffuse huge ulcerative lesions and ischemic changes on mid rectum and sigmoid colon. emergent subtotal colectomy and temporary loop ileostomy were done, and pathologic findings revealed diffuse mural infarct with serosal abscess formation in whole colon and transmural infarct in terminal ileum. conclusion: surgical approach could be one of the treatment options, though it depends on severity of the symptoms and the patients' conditions. colonic lipomas are extremely uncommon benign tumours, with an incidence ranging between . % and . %. although they are most frequently asymptomatic, when colonic lipomas are [ ?cm, they may present symptoms such as constipation, abdominal pain or rectal bleeding. most colonic lipomas typically occur in middle aged women and are located in the ascending colon and the caecum, while occurrence in other parts of the colon and rectum is rare. in this case report, we describe a lipoma that caused descendent bowel intussusception. a -year-old male presented with longstanding history of constipation. personal history of interest included active smoker, hypertension, hypercholesterolemia, psoriasis with joint affectation and reiter syndrome. he had had no previous surgery. he attended the emergency services on th july with a two-day bowel obstruction, without fever or nausea, being attended by our surgical emergency unit. he had been assessed during the previous months by gastroenterology, with a colonoscopy that showed a cm submucosal lesion that partially occluded descendent bowel, with inconclusive biopsy. an abdominal contrast-enhanced computed tomography (ct) was performed, confirming a welldefined mass located in splenic flexure of descendent bowel, conditioning a large bowel intussusception, nevertheless with no obstructive acute signs. the surgery was scheduled a few weeks later, performing a laparoscopic segmental resection with primary anastomosis including oncologic margins. the patient evolved satisfactorily in the postoperative period and was discharged six days after the surgery without any complications. likewise, he was monitored on a regular basis at our outpatient department and was free of symptoms at the -month follow-up visit. the histological analysis revealed a cm ulcerated lipoma affecting % of bowel circumference. the molecular study, using fluorescent in situ hybridation (fish) showed no mdm gene amplification. laparoscopic segmental resection of the large bowel is a safe and feasible technique for the treatment of large bowel intussusception caused by a colonic lipoma. the complete removal of the lipoma will condition the prognosis. furthermore, in the future, endoscopic surgery using colonoscopy could be employed when having a certain preoperative diagnosis of lipoma. surg endosc ( ) introduction: acute appendicitis is one of the most common abdominal surgical emergency, the diagnosis of which mostly relies on conventional methods such as physical examination and blood tests. the use of ultrasonography and ct abdomen aids in more precise diagnosis especially in patients with atypical presentation or in elderly. aim: this study aims to evaluate the ability of the neutrophil/lymphocyte ratio (nlr), platelet/lymphocyte ratio (plr) and mean platelet volume (mpv) in predicting the diagnosis of acute appendicitis. methods: retrospective analysis of prospectivly maintained data of all patients ( ) admitted with acute appendicitis to the emergency department at a tertiary hospital in the middle east between january till september . medical records and database of patients,who had appendicectomy for clinically and radiologically proven appendicitis, were reviewed. the retrieved data included patient's demographic and laboratory values of white blood cells (wbc), neutrophil (n), lymphocyte (l), and platelet (p) along with their ratios for comparison. results: spss version was used for tabulating the data. the recommended cutoff value of the nlr, plr and mpv in predicting the diagnosis of acute appendicitis was decided by using receiver operating characteristic (roc) curve analyses. at least for nlr, the confidence interval (ci) was . which is percentage of the positive values, since the confidence limit was between to %. our results showed that the laboratory parameters were fairly significant since the confidence interval was . in predicting the diagnosis in our population. conclusion: although appendicitis is a clinical diagnosis but laboratory parameters specially nlr, plr and mpv can be used as an adjunct in the diagnosis of acute appendicitis. literature is scarce concerning the validity of such parameters in our part of the world and prospective randomized controlled trials are needed to prove the efficacy of such rationale. objective: tumors of the cecal appendix represent a subset of colonic neoplasms whose early diagnosis is a real clinical challenge. correspond to . % of all gastrointestinal tumors and their prognosis depends on the type of injury, being the most frequent variety the carcinoid type. appendix involvement in endometriosis is rare, accounting for % of all endometriosis cases, and sometimes mimicking cecal tumors. methods: a -year-old woman with a history of hypothyroidism due to autoimmune thyroiditis and atrophic gastritis with gastric neuroendocrine tumors resected by endoscopy that in the digestive unit reviews, tac with double contrast was requested, showing a lobulated lesion in the cecum adjacent to the ileocecal valve, with contrast enhancement of approximately mm, suggestive of tumor. the colonoscopy evidenced a protruding appendicular osteum with inflammatory aspect that was biopsied. the pathological anatomy of the biopsy reports chronic congestive colitis with edema of the own blade and minimal acute activity, with moderate local eosinophilia.the case was presented in the multidisciplinary oncology committee and it is decided, due to the patient's background, to perform surgery on the lesion. laparoscopic right hemicolectomy was performed, with extracorporeal latero-lateral mechanical anastomosis with endogia signiaÒ mm. results: the patient evolves favorably, with good oral tolerance and depositional habit. she is sent home at the sixth postoperative day. the pathological anatomy reports tumor injury in the appendicular ostium compatible with endometriosis at the base of the cecal appendix implantation, ruling out malignant tumor pathology. conclusions: gastrointestinal tract endometriosis represents - % of cases, being most frequently located in the rectal-sigmoid region. appendix involvement in endometriosis is rare, accounting - % of all endometriosis cases and presents a preoperative diagnostic challenge, because sometimes mimicking a carcinoid cecal tumor. in our case, due to the patient's history, we assumed that the cecal lesion was a carcinoid tumor, so we performed a laparoscopic right colectomy, but if we had known that it was an endometriosis, we could have performed an appendectomy, although in both cases the laparoscopic approach gives us some benefits compared to the open approach aims: the natural history and predictive factors associated with chronic anastomotic complications have not been clearly studied. the aim of this study was to evaluated the predictive factors related to chronic anastomotic complications methods: from january to december , a total of patients who underwent anastomotic leakage were enrolled in this study. all patients underwent anterior resection with or without defunctioning stoma due to colorectal cancer. the patients received follow-up by clinical examination and abdominopelvic computed tomography (ct). they underwent a follow-up ct every months for the first year and then every months for the next years after that. complicated group (cg) underwent chronic anastomotic complications. normal group (ng) didn't underwent chronic anastomotic complications like stricture, fistula, chronic sinus, etc. results: there were no significant differences in gender, age, preoperative chemoradiotherapy and operation type between two groups. low rectum lesion and defunctioning stoma at the time of primary surgery were more frequent in cg (p = . , . ). there were no significant differences in type of anastomotic leakage, international leakage grade and ct findings at the time of diagnosis of anastomotic leakage. however, abnormal ct findings at the time of month were more frequent in cg group (p \ . ). in multivariate analysis, abnormal ct finding at the th months was only significant factor related to chronic anastomotic complications. conclusions: abnormal ct findings at the th month associated with prediction of chronic anastomotic complications. aims: acute appendicitis is the most common cause of acute abdomen requiring surgical intervention in the world. nowadays, standard treatment of acute appendicitis involves a surgical approach, eitherlaparoscopic or open.the purpose of the present study is to evaluate the safety of a discharge within less than h after performing appendectomy as a result of an uncomplicated acute appendicitis. conclusions: patients who undergo appendectomy (open or laparoscopic) for acute uncomplicated appendicitis, without surgical incidents and an adequate social/family network, can be discharged in less than h without a higher risk of post-operative complications or readmissions than patients with longer postoperative stays. it will be necessary to conduct more prospective studies with higher level of evidence that could corroborate our results. aims: median arcuate ligament syndrome (mals), also known as the celiac axis compression syndrome, is a rare condition caused by to the compression of the celiac trunk and the nerves located in this area (celiac plexus) by the median arcuate ligament. it is believed that mals is caused by the median arcuate ligament compression of the celiac plexus nerves over the celiac trunk, but another probably cause may be the lack of blood flow to the organs supplied by the celiac artery, however, this theory is controversial. the first clinical sign of mals is the apparition of postprandial abdominal pain in the upper abdomen. this typical pain forces patients to avoid eating, which can lead to loss weight (often more than pounds). other associated symptoms may include nausea, diarrhea, vomiting and delayed gastric emptying (a delay in food moving from the stomach into the small intestine). in relation to this uncommon condition, we present a clinical case of laparoscopic management of mals. methods: we present a -year-old patient with complaints of recurrent epigastric pain, postprandial vomiting and loss weight. blood tests and gastroscopy were performed to help ruling out more common causes of his symptoms, such as gastroesophageal reflux disease (gerd), gastritis or gastroparesis. as a part of the differential diagnosis, mals was suspected and a mesenteric doppler ultrasound was ordered to check blood flow through the celiac trunk and evaluate a possible compression of the celiac plexus. also, an angio-ct scan was also performed to confirm the diagnosis. once the mals was diagnosed, we decided to perform a laparoscopic approach as definitive surgical procedure. results: the patient was discharged h after surgery with no remarkable events during his postoperative stay. he has been followed up during months, remaining asymptomatic. conclusions: laparoscopic approach in mals offers a superior visualization during the surgery and involves lower morbidity in compare to open approach, which makes it an optimal treatment for this condition. aim: pilonidal sinus is a common disease with annoying and often painful symptoms. traditional surgical techniques for its treatment are characterized by either intense postoperative pain and prolonged wound-healing periods (wide resection, marsupialization) or unsatisfying aesthetic results (advancement or rhomboid flaps). 'endoscopic pilonidal sinus treatment' (epsit) is a new minimally invasive technique which utilises the meinero scope, primarily designed for the endoscopic treatment of complex perianal fistulas in a technique known as vaaft. we present our experience and outcomes in three treatment centers in northern greece. methods: between july and november we treated patients with pilonidal sinus using the epsit technique. the mean age of patients was , and % of them were male. patients were treated in the acute phase with the presence of pilonidal abscess. all operations were performed by two laparoendoscopic surgeons specifically trained in the technique. most patients were treated on a day-case basis. postoperative wound care included daily tract irrigation with ml of saline for a total of days. results: there were no immediate postoperative complications. medium postoperative pain was . on a vas scale. % of patients were discharged on the same day, patients remained in hospital for one day mainly due to social reasons. return to daily activities was immediate. in a maximum follow-up of months we observed recurrences. conclusions: epsit is a promising minimally invasive technique for the treatment of pilonidal sinus. what makes it mostly attractive is the minimal amount of postoperative pain, the excellent cosmetic result and the fast recovery with return to daily activities. introduction: isolated acute chylous peritonitis is a rare event. when presented as an acute abdomen warranting surgical intervention, it is often difficult to determine the cause pre-operatively. here, we report a case of acute chylous peritonitis due to meckel's diverticulitis presented with the clinical features suggestive of acute appendicitis. presentation of the case: a -year-old female presented with abdominal pain and clinical features consistent with acute appendicitis underwent diagnostic laparoscopy. she was found to have four-quadrant chylous peritonitis and ileus caused by an inflamed meckel's diverticulum adhered underneath a loop of small bowel and mesentery leaking chyle. after uneventful postoperative recovery, she was discharged at post-operative day two with oral antibiotics and was advised to take a low-fat diet. aims: perforated diverticulitis with purulent peritonitis (hinchey iii) has traditionally been treated with surgery including colon resection and stoma (hartmann procedure) with considerable postoperative morbidity and mortality. laparoscopic lavage has been suggested as a less invasive surgical treatment. methods: a -year-old woman with a -day history of abdominal discomfort exacerbed during the last h. ct scan showed neumoperitoneum accompanied by free fluid and a cm collection adjacent to descending colon showing diverticula suggestive of covert perforation. after h of non-response to medical treatment, associated with the impossibility of percutaneous drainage through interposition of intestinal loops, colon and lumbar vessels, urgent surgical intervention is decided. results: laparoscopic lavage of all quadrants was performed with saline, l or more, of body temperature, until clear fluid was returned. two non-suction j-pratt drains were placed. intravenous antibiotics were continued for a minimum of h, then oral antibiotics were continued for week. oral fluids were commenced on the first postoperative day and solids were subsequently introduced, depending on clinical progress. conclusion: laparoscopic management is reasonable alternative to the traditional open resection for hinchey grade ii-iii perforated diverticulitis with generalized peritonitis. this approach has a low mortality rate despite patient co-morbidity and disease severity. benefits include stoma avoidance and minimal wound infection. subsequent elective resection is probably unnecessary and readmission in the medium term is uncommon. background: constipation and fecal incontinence are common annoying complications after pull through procedures for hirschsprung disease (hsd). many causes could be the etiology of these problems. perineal descent syndrome could be the major hidden cause of these complications. the aim of this study is to evaluate the role of perineal descent syndrome in the development of post pull through constipation and fecal incontinence in addition to evaluate the role of laparoscopic rectopexy for treatment of these problems. \ b[patient and methods: \/b [ patients treated with pull through for hsd over the period of five years. out of the patients presented with constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. rectal exam, anorectal manomety, defecography, contrast enema, rectal biopsy, emg, proctoscopy and endorectal ultrasound were performed to all patients. patients with stricture, missed aganglionic segment, injured internal anal sphincter, and loss of the sensory mucosa above the dentate line were excluded from the study. anterior wall rectopexy was performed for anterior wall rectocele. posterior wall rectocele was treated by retro rectal mesh rectopexy. emg is repeated weeks and months after surgery. outcome measurements included constipation, fecal incontinence and pudendal nerve latency. results: cases of post pull through constipation and fecal incontinence. patients with constipation and patients with fecal incontinence. patients with stricture, patients with missed aganglionic segment, patients with loss of anal sensory sensation and patients with injured anal sphincter were excluded from the study. defecography showed patients with anterior rectocele ( males and females) and patients with posterior rectocele ( males and females). the patients mean age . ± . years . emg showed prolonged pudendal nerve conduction in all cases. anterior wall and retro rectal rectopexy were performed laparoscopically without complications. constipation was resolved in all patients after surgery. all patients showed fully control in defecation. pudendal nerve latency decreased in all patients. conclusion: perineal descent syndrome proved to be a major hidden cause of post pull-through constipation and fecal incontinence. laparoscopic rectopexy showed a good solution of these complications. cystic lymphangioma is a rare entity. the surgical indication is determined by the size and symptomatology, and consists of the complete exeresis of the tumor. the laparoscopic approach is feasible in these cases, allowing a broad visualization of the anatomy, accessibility to the retroperitoneum in the context of a minimally invasive approach and a better recovery of the patient, without providing an increase in morbidity compared to the conventional. in this way we defend as a technique of choice laparoscopic surgery against these rare tumors for the general surgeon in the abdominal cavity, betting on a minimally invasive surgery. aims: laparoscopicposterior sutured rectopexy is one of the accepted treatment options for fullthickness rectal prolapse. recently, reduced port surgery(rps) has beenan emerging concept that, compared with conventional multiple port surgery (mps), yields reduced postoperative pain and improved cosmesis. the aim of the study is to evaluate the feasibility and safety of rps for fullthickness rectal prolapse. methods: rps was performed by single-incision plus one puncture, using internal organ retractor(ior) to secure operative field. straining one ior by - strings in - directions makes it possible to retract the internal organs three-dimensionally. this multi-directional flexible retraction could secure good operative field. from to , patients (rps: cases, mrs: cases) underwent laparoscopicposterior suture rectopexyfor total rectal prolapse. shortterm outcomes were compared between the two procedures. results: there was no significant difference between rps and mps in median operative time ( vs . min, respectively, p [ . ). the median blood loss volume was not significantly different between rps and mps groups ( . vs . ml, p \ . ). the duration of median hospital stay after surgery was not significantly different between two groups ( . vs days, respectively, p [ . ). the frequency of complications after surgery were not different between them. conclusions: reduced port lap-rectopexy can be a good therapeutic option for total rectal prolapse. a prospective, randomized, controlled trial should be conducted to confirm the superiority of this procedure over mps. the piccolo project proposes a new compact, hybrid and multimodal photonics endoscope based on optical coherence tomography (oct) and multi-photon tomography (mpt) combined with novel red-flag fluorescence technology for in vivo diagnosis and clinical decision support. for its development it includes different phases of validation. within this framework, the present study has as main objective: to characterize a model of rat colonic hyperplasia, which will be used for the development and validation of the previously mentioned endoscopic technology. secondary objectives: procure the reproducibility of the model chosen and determine the optimal time, after induction of the model. material and methods: animals (rattus norvegicus), wistar, males and females \ -yearold, randomly distributed. group (n = ): by laparotomy, a non-resorbable suture (silk / ), not stenosing, is placed through the wall of the colon. group (n = ): by endoscopy, a . mm long segment of a polymeric catheter is inserted, which is fixed to the wall of the colon by means of a suture. group (n = ): by means of endoscopy, a self-expanding and uncoated metallic stent are placed in the colon. group (n = ): a superficial laser resection of the colonic mucosa is performed by endoscopy. group (n = ): as an extension of the most optimal model. weekly, the animals were anesthetized again to perform a colonoscopy, which determined the degree of mucosal growth in descending colon and colonic biopsies were extracted weekly ( weeks). results: group . growth around the sutures after the second follow-up, diagnosed as hyperplastic polyps after a histopathological analysis. aim: the role of laparoscopy in the management of generalized appendicular peritonitis is controversial. this is due mainly to the lack of scientific data. through this study and a laborious bibliography research, we proposed to report our experience in terms of postoperative results, in the laparoscopic treatment of generalized appendicular peritonitis and to try to identify the risk factors associated with the occurrence of global morbidity and conclude on the feasibility of this technique in its treatment. methods: we conducted a retrospective study including all cases of generalized appendicular peritonitis managed laparoscopically, in the general surgery department of charles nicolle hospital between january and december . results: we identified patients. the mean age was . years. one fifth of the cases required a midline conversion ( . %). the mean operative time was . ± , min. the overall morbidity rate was % including surgical complications. there were no deaths. in uni-variate analysis, comorbidity, crp [ mg /l, operative time exceeding min and midline conversion were significantly associated with postoperative morbidity. co-morbidity, diabetes, asa score [ , delay of consultation [ days, intra-abdominal abscess and operative time exceeding min were significantly associated with medical complications. the univariate analysis also revealed that crp [ mg /l and midline conversion were predictive of surgical complications.the multivariate analysis identified the midline conversion as the only independent factor significantly associated with post operative morbidity (odds ratio = . , % confidence interval [ . - . ] ). conclusion: based on our results, it appears reasonable to continue the laparoscopic management of diffuse appendicular peritonitis. however, enhance this technique is basic in order to reduce midline conversion rate and to shorten operative time, which can lead to post operative complications. aims: currently, acute appendicitis is the most common surgical emergency. laparoscopic appendectomy is the usual procedure to treat acute appendicitis. the aim of this study is to evaluate the safety of electrocoagulation in the treatment of mesoappendix in laparoscopic appendectomy. methods: we have retrospectively studied a prospective database of operated patients of appendecectomy in emergency surgery unit. we have reviewed laparoscopic appendectomies from june st, to december st, . the mesoappendix was electrocoagulated in every laparoscopic appendectomy. the statistical analyses has been done with spss . version. results: our group consists of patients of which . % were male and . % were female. the average age was . years with a standard deviation of . % and p was . years. the most common total stay was day ( patients). the usual post-operative stay was one day ( ). we classified the diagnosis in complicated apendicitis ( patients) and no complicated apendicitis ( patients). the conversion rate was . % ( ). the main surgical complications were: surgical wound infection ( . %); intraabdominal abscess ( . %); and bleeding ( %). only one of the patients that suffered bleeding had complicated appendicitis. the medical complications were catheter sepsis ( . %); respiratory infection ( . %); cardiologicals ( , %); and paralytic ileus ( . %). the treatment of mesoappendix with electrocoagulation is safe and effective since the complications rate is very low. even so, it would be necessary to conduct more prospectives randomized studies in order to get enough evidence about the treatment of mesoappendix with monopolar electrocoagulation. introduction: the difficulty of resection of the rectum is determined by its anatomical relationships, intimately in contact with the bladder, seminal vesicles, prostate and urethra in the case of the male, vagina in the woman and nerve structures that will give defecatory, genital and urinary functionality. this structure creates a big impediment due to problems of visualization and difficult dissection, in such a way that conventional surgical techniques instigates a series of complications derived from this difficulty. we propose a new approach in rectal surgery in patients with inflammatory bowel disease. material and methods: a -year-old man with a history of ulcerative colitis developed a severe acute outbreak refractory to treatment. a total laparoscopic colectomy with a terminal ileostomy was performed in . in he was notified for reconstruction. we evidenced a rectal stump of about cm with signs of inflammatory disease at the mucosal level. a transanal proctectomy was performed with confection of 'j-pouch' and ileoanal anastomosis about cm from the anal margin by laparoscopy. the postoperative courses favorably, being discharged on the sixth day. currently in follow-up in digestive and general surgery, he is asymptomatic and he has an optimum level of quality of life valued by the sf- weeks after the intervention. conclusions: our service introduces the transanal approach to the performance of proctectomy in cases of inflammatory disease, a technique that provides clear advantages by improving visualization and the identification of anatomical structures. in this way, a safe dissection of the pelvis is achieved, adjusted to the serosa of the rectum, with preservation of the mesorectum and the hypogastric plexus, and with the consequent improvement of the genital and urinary function. the result is an equally safe surgery, which implies little increase in operative time and with better and shorter postoperative recovery.the conservation of the pelvic innervation avoids disorders of ejaculation, vaginal lubrication and bladder and rectal motility. the transanal approach for the performance of proctectomy provides benefits in terms of the preservation of the hypogastric plexus, minimizing the anatomical difficulties involved in rectal surgery and maintaining urinary and sexual function. aims: to evaluate the feasibility and outcomes of laparoscopic appendicectomies in both simple and complicated appendicitis, given the increasing trend towards a laparoscopic approach in the last four decades for the treatment of acute appendicitis. we present data from a district general hospital over a -year period. methods: we retrospectively analysed a single consultant's continually updated database of laparoscopic appendicectomies between / / and / / ( months). patient demographics, investigations, intraoperative findings and postoperative outcomes were recorded and analysed. complicated appendicitis was defined as the formation of appendiceal mass or abscess with or without perforation and peritonitis. results: cases of laparoscopic appendicectomies were identified during the specified period. the median patient age was (range - years). true positive rates for uss and ct were % and %, respectively. the rate of negative appendicectomies was %. transanal minimally invasive surgery (tamis) has been used for the treatment of rectal neoplasms such us benign polyps and early rectal cancer. when the tumour is located in the upper rectum or close to the rectosigmoid junction, this approach may be technically dificcult.we present a video of a tamis resection of a large polyp located cm from the anal verge. after properative examination and ct and mri were performed, the patient was prepared for surgery, and a trasnanal minimally invasive surgery was proposed.resection of the polyp was performed with the aim of an endogia and conventional laparoscopic materials. total resection of the polyp with free margin was possible. the postoperative pathology report confirmed a high grade displasia villo-tubular adenoma with a lesion free margin. tamis resection of tumours located above the rectosigmoid junction may be a safe and feasible technique in selected patients. aims: pelvic organ prolapse (pop) is a very relevant problem for women's quality of life and has a prevalence of about % defined by symptoms and up to % when established by physical examination. nowadays, sacrorectopexy for posterior pop and sacrocolpopexy for apical pop are considered the gold standard techniques. recently, we have seen that laparoscopic lateral suspension is a feasible procedure for apical pop, obtaining a success rate higher than % at one year. these results are similar to what we can achieve with sacrocolpopexy. methods: we herein present the case of a -year-old woman with apical and posterior pop, this was provoking an important impact on her quality of life, with obstructive defecation (needing digitations) and urinary incontinence. we proposed sacrorectopexy for her posterior pop and laparoscopic lateral suspension for her apical pop. in the video we can see how we perform a ventral mesh sacrorectopexy, following d'hoore technique; and a laparoscopic lateral suspension with preperitoneal dissection, following the technique described by the team headed by dubuisson and veit-rubin. we used laparoscopic ports ( , , . and . mm). results: patient was discharged home on the second postoperative day and has not had any sign of recurrence or extrusion after more than two years of follow-up. in addition, she has not suffered lower urinary tract symptoms, constipation or pain. conclusions: we present a case in which we have carried out a laparoscopic lateral suspension instead of a sacrocolpopexy for an apical pop, obtaining good short-term and long-term results. we consider it is very soon to assess this technique's efficacy and it has to be validated in studies with larger source of patients. nevertheless, we think this procedure might become an excellent alternative to sacrocolpopexy for apical pop. aims: laparoscopy is a minimally invasive approach with low morbidity. the aim is to show the usefulness of the laparoscopic approach for massive intra-abdominal abscesses, which it is controversial. we report three patients who underwent emergency laparoscopy for peritonitis or massive intra-abdominal abscesses not amenable to percutaneous approach that were suspected to be caused by acute diverticulitis. methods: all patients had diagnosis of acute diverticulitis (hinchey ii-iii grade) with pelvic abscesses situated between sigma and bladder or diffuse peritonitis. the patients with hinchey ii grade had failed conservative management with antibiotics. they underwent emergency laparoscopy under general anaesthesia, with three abdominal ports. intra-abdominal abscess cavities were exposed and the purulent exudate was sampled and aspirated. copious irrigation was performed under direct vision and thorough examination without other findings. the procedure was completed laparoscopically in all cases. results: all patients had favourable evolution. one of them had a properly drained faecal fistula which changed to a purulent fistula on the twentieth postoperative day. this patient underwent laparoscopic left colectomy three months later because he had have a new episode of acute diverticulitis. other two cases showed very good clinical evolution, without evidence of fistula in postoperative period and they were complete asymptomatic one month later. conclusion: in our experience laparoscopic drainage is a feasible, safe, and effective for the treatment of pelvic abscesses and diffuse peritonitis secondary to acute diverticulitis. n. pinheiro, a. ziegler introduction: solitary rectal ulcer syndrome (susr) is characterized as a rare disease whose pathophysiology remains uncertain. it was first described in by cruveilhier and his clinicopathological feature was reported in by mandigan and morson, where he is associated with defective disorders, internal rectal prolapse, and psychological changes. according to works about % of the patients are asymptomatic. when symptomatic the diagnosis can be made through physical examination, clinical history and, often, confirmed by endoscopy with biopsies. treatment depends on the severity of the symptoms and the existence of associated rectal prolapse. according to the literature, conventional surgical options include local excision, rectal mucosectomy, retopexy, and segmental colonic resection. rolato: a -year-old male complaining of anal bleeding at bowel movements years ago. he performed, several times, conservative treatment, but without improvement. he sought proctological care and underwent colonoscopy, in which he showed an ulcerated lesion on the anterior wall of the distal rectum. new investigation with videodefecogram revealed colorectal intussusception with associated mucosal prolapse, being considered the factor causing the ulcer. elected by the sacropromontofixação. evoluiu with improvement of anal bleeding, mucorrhea and anal discomfort. after a proctological examination, which was normal, a control colonoscopy performed after months of surgery revealed rectal mucosa, with residual scarring and disappearance of the submucosal nodule present in the initial examination. reassessed after months, the patient is asymptomatic. conclusion: rectal solitary ulcer whose causal factor was a colorectal prolapse (intussusception) with mucosal exteriorization through the anal canal, which was individually treated with sacropromontofixation. j.p. mali, p.j. mentula, a.k. leppäniemi, v.j. sallinen approximately - % of patients diagnosed with colonic diverticulitis have an intra-abdominal abscess as a complication. abscess diameter of - cm is generally accepted as a cut-off determining the choice of treatment between antibiotics alone and percutaneous drainage. the aim of this study was to analyze the treatment choices and outcomes of patients with diverticular abscesses. this was a retrospective cohort study which was conducted in helsinki university hospital, an academic teaching hospital functioning as secondary and tertiary referral center. patients with computer tomography-verified acute left-side colonic diverticulitis with intra-abdominal abscess were collected from a database containing all patients treated for colonic diverticulitis in our institution during - . altogether, suitable patients were included in analyses. those treated primarily with percutaneous drainage or antibiotics alone ( and patients, respectively) were further compared in regards to treatment results. the main measured outcomes were need of emergency surgery and -day mortality. abscesses under mm were mostly treated with antibiotics alone with high success rate ( out of , %). in abscesses over mm, the use of emergency surgery increased and use of antibiotics alone decreased with increasing abscess size, but the proportion of successful drainage remained at - % regardless of abscess size (figure ). there were no differences in failure rate, -day mortality, need of emergency surgery, permanent stoma, recurrence, or length of stay in patients treated with percutaneous drainage versus antibiotics alone, even when groups were adjusted for potential confounders. white blood cell count = . * /l, abscess diameter = mm, and corticosteroid medication were independent risk factors for failure of treatment with antibiotics alone. patients without these risk factor had % and patients with one risk factor had % success with antibiotics alone. percutaneous drainage as treatment for large abscess does not seem to be superior to treatment with only antibiotics. majority of patients with abscesses over mm in diameter undergo surgery as primary intervention. introduction: even today, 'chronic appendicitis' is a clinical term that is not widely accepted nor well documented amongst the medical community. its etiology is the presence of a mass (e.g. fecal mass, hyperplasia of lymphatic tissue, etc.) that continuously and partially obstructs appendix lumen. it is presented as a low intensity, intermittent, with exacerbations and remissions, abdominal pain that is located at the right iliac region. the pain lasts up to several months and it is usually underestimated by the patient. its diagnosis is based on imaging examination. appendectomy is the treatment of choice for chronic appendicitis. the operation is challenging for the surgeon who has to cope with an intensively inflamed area around the appendix without the ease of access to that area. purpose: to present our laparoscopic approach to a chronic appendicitis case and to review the literature. case report: a -year-old woman is hospitalized due to chronic appendicitis. the patient was treated conservatively with the use of intravenous antibiotics in two separate hospital admissions dated and months back respectively. eight weeks after the last exacerbation, she underwent a laparoscopic appendectomy. results: even though the procedure was planned six months after the first episode, the laparoscopy revealed a severe inflammation of the appendix, which was extended to the caecum and the surrounding preperitoneal tissues. although the difficultness of the operation it was completed successfully laparoscopically. the histological examination confirmed without any doubt the existence of 'chronic appendicitis'. the patient was discharged uneventfully the third postoperative day. conclusions: chronic appendicitis is an existing clinical entity that the surgeon may come through during his career. in the hands of experienced laparoscopic surgeon, the laparoscopic approach is feasible and safe. introduction: ventriculo-peritoneal shunting (vps) used in the treatment for hydrocephalus is associated with several complications.the exact cause of such extrusion is not known. visceral perforation is an unusual but serious complication with consequeces such as peritonitis, meningitis or encephalitis. management involves prompt removal of shunt, intravenous antibiotics, an adequate recovery gap so that cerebrospinal fluid culture is sterile and then followed by shunt replacement on opposite side. aim: multidisciplinary approach of extrusion of vps through anus by laparoscopic and external ventricular drainage. case exposure: a -year-old woman had a vps inserted year ago after excision of gangliocytoma due to lhermitte-duclos disease. she was admitted in the emergency department without symptos after trans-anal protrusion of vps catheter. the neurological and abdominal evaluation was normal. laboratory tests did not reveal disorders and abdominal ct-scan suggested perforation, itshowed the insertion of the end of the catheter in sigma, without pneumoperitoneum or intraabdominal free fluid. cranial ct-scan did no describe sings of hydrocephalus. the patient underwent an emergencysurgical intervention. first of all, antibiotic therapy was initiated and neurosurgery's team was performed an external ventricular drain and they disconnected the proximal catheter side. after that, an exploratory laparoscopy was performed. it revealed a microperforation and collection beside to an appendix' base due to the proximity with the catheter. additionally, the catheter was freed from adhesions at the point of entry into the colon and after careful dissectionwe release the vps from colon with a . cm transmural trajectory at the sigmoid level. no free fluid was seen and rest of the bowel appeared normal. the distal end was removed through the anus and the proximal end through a laparoscopic port. we performed a laparoscopic segmental cecum resection and an extracorporeal colo-colonic anastomosis was performed for a mini-pfannestiel laparotomy of assistance. there were no complications in the postoperative period, being discharge on the th day. conclusion: the multidisciplinary approach and the laparoscopic support in the diagnosis and treatment of patients with colon perforation caused bay vps catheter is a feasible and safe option in third level centers. background: acute appendicitis continues to be the most common source of complicated intraabdominal infection worldwide. the high incidence of postoperative complications and dissatisfaction with the results of treatment in cases of complicated appendicitis and peritonitis gave the reason for conducting this study. aim: to evaluate the effect of different laparoscopic trocars position in case of laparoscopic appendectomy for diffuse appendicular peritonitis for the incidence of postoperative complications methods: the results of laparoscopic treatment of patients with acute appendicitis complicated by diffuse peritonitis were analyzed. the first group consisted of ( %) patients operated by triangulation access (type trocar placement according sages guidelines for laparoscopic appendectomy (sages qla). the second group consisted of ( %) patients operated by sectorisation access (type sages qla). postoperative complications were classified by clavien-dindo classification. results: the duration of the operation for the analyzed groups was . ± . vs . ± . min. there were no deaths among this group of patients. the incidence of postoperative complications for both group was . %. postoperative complications in the triangulation and sectorisation group were % and . % respectively (p . ). clavien-dindo iiib complications were noted in . % (n- ) patients and presented with intra-abdominal abscesses (iaa). all patients with iaa were operated in sectorisation group. conclusion: sectorisation trocar placement increases the incidence of intra-abdominal complications for laparoscopic appendectomy for diffuse appendicular peritonitis. introduction: the diverticular disease of the colon is a chronic entity with a variety of abdominal symptoms that can present with recurrent episodes of acute diverticulitis (ad). the prevalence of diverticulosis is not influenced by gender and increases with age, which, according to the increase in life expectancy, explains the accumulation of cases in western countries. the classic diagnostic-therapeutic algorithm of the disease is it has been based on the hinchey classification, the use of antibiotics and the intervention of hartmann (ih) at the acute time and elective colectomy in the multirecurrent cases. the use of laparoscopy with washing and drainage is actualymore extended in cases with peritonitis. objectives: to demonstrate the safety and efficacy of the laparoscopic approach, in cases with diverticular disease complicated by severe inflammatory plastron with 'covered' perforation, with several recurrent episodes. material and method: case report: a -year-old man with ap-diverticulitis years ago with complete resolution and normal control colonoscopy. he presents in the last two months three compatible episodes of acute diverticulitis, exploration with plastron-mass in hypogastrium without defense, tac-marked thickening of a segment of cms. of medium sigma, collection not drainable in mesosigma, of cm, which loses the plane of cleavage with loops of thin neighbors with a linear tract that suggests fistulization. evidence of interest is exposed. given the evolution, it is decided surgical elective treatment. result intervention: preoperative ureteral double catheterization, laparoscopic approach, is exposed by video, rectosigmoid resection by diverticular plastron, with negative io biopsy, mechanical colorectal anastomosis. good postoperative course, discaharge at th day. defini-tive ap: perforated diverticulitis, absence of malignancy. the laparoscopic approach is a valid and effective alternative in cases of complex and severe diverticular disease. aim: tamis resection has been described for the treatment of rectal neoplasms, wether benign or early malignant tumours. since tamis appearance, many different indications have been reported.we aim to show an special indication as seen in this video of a tamis resolution of a rectal stenosis non treatable by endoscopy. method: we present a video of a female patient, previously treated for a large rectal adenoma treated by trasnanal apporach, with a postoperative sepsis which required lateral colostomy and trasanal drainage. after surgery, the patient suffered from a rectal stenosis which couldn' t be solved by endoscopy, so the patient was sent back for a surgical treatment.we decided to performed a trasnanal apporach by tamis and a long and circunferiential stenosis around cm from the anal verge was seen.we performed a rectotomy by electrocautery in the posterior rectal wall until the perirectal fat was seen and the stenosis was passed. a dilatation with a foley catether was also performed. results: postoperative course was uneventful and after months she was prepared for colostomy closure with no complications and remains asymptomatic nowadays. conclusion: tamis approach of rectal stenosis may be a safe and feasible technique in selected cases if conservative treatments fail. iatrogenic endoscopic colon perforation it is a severe, but rare complication of colonoscopy. the incidence of this complication is estimated to be . - . % for diagnostic colonoscopies and . - % for therapeutic colonoscopies. the management of these complications depends on the size of the lesion,the time elapsed between the lesions were produced and diagnostic of the lesions and associated pathology. the treatment can be consevative,endoscopic or surgical(clasic/ laparoscopic) in our sevice in last years we treated cases with iatrogenic colon perforation after diagnostic colonoscopies. all lesions were at sigma level. one case was admission in our service at days after a diagnostic colonoscopy.the pacient was operated clasic,in emergency,we found a fecaloid peritonitis,a perforation at sigma level.we made a colostomy, lavage, drainage but the pacient died after days. in cases we made the operation at maximum h after the lesion was diagnosticated by the endoscopist(directly visualisation).we didn't made radiologic investigation.the pacients were operated laparoscopic,we made suture,lavage,drainage. evolutions of the pacients were good. conclusion: iatrogenic colonic perfortion are rare,but severe complication. laparoscopic surgery can be a choice in treatment of this complication introduction: complicated diverticulitis with fistula is responsible for about % of surgical procedures in diverticular disease and is commonly found in patients with diverticulitis of the sigmoid colon. colovesical fistulas are the most frequent ( %), with highest incidence in males. only a third of these patients have a history of diverticulitis. in most cases, treatment is surgical, and colectomy is performed, whether or not in association with vesical recession. case report: year old male with pneumaturia and fecaluria for the preceding months. the colonoscopy identified a diverticulitis of the sigmoid colon and the subsequent pelvic mri suggested a colovesical fistula. the cystoscopy was not able to identify any fistulous opening, but a double j catheter was placed in the left ureter, as surgical treatment had been proposed. a subsequently abdominal pain motivated a preoperative ctscan which revealed a pneumoretroperitoneum and a fluid collection near the left ureteral tract. the multidisciplinary team on the case decided to perform a percutaneous nephrostomy, followed by an exploratory laparoscopy. the fistula tract was identified and a laparoscopic sigmoidectomy with partial cystectomy was performed, as well as a ureterorenoscopy (with double j replacement). there were no intra or postoperative complications, and the patologic repport had no signs of malignancy. video of the surgical procedure is presented. conclusion: a laparoscopic approach to complicated diverticulitis with colovesical fistula is safe and effective when performed by experienced colorectal surgeons. introduction: diverticular disease is characterized by its high prevalence, being one of the most frequent causes for hospital admission when it comes to gastrointestinal pathology. even though it is more frequent in older patients, there has been an increase in incidence amongst lower age groups. the approach to the disease has also suffered changes in the last few years, showcasing a tendency for less invasive options, deferring elective surgery to later in the course of the disease. this study examines the therapeutic approach to diverticular disease in our hospital. methods: retrospective analysis of demographic data, therapeutic options and surgical outcomes in patients admitted for diverticular disease between january and june . results: patients(n = )were included in the study: ( %)were male and ( %) were female, with an average age of years. patients( %) underwent medical treatment, with surgery reserved for the remaining patients, of which were emergencies, with the other being elective( %).about % of patients were only admitted once, %were admitted twice and % had or more episodes. for the single-admission group, the most common treatment was medical( %of cases), as was the case for the group with or more episodes(in which %of cases were subjected to medical treatment). in the patient group with episodes, %were submitted for surgery, most of which elective.as far as the surgically operated group is concerned, no statistically significant differences were found with regards to patient sex. age, however, was significantly greater for the group that underwent emergency surgery vs that submitted to elective ( . years vs . years,p \ . ).the most common procedure overall was colic recession, with hartmann's operation standing out as the most frequent for the emergency surgery group. length of hospital stay was again higher for the emergency group (vs elective; vs ,p \ . ),as well as the morbidity rate. no statistically significant differences were found with regards to mortality rate. conclusion: knowledge of the natural history of diverticular disease led to changes in the approach to treatment, with a tendency to adopt a less aggressive therapeutic. despite controversy around aspects such as selection of patients for elective surgery, among others, it is key to the approach to diverticular disease that existing recommendations are taken into account, treatment is individualized and outcomes are closely monitored. surg endosc ( ) aims: the objective of this analysis is to establish if there is differences after the procedure of laparoscopy appendicectomy comparing the use of endoloop (el) vs. endostapler (es) in complicated and non-complicated acute appendicitis. methods: we performed a retrospective analysis of a prospective database of patients from february to june . we divided the patients in two groups: depending on that the procedure of the appendicectomy was with endoloop or endostapler. the groups were created selecting patients in order to be homogeneous as to perforation appendix rate, thus a propensity score was performed for sex, age and perforation rate. an univariant analysis was carried out in regard to the differences in the use of el vs es in the apparition of abdominal complication, as well as hemorrhage, ileo, surgical wound infection, collection, reintervention or hospital readmission.qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. introduction: intramural haematomas can develop anywhere within any the gastrointestinal tract . these are most frequently associated with blunt trauma above the level of the sigmoid colon and very rarely occur in the rectum . spontaneous, non-traumatic haematomas are a rare clinical condition usually secondary to haematological blood disorders or anticoagulant therapy . case summary: a -year old gentleman presented to the emergency department with a day history of worsening lower abdominal pain and bloody stool. he presented twice within the previous week with worsening, generalised abdominal pain. the patient had been taking regular aspirin and clopidogrel following insertion of coronary artery stents. on clinical examination, he was guarding with a distended, generally tender lower abdomen but all observations were stable, afebrile. an initial computer tomography of the abdomen reported pneumoperitoneum with haemorrhagic ascites; a differential diagnosis being perforated sigmoid colon with a large localised haematoma. the patient underwent an emergency laparotomy and hartmann's procedure (appendicectomy, sigmoid colostomy and rectal stump). he recovered well with no significant post-operative complications. histology reported the rectal perforation macroscopically associated with an opened haematoma and no evidence of malignancy. the appendix shows reactive appendicitis with serousal inflammation background: ulcerative colitis (uc) is one of the risk factor of developing sporadic colorectal cancer. approximately % of uc patients develop an acute attack of severe colitis, and % of these patients require colectomy. one third of the patients will not respond to steroid therapy. thus, a long-term follow-up has been recommended. case report: we reported a single case of completed years follow of colorectal cancer related ulcerative colitis on years old female patient undergoing emergency operation ( staged total colectomy and j-pouch ileo-rectal anastomosis) after year no responsed of medical treatment before, presenting with bloody diarrhea and anemia. there was no post operative complication reported. pathologic finding was early adeno carcinoma, closed follow up was done each year and for another five years later, no progression of the disease was found in this period and the patients has good quality of life after this procedures. is becoming a standard and feasible surgical method worldwide. % of patients with crohn's disease (cd) and % patients with ulcerative colitis (uc) will require an operation during their life. over the last decade, there have been many studies documenting the safety and feasibility of the laparoscopic approach for ibd in well-selected patients. methods: patients with a cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon and rectum stenosis, patients with uc with ineffective medical therapy, steroid dependence or dysplasia underwent the lcs. from to , ileocolic resections, hemicolectomies, subtotal colectomies and restorative proctocolectomies with ileopouchanal anastomosis were performed either totally laparoscopically or laparoscopically assisted (n = ).the average time of the procedure was min ( - min), average blood loss ml ( - ml) and the conversion to laparotomy was in . %. average return time of the bowel function was . days ( - days) and the average hospital stay was . days ( - days). complications occurred in patients ( . %). cases of the early ileus due to adhesions, cases of the anastomotic bleeding threated conservatively, case of the instrumental perforation of the small bowel, cases of the incisional hernia in minilaparotomy and wound infections occurred. conclusion: in well-selected patients with ibd, thanks to superior short-and long-term outcomes, the laparoscopic approach should be considered a safe and effective method when performed by experienced surgeons. supported by mo . aim: over % of patients with crohn's disease (cd) will require a surgical resection within years of their diagnosis and one quarter will have another resection for disease recurrence. laparoscopy should by preferred approach in surgery in cd due to reduced morbidity, faster recovery time, shorter hospital stay and reduction in adhesions and hernial formation. methods: patients with cd with the tight stenosis in the distal ileum and/or ileo-colon or various colon stenosis were indicated for the laparoscopy. from january to november we performed ileocolic resections, hemicolectomies and subtotal colectomies either totally laparoscopically or laparoscopically assisted. the average time of the procedure was min ( - min). the average return time of the bowel function was . days ( - days) and the hospital stay was from to days. complications occurred in patients ( . %) . in cases early ileus developed due to adhesions, in case was anastomotic bleeding threated conservatively, the incisional hernia in minilaparotomy occurred in cases and wound infections occurred. conclusion: minimally invasive surgery is becoming a gold standard in cd. it is safe and feasible in well-selected patients thanks to short-and long-term outcomes. laparoscopic approach for recurrent disease is still in debate. supported by mo . aim: the aim of the study was to observe when laparoscopy is avoided when treating surgical complications of crohn disease. methods: we did a retrospective study which included all of the patients diagnosed and operated in our clinic for complications of crohn disease during a period of years. results: we identified a number of patients operated for complications of crohn disease. of these were operated by minimally invasive procedures. we observed that laparoscopy was avoided in the case of intestinal fistulas (p = , ). also when sepsis associated the surgical complication-laparoscopy was avoided (p = , ). age under years represented another factor to avoid laparoscopy (p = , ). conclusions: although laparoscopy offers numerous advantages careful selection of the patients is of utmost importance so the safety of the procedure can be ensured. retroperitoneal sarcoma represents approximately - % of all sarcomas and less than . % of all neoplasia. radiotherapy and chemotherapy still do not represent valid therapeutic alternatives; therefore radical surgery remains the only valid option. complete surgical resection is the only potential curative treatment modality for retroperitoneal sarcomas. the ability of complete resection of a retroperitoneal sarcoma with tumor grading remains the most important predictor of local recurrence and disease-specific survival. hypoglycemia is a rare but potentially lifethreatening presentation of soft tissue tumors the etiology of hypoglycemia may be difficult to diagnose, assays for insulin-like activity (ila) were found to be high in the extract of tumor tissue, while insulin was not detected in significant concentration neither in the same extract nor in his serum. the most likely mechanism of hypoglycemia appears to be production of insulin like substance and increased utilization of glucose by the tumor. laparoscopic surgery represents an alternative technique for radical resection of such tumors rather than traditional surgery. only few cases of retroperitoneal tumors resected laparoscopically were reported in the literature. we report a rare case of years old male presented to ed unconscious due to hypoglycemia.he was resuscitated and admitted for further investigations. hypoglycemic attack recurred again during the same evening of admission. initial investigations were within normal except for serum glucose mg/dl ( . mmol/l). his tsh, glucagon & cortisol levels were within normal, insulin and c-peptide levels were undetectable. only hypokalemia ( . meq/l). he tested negative for the anti-insulin antibodies. his abdominal ultrasound as well as his ct scans showed the presence of a large retroperitoneal tumor ( cm cm cm) with a heterogeneous contrast effect. a glucose supplement was required to maintain the plasma glucose level within normal limits during which complete resection of the tumor which was performed laparoscopically. diagnosis of such hypoglycemia inducing retroperitoneal fibrosarcoma represents great challenge especially when patients presents only with hypoglycemia and no other abdominal symptoms, management using minimal invasive technique to resect and remove such tumors from the retroperitoneal region shows superiority in recovery and limitation of complications when done by experienced surgeons. solitary fibrous tumor (sft) is a rare fibroblastic mesenchymal neoplasm, tipically arising from the pleura, less frequently from other anatomic sites. sft is an indolent neoplasm, but it have been described cases of greater aggressiveness in terms of local recurrences and more rarelly of distant metastases. among the various extrapleural sites, intrabdominal, retroperitoneal localization is the most common site, followed by the pelvis soft tissues and parenchymatous organs. the most common clinical finding of intraabdominal localization is a palpable mass, and the pain is the most frequently associated symptom. the diagnosis is performed by imaging, but the histological as well as immunohistochemistry characterization of the lesion is the latest goal. furthermore, histological features are used to attempt to identify the patient with a hight risk of malignant evolution of the tumor. the gold standard treatment is surgical approach, meanwhile there are no evidences about the efficacy of any adjuvant treatment. we present the case of a -year-old man affected by symptomatic tfs arising from mesosigma treated by surgical radical excision. finally, we propose a review of the literature of last decade. background: laparoscopic right hemicolectomy involves making an additional incision to remove the specimen and perform the anastomosis. recently, natural orifice specimen extraction surgery (noses) has been reported as an alternative approach without any additional incisions or extensions, may lead to better outcomes compared to conventional laparoscopic right hemicolectomy. in this video, we aimed to evaluate the safety and feasibility of noses for laparoscopic right hemicolectomy. methods: we describe the technique with transvaginal specimen extraction and d lymph node dissection in laparoscopic right hemicolectomy by this video. we performed intracorporeal anastomosis combined with a transvaginal route of specimen extraction after medial-to-lateral mobilization. transverse transvaginal posterior colpotomy was performed under aid with visualization. the specimen was pulled into the sterilized plastic bag, passed transvaginally. the vaginal incision was then closed with a running suture. results: the operation time was min and the hospital stay was days. an excellent postoperative recovery was demonstrated and has shown future potential for less incision. the pathologic tnm stage is t n m . conclusions: this video has shown that laparoscopic right hemicolectomy with the noses technique is feasible and safe for selected cases. the long-term benefits of this procedure need to be more evaluated. recently, indocyanine green (icg) fluorescence has been introduced in laparoscopic colorectal surgery to provide detailed anatomical information.the aim of our study is the application of icg imaging during laparoscopic colorectal resections: to identify the sentinel lymph node (sln) to search micrometastases that can be missed with the conventional pathological exam, and to assess anastomotic perfusion to reduce the risk of anastomotic leak. after tumor identification ml of icg solution ( . mg/kg) is subserosal peritumoral injected. a full hd image s camera, switching to nir mode, in about min displays fluorescence: the sln is identified and the sln biopsy (slnb) is performed.after the transection ml of icg solution is injected to confirm the stumps perfusion. if there is an ischemic area, a new resection is performed.after the anastomosis is performed, another bolus of icg is intravenous injected to confirm the anastomotic perfusion.when the sentinel node is negative for cancer metastases by conventional histological examination, ultrastaging is performed by serial sections. when no micrometastases are identified on these sections, immunohistochemical techniques are applied. from november , patients were enrolled: left colectomy, right colectomy, transverse resections, and splenic flexure resections. in two cases, one left colectomy and one right colectomy, the anastomotic perfusion wasn't good and the surgical strategy was changed. four postoperative complications occurred, of which one anastomotic leak, due to a mechanical problem. from november , patients were enrolled to perform the slnb: right colectomy, left colectomy, transverse resection and splenic flexure resections. the sln was identified in cases. cases were found to be n to the conventional examination and were subjected to ultrastaging. the serial sections showed micrometastases in two cases. in the other cases the immunohistochemistry was performed but the exam is still in progress. icg-enhanced fluorescence imaging is a safe, cheap and effective tool to increase visualization during surgery. it's recommended to assess the anastomotic perfusion in order to reduce the incidence of anastomotic leak, and to perform the slnb for the sln ultrastaging in order to identify micrometastases. methods: for the last years, tem was performed on patients' with early rectal cancer. there were women and nine men, age to . localization of tumors was - cm from anus. mean size of tumors was . cm. full thickness excision was performed in all patient with suturing of mucosa. during follow-up in three patients' metastasis in lymph nodes of mesorectum were detected. all of these patients were re-operated: laparoscopic colectomy with total mesorectal excision (tme) was done. for the last year in patients with early stage rectal cancer we used indocyanine green (icg) with fluorescent imaging for mapping sentinel lymph node. icg was injected in four quadrants to submucosa around the tumor. during the laparoscopy, sln was detected and removed with morphological examination. results: among nine patients in patients, sln was negative. tem was performed in these patients with good results. after - months no recurrence or metastasis were detected in these patients. in two patients with positive sn laparoscopic tme was performed with low colorectal anastomosis. anastomotic complication was occurred in one patient. conclusion: tem procedure is highly effective in selected group of patients with early rectal cancer. mapping and examination of sln can clarify indication for the tem in the patients with early rectal cancer. purpose: laparoscopic surgery for colorectal cancer provides better short-term benefits and similar long-term outcomes compared with conventional open surgery. unlike minimally invasive surgery, natural orifice specimen extraction (nose) can provide additional advantages by reducing morbidity and postoperative pain related to the surgical extraction site. this study aimed to evaluate the efficacy and safety of a nose procedure using needlescopic instruments for colon cancer surgery. methods: between november and february , patients underwent laparoscopic nose using needlescopic instruments. the first port for the camera was placed at the umbilicus. a -mm or -mm port was inserted in the right lower quadrant. a -mm or -mm port was inserted in the right upper quadrant. individual needlescopic forceps for the assistant were inserted into left upper and lower quadrant ports. thus, a total of ports were placed. the superior rectal artery and inferior mesenteric vein were ligated with clips, and colonic mobilization was performed using a medial to lateral approach. after rectal stump irrigation, the distal rectum was transected using an endoscopic linear stapler. the proximal colon and associated mesentery were transected. after the rectal stump was opened, a wound retractor was pulled through the anus and inserted in the rectal lumen. the resected specimen was transanally extracted through this route. an anvil was intracorporeally attached to the proximal colon, and the open rectal stump was reclosed using an endoscopic linear stapler; colorectal anastomosis was then performed using a double-stapling technique. results: of the patients, were male and were female, with a median age of years ( - years). median body mass index was . . the tumor site was in the sigmoid colon in patients and rectosigmoid colon in patients. median operative time was min and blood loss was ml. there was no conversion to open surgery. no postoperative complication was observed. median postoperative hospital stay was days ( - days). conclusions: nose surgery using needlescopic forceps is an easily performed type of reducedport surgery with a conventional port arrangement. this procedure is feasible for the selected patients. introduction: splenic flexure colon cancer accompanying obstruction is usually managed stent insertion as a bridge to surgery and left hemicolectomy, or subtotal colectomy. however, stent insertion can fail more often than in sigmoid colon because it requires longer colonoscopic approach in the circumstance of impossible bowel preperation. although subtotal colectomy has advantage in the aspect that it is -stage treatment, it needs open surgery in most cases, right colon has to be sacrificed without oncologic neccesity, and preoperative staging and evaluation can be insufficient. despite colostomy is reluctant procedure when considering quality of life, in splenic flexure colon cancer obstruction, we can obtain prompt stabilization of patient state, suffient time to preoperative staging and evaluation, and also we can achieve minimally invasive surgery by using colostomy site as mini-laparotomy and close colostomy before discharge. colostomy site, tumor location, and minilaparotomy site for next radical surgery have to be considered comprehensively before making colostomy incision. colostomy site has to be appropriate as mini-laparitomy site for feasibility of laparoscopic left hemicolectomy and the colostomy has to be included in the specimen with caution to prevent unneccessary lengthening of the specimen. we experienced cases which were treated succefully in this strategy and report them. result: temperary loop transverse colostomy and laparoscopic left hemicolectomy via colostomy site in splenic flexure colon cancer obstruction has advantage of quick stabilization of patient's status, suffient preoperative staging and evaluation, achieving minimally invasive surgery, and also rapid colostomy closure before discharge. our tatme procedure for locally advanced low rectal cancer following chemoradiotherapy y. nakamoto , r. okamoto , f. kimura , h. yanagi , t. nakajima , h. yoshie , n. yamanaka surgery, meiwa hospital, nishinomiya, japan, surgery, yoshie clinic, itami, japan background: short-course chemoradiotherapy using hyper-fractionation method (scrt; gy/ fraction/ days ? s- or xeloda) is performed to secure circumferential resection margin (crm) due to tumor shrinkage, reduction of cancer cells with viability, reduction of radiation hazard for resectable locally advanced lower rectal cancer (more t or n ). the patient underwent radical surgery after one month of scrt. for more locally advanced lower rectal cancer (t or n ), induction chemotherapy is performed before scrt. for patients with poor efficacy of chemotherapy, we also do normal fraction gy radiotherapy. methods: we introduced tatme from last august, and cases were performed so far. in all cases temporary stoma has been constructed, and intersphincter resection (isr) is based on partial isr avoiding total isr considering postoperative anal function. if possible, colonic j-pouch is added, and pelvic floor repair may be added for esr cases and older people. at first one team preceded with the anal operation and shifted to the abdominal procedure, now it is done with two teams with the advantage of getting good visual field from both sides when there is difficulty identifying the right dissecting layer. tatme is very useful in cases such as large tumor, obesity, and narrow pelvis. furthermore, when it is difficult to identify the dissecting layer by scarring after crt, it is more possible to control the crm/drm of cancer. results: cases of isr, case of apr, case of tpe were performed, and in of these cases lateral lymph node dissection was also performed (one side , both sides ). postoperative complications were anastomotic leakage, pelvic floor infection, perineum infection, and bowel obstruction. conclusions: tatme for locally advanced lower rectal cancer is useful even after chemotherapy and scrt. background: although many studies have demonstrated similar perioperative outcomes for single-incision laparoscopic surgery (sils) and conventional laparoscopic surgery (cls) for colon cancer, few have directly compared the costs of them. we aimed to compare costs between sils and cls for colon cancer. methods: we analyzed the clinical outcomes and overall hospital costs of patients who underwent laparoscopic surgery for colon cancer from july to september at severance hospital; were used for analysis after propensity score matching. the total hospital charge, including fees for the operation, anesthesia, preoperative diagnosis, and postoperative management was analyzed. results: the total hospital charges were similar in both groups ($ . vs. $ . , p = . ). however, the patients' total hospital bill was higher in the sils group than in the cls group ($ . vs. $ . , p \ . ) mainly due to the difference of the cost of access devices. there was no difference in the additional costs associated with readmission due to late complications between the two groups ($ . vs. $ . , p = . ). conclusions: sils for colon cancer yielded similar costs as well as perioperative and long-term outcomes compared with cls. therefore, sils can be considered a reasonable treatment option for colon cancer for selective patients. aims: technology improvements in medicine allow the development of new minimally invasive approaches. despite every single advantage of these new devices they also can cause technical problems and difficulties for the surgical team. well known from last few years-laparoscopic assisted transanal total mesorectal excision for distal rectal cancer is perfect example for a quite new procedure, based on the combination of forgotten old surgical principles and technology advances. the aim of the study is to analyze the rate of technical problems during the procedure and to measure the impact of them on the operative time. methods: we conducted prospective observational study related to technical problems during the procedure. for the period between september and november in the department of endoscopic endocrine surgery and coloproctolgy at military medical academy-sofia have been performed laparoscopic assisted transanal total mesorectal excisions. we used standard local preoperative work up and postoperative care protocols. we defined technical problem as intraoperative event different from complication leading to delay in operative time. every technical problem during the procedure was recorded and time for resolving the problem was measured in seconds. results: overall technical problems occurred in of the cases. most of them were related to the insufficient smoke evacuation during the cases. the second most common technical problem were the excessive rectal stump spasms during the procedure-this complication occurred in of the patients. mean delay of the procedure related to technical problems is min. in our series we experienced only one intraoperative complication which was specimen perforation during the dissection. three complications occurred in postoperative period-two urinary retentions and one perianastomotic abscess, without need of reoperation. conclusion: technical problems during the procedure can be source of delay in operative time. correct use of devices in operating room is the key to reduce technical issues. technical problems can increase the rate of intraoperative near miss events and complications during the transanal total mesorectal excision. surg endosc ( ) aims: anastomotic leak after rectal cancer surgery constitutes a severe complication associated with poorer oncologic outcome and quality of life. preoperative assessment of the risk for anastomotic leak is a key component of surgical planning, including the opportunity of creating a defunctioning stoma. methods: studies on rectal cancer surgery published between and were systematically reviewed according to the preferred reporting items for systematic reviews and meta-analyses of individual participant data (prisma-ipd) guidelines. with the aim to generate a score for anastomotic leak, all available per-operative covariates were used as independent factors in a logistic regression model with anastomotic leak as dependent variable. a receiver operating characteristic curve (roc) analysis was generated. we selected as threshold the value that allowed a missing rate of anastomotic leak \ %. the predictive power of the previously selected cut-off was validated in an independent set of patients. results: twenty-six centers provided individual data on patients. with a threshold value of the roc corresponding to . in the training set, the area under the roc curve (auc) was . (p \ . ). sensitivity and specificity of the model's probability [ . to identify anastomotic leak were . % and . %, respectively. accuracy of the threshold value was confirmed in the validation set with . % of sensitivity and . % specificity. conclusions: we trust that, with further refinement using prospective data, this nomogram based on preoperative risk factors may assist surgeons in decision making. the score is now available online (http://www.real-score.org). in ( . %) cases laparoscopic interventions were performed in patients with diverticular colon disease. in the group of patients with colorectal cancer localization of the tumor in the right parts was observed in ( %) patients, in the left-in ( %), in the rectum- ( %). results: in the adenocarcinoma of the sigmoid colon, performed a left laparoscopic hemicolectomy ( cases) and resection of the sigmoid colon ( ). was executed high clipping and intersection of the lower mesenteric vessels, aorto-iliac lymphatic dissection. in the standard scope, lymph node dissection was performed with removal and testing of not less than epi-, para-and mesocolical lymph nodes (max ). the average length of the laparoscopic stage is ± min. laparoscopic right hemicolectomy ( cases) was performed in accordance with the principles of cvl (central vascular ligation) and cme (complete mesocolic excision). intracorporal ileotransversoanastomosis was formed by a semimanual method with endogia universal and v-lock suture material. the average length of the laparoscopic stage was ± min, the open phase was ± . in the tumor of the lower and middle ampullary parts of the rectum ( cases) after neoadjuvant chemoradiotherapy, was executed a laparoscopic total mesorectumectomy. conclusions: the use of minimally invasive technologies in colorectal surgery provides a complete revising of the abdominal organs, adequate scope of resection and lymph nodes dissection in surgical interventions. background: it is thought that complete mesocolic excision (cme) improves the oncologic outcomes for colon cancer. but, precise mesenteric mobilization from retroperitoneum and safe ligations at the origins of central vessels are considered to be technically difficult in single port surgery(sps). to resolve this problem, we utilize retro-mesenteric medial approach for right side colon cancer. herein, we introduce this technique and assess its outcomes. operative procedure: the multi-trocar platform is placed in the umbilical site. d laparosopy is inserted from one of this channels. the surgeon manipulates instruments via the other channels. st step: right colonic mesentery is mobilized medial to lateral from the head of the pancreas and retroperitoneum along the embryonic plane. nd step: the origins of ileocolic and right colic vessels are divided and central lymph node dissection is achieved. rd step: hepatic flexure is taken down from cranial. and right lateral attachment is dissected away and cme is achieved. th step: specimen is extracted and anastomosis is performed using a functional end to end anastomosis extracorporealy. results: from april to december , consecutive patients underwent sps-cme with right side colon cancer. there were in stage i, in stage ii, in stage iii and in stage iv. the mean operative time was min. the mean estimated blood loss was ml. there was no conversion to open surgery. additional port was placed in patients ( . %). intraoperative bleeding was occurred in patient. anastomotic leakage was observed in patient ( . %), intestinal obstruction ( . %) and wound infection in ( . %). conclusion: these results suggest that retro-mesenteric medial approach in single port surgery with right side colon cancer is useful and safe technique. aims: this multicenter, randomized controlled trial (simple trial) aimed to investigate the quality of life (qol) and patient satisfaction of single port laparoscopic surgery (spls) for colon cancer, compared with multiport laparoscopic surgery (mpls). methods: patients with histologically diagnosed adenocarcinoma in cecum, ascending and sigmoid colon were eligible for this trial. eligible patients were randomly assigned to the spls or mpls group at a ratio of : . qol was measured with the eortc qlq-c third edition (korean version) preoperatively and postoperatively at month , , and . in addition, patient satisfaction was surveyed with a five-point questionnaire at postoperative month. to exclude the impact of adjuvant chemotherapy on qol, subgroup analysis for patients with or without adjuvant chemotherapy were carried out. (clincaltrials.gov identifier: nct ) results: total patients were randomly allocated into the spls group (n = ) and mpls group (n = ). in total patients, global health status and five functional scale steadily increased and nine symptom scales also gradually improved over time. but, nausea/ vomiting and appetite loss temporally deteriorated at postoperative month. pain score was significantly worse in the mpls group ( . in the spls group vs. . in the mpls, p = . ) at postoperative month and appetite loss score was significantly worse in the spls group ( . vs . , p = . ) at postoperative month. except for that domains, all the other items of qol between groups were not different until postoperative months. patient satisfaction was significantly higher regarding the operation (p = . ) and the abdominal wound (p = . ) in the spls group. in patients without adjuvant chemotherapy, some items of qol (global health status, physical functioning, role functioning, emotional functioning, fatigue and pain) were significantly better in the spls group at postoperative month. since postoperative month, all of qol domains (except pain score) were similar between groups. conclusion: although postoperative pain was temporarily better in the spls, most of qol domain were similar between the spls and the mpls group until postoperative month. in patients without adjuvant chemotherapy, spls showed better outcomes in some of functional scales and symptom scores at postoperative month. coloproctological surgery, juntendo university, tokyo, japan; gastroenterological surgery, juntendo university, tokyo, japan introduction: laparoscopic surgery causes less postoperative pain compared with pain after laparotomic surgery, and its low invasiveness should be considered for pain control. we have previously controlled postoperative pain by epidural anesthesia. in this study we compared postoperative multimodal analgesia centering on acetaminophen in patients who underwent laparoscopic colorectal cancer surgery with the conventional method. subjects: the subjects were patients who underwent laparoscopic colorectal cancer surgery between january and june . surgery was performed under epidural anesthesia in patients and multimodal analgesia in : periodic acetaminophen administration ? transverse abdominis plane (tap) block in , periodic acetaminophen administration ? local anesthesia of the wound in , and periodic acetaminophen administration ? intravenous patient-controlled analgesia (ivpca) in . the operating roomoccupying time, postoperative pain (nrs), frequency of taking analgesics as needed, and postoperative nausea were investigated for days after surgery and the duration of urethral catheter placement and postoperative intestinal movement were investigated in the epidural anesthesia and multimodal analgesia groups. results: while the time from entering the operating room to initiation of surgery was significantly shorter, the time from completion of surgery to leaving the room was significantly longer in the multimodal analgesia group. there was no difference in the operating room-occupying time. the frequency of postoperative pain was significantly lower in the multimodal analgesia group on postoperative day (pod) . the frequency of taking analgesics as needed was significantly lower in the multimodal analgesia group on pod , , and . no significant difference was noted in the duration (number of days) of urethral catheter placement or postoperative nausea between the groups. regarding postoperative intestinal movement, discharge of gas occurred significantly earlier in the epidural anesthesia group. the total number of incidents of complications in the epidural anesthesia group was . discussion: in laparoscopic colorectal cancer surgery, the effect of multimodal analgesia centering on periodic administration of acetaminophen without epidural anesthesia for postoperative analgesia was sufficient compared with the effectiveness of epidural anesthesia. this approach to analgesia may be useful because none of the potential complications of epidural anesthesia occur. surg endosc ( ) in the last years the application of new technologies like d vision or virtual reality have provided to surgeons the possibility of establish a preoperative surgical plan of each surgery and of each patient. these advances are specially useful in minimally invasive colorectal surgery due to the variability in location, anatomical relationship with other organs and vascular variants of these type of surgeries. the aim of our work is to built a digital -dimensional virtual model of the colorectal ct scan imagen of patients with colorectal cancer. the virtual models are obtained from the preoperative ct scan. the ct scans that we use to this work are general electric healthcare revolution gsiÒ and siemens somatom perspective Ò and the size of each image is mm. a medical software let us build a reconstruction of colorectal digital images where a radiologist has marked the exact image of the tumor so we obtain a d reconstruction which can provide an enhanced understanding of crucial anatomical details like the exact location of the tumor and the relationship with other organs and structures of the patient which can be selectively displayed or hidden. this information has an important applicability into clinical practice since it lets surgeons estimate the colorectal anatomy, tumor size and relationships, providing key landmarks to choose the most appropiate surgery, the best trocar location and a safer dissection specially in some cases whose location can change the kind of surgery radically. we present some cases where virtual models were crucial for the preoperative and intraoperative surgical plan, showing the potential interest of these d reconstructions in colorectal surgery. in conclusion the ct scan colorrectal image reconstruction can provide an enhanced understanding of crucial anatomical details of the colon and tumor location and relations which could contribute to choose the best surgical option and to improve safety in colorectal surgery. background: anastomotic leakage (al) after colorectal procedures are a common surgical experience and represents a significant burden both for patients and surgeons. the incidence of al has been reported to vary between . % to up to %, with rates for the colon and rectum of - % and - %, respectively. they, not only add to potential postoperative patient morbidities and to overall costs of postoperative patient care, but also are considered a quality indicator in colorectal surgery. aim: we aimed to evaluate the clinical burden associated with anastomotic leaks following colorectal surgery. methods: we conducted a retrospective analysis of colorectal patients who underwent conventional or laparoscopic colorectal surgery for colorectal cancer (crc), from january st, to december st, in a single colorectal centre (centro hospitalar de leiria). patient demographics, intraoperative and postoperative aspects were collected and analysed. all statistical analysis will be conducted using stata software (statacorp lp). results: in our cohort of pts, developed a clinical al ( . %), mostly males ( %), with an average age of ± . . male gender and conversion were independent risk factors. the group with al had a higher lohs ( . days vs, . -p \ . ). out of al have been detected after the discharge. the mean diagnostic day was the eighth, and mode estimated at day . when compared with a control group, wcc, eosinophils and crp were statistically significant different in al group, at day and . conclusion: in the present study, no statistically significant risk factors for al in crc surgery were detected, except for male gender and conversion. clinical methods and biomarkers were useful for early diagnosis. technology combined with experience and common sense may be the embodiment of the clinical method. conclusions: our regional screening program has significantly improved early diagnosis and quickened surgical treatment of crc. thanks to this, we obtained an earlier stage at diagnosis, a less invasive surgical approach, and a lower rate of complications and emergency surgery need were obtained also with an improvement in both os and dfs. introduction: surgeons are increasingly being faced with the problem of treating elderly colon cancer patients. we evaluated the outcome of silc in patients of over years with colon cancer with a propensity score matched comparison to assess its perioperative and long-term oncological outcomes. methods: this retrospective cohort study analyzed our experience with silc for colon cancer over years. eighty-seven patients of over years with colon cancer who electively underwent silc were included in this study (elderly group). eighty-seven patients were then chosen out of a collective of patients less than years old in a propensity score matched design (younger group). short-term clinical outcomes in both groups were compared and verified its long-term oncological outcome. results: american society of anesthesiologists score and post-operative complication rate were significantly higher in elderly group. however, the other short-term clinical outcomes including post-operative hospital stay were equivalent in two groups. the rates of -year cancer specific survival were . % in elderly group and . % in younger group, respectively, and the -year overall survival rates were . % and . %, respectively. no significant differences were seen between two groups. conclusions: our initial experiences suggested the oncological and clinical safety of silc in patients of over years with colon cancer. however, further studies are needed to demonstrate the advantages of this procedure compared to conventional laparoscopic colectomy. aim: some clinical trials have reported the safety and efficacy of laparoscopic colectomy for colon cancer. on the other hand, transverse colon cancer was excluded in these trials because of the difficulty of laparoscopic colectomy for transverse colon cancer. in this presentation, we report the tips for laparoscopic colectomy for transverse colon cancer. tips: in our department, transverse colon cancers has been resected by laparoscopically so far. to complete cvl and cme, lymph nodes around middle colic artery should be resected, however many important structure, duodenum, pancreas, superior mesenteric vein (smv) and so on, may be obstacles. this is most difficult point for this surgery. our surgery is as follows. mobilization of ileum and ascending mesocolon from caudal sideconfirm duodenum and pancreasexpose smv and ligation root of ileocecal artery and veindissect lymph nodes around smv and ligation of middle colic artery and accessary right colic veinconfirm pancreas from caudate side of transverse mesocolon and incise the peritoneum along the caudal side of the pancreasdissect lymph nodes sufficiently by dissection from both side of transverse mesocolonmost important point. to dissect lymph nodes safely, confirmation from both side of transverse mesocolon is necessary and dissection should be performed along important structure, smv, pancreas and so on. introduction: we have developed and previously reported single-incision plus one port laparoscopic anterior resection of the rectum (sils ? -ar) as a reduced port surgery in which we can utilize the incision for drainage as an additional access route for laparoscopic procedures including the transection the lower rectum. a consecutive experience from its introduction of sils ? -ar for rectal cancer is reviewed, and its -year oncological outcomes are evaluated retrospectively. methods: one hundred and forty-one patients ( female) with a mean age of . years adopted the sils ? procedure for rectal cancer. a lap protector (lp) was inserted through a . cm transumbilical incision; an ez-access was mounted to the lp and three -mm ports were placed. a -mm port was inserted in the right lower quadrant. results: one hundred and thirty-six patients ( . %) completed with sila ? -ar. the tumor locations in the rectosigmoid, rectum above the peritoneal reflection (ra), and rectum below (rb) were , and , respectively. the median follow-up interval was months. aims: colovesical fistulae came from inflammatory disease or cancer and do have a significant morbidity. the most common location is the sigmoid colon and the most common aetiology is diverticulitis. the treatment of choice is a surgical procedure. the aim was studying compare laparoscopic approach in patients diagnosed by benign (diverticulitis) and malignant (colon adenocarcinoma) colovesical fistulae. methods: from january to march all characteristics of surgical patients with diverticular and colon adenocarcinoma colovesical fistulae were reviewed. patient details (sex, age, symptoms, diagnosis, medical history and anaesthetic risk), surgical approach, hospital stay and complications were recorded. both groups were compared with significance level set at p \ . . results: nine laparoscopic ( %) and open approaches ( %) in diverticular colovesical fistulae were performed, with a conversion rate of %. the procedure done was sigmoidectomy. there were also performed laparoscopic ( %) and open approaches ( %) in colon adenocarcinoma colovesical fistulae. the procedures done were sigmoidectomy, pelvic exenteration, left colectomy, low anterior resection and loop colostomy. comparison between the two groups didn't show significant differences in characteristics but did show significant differences regarding the approach, with more cases performed by open approach in colon adenocarcinoma colovesical fistulae (p = . ). conversion rate didn't show significant differences. patients diagnosed for malignant colovesical fistulae had more complications, cases ( %), ( %) i-ii and ( %) iii-iv-v according to clavien dindo classification, manifesting significant differences (p = . ). laparoscopic approach didn't show significant differences regarding complications. conclusions: generally, surgical approach with colonic resection and partial or total cystectomy is the treatment of choice in colovesical fistulae, although vesical resection can be avoided if it is suspected benign aetiology. whenever laparoscopic approach is performed by experienced surgeons, is feasible in colovesical fistulae and the morbidity and mortality numbers are acceptable. laparoscopic approach allows the advantages of a minimally invasive treatment but implies clinical trials to stablish stronger evidence. aims: laparoscopic right hemicolectomy became the standard of care for treating cecum, ascending and proximal transverse colon cancer in many center. most centers use multiport laparoscopic colectomy with extracorporeal resection and anastomosis (mce). single-incision laparoscopic colectomy with intracorporeal resection and extracorporeal (sci) remains controversial. the aim of the present study is to compare these two techniques using propensity matching analysis. methods: this study analyzed patients who underwent laparoscopic right hemicolectomy including mce surgeries and sci surgeries from december to december . short-term outcomes were recorded. postoperative pain was evaluated using a visual analogue scale (vas) and postoperative analgesic use as outcome measure. results: the length of skin incision in the sci group was significantly shorter than in the mce group: median (range) ( - ) cm verses ( - ) cm (p \ . ). the vas score after surgery was significantly less in srhi than in mrhe. significantly fewer patients required analgesia after srhi after surgery. there were no significant differences in operative time, intraoperative blood loss, the number of lymph nodes removed and postoperative courses between the groups. the cost effectiveness was significantly cheaper in srhi than in mrhe. conclusions: sci for right colon cancer is safe and technically feasible. sci reduces the length of skin incision and postoperative pain compared with conventional mce. aim: this study was designed to clarify the utility of laparoscopic surgery for advanced lower rectal cancer after neoadjuvant chemoradiotherapy (ncrt). patients and methods: we investigated -year disease-free survival rate, operative outcomes and recurrence risk factor in patients with lower rectal cancer (ct - , n - ) who underwent laparoscopic surgery after ncrt from to december in kitasato university hospital. results: of patients, patients underwent low anterior resection (lar), patients underwent intersphincteric resection (isr) and abdominoperineal resection (apr). there were anastomotic leakage, and urinary disorder and sexual dysfunction. ypcr rate was . %, but patients ( . %) had recurrence ( liver, lung and lymph node and local recurrence; there is some overlapping). ypt and lymph node metastasis were detected as a recurrent risk factor. the -year relapse-free survival rate (rfs) was . % and the -year overall survival rate (os) was %. conclusion: in this examination, ypt and lymph node metastasis were risk factor for recurrence. the operative outcomes, -year rfs and the -year os are relatively good results. we will conduct further follow-up, and it is necessary to investigate a long term prognosis. laparoscopic surgery is warranted for rectal cancer after ncrt. surg endosc ( ) introduction: synchronous colorectal neoplasia presents an incidence ranges from % and %. classically its surgical treatment consisted in the realisation of a subtotal colectomy (stc), however, several authors have proposed that in certain occasions the realisation of two segmental resections with two anastomoses was not accompanied by an increased risk of anastomotic failure. the objective of this study was to compare the feasibility and safety of the laparoscopic approach of synchronous colorectal neoplasia using two different techniques: stc versus two segmental resections with two anastomoses. methods: we retrospectively reviewed the clinical data of patients over years of age who underwent colorectal surgery between and at a single center. we included patients with a synchronous colorectal neoplasia who underwent laparoscopic surgery, either stc or double resection (dr). results: a total of patients met the inclusion criteria. mainly males ( %) with an average age of years, with a scale of the american association of anesthesiologists superior to ii in % and with an average body mass index of kg / m . the mean operative time was min in the dr and min in the stc, the stc resulted in a higher conversion rate ( % vs %) and intraoperative bleeding ( % vs %), in addition to a postoperative period with more complications, only % of the patients undergoing stc didn't present any complication while % of the patients with a dr didn't present any complication. % of the stc presented anastomotic failure and only % of the dr. the mean hospital stay was days in the dr and . in the stc. in the dr, an average of cm of colon was resected with an average of . lymph nodes, while in the stc, cm of colon was resected with an average of . resected nodes. conclusions: the double resection with two anastomosis is a less aggressive surgery, with fewer complications and a shorter hospital stay, providing similar oncological results. there were no differences in morbidity, re-operations or hospital stay. regarding tumor stage there were no differences between the three groups. as for the resected nodes, we found a mean of in stc, in lc and sr with no statistical difference. there were no differences in the affected nodes among groups. in our patients we didn't find differences in the recurrences rate or in the distant metastases rate.the average follow-up was months (range: - ), with no differences in overall survival. conclusion: segmental resection of splenic flexure neoplasias is safe and feasible, with no differences in morbidity or in the oncological outcomes compared with more aggressive surgeries. introduction: the evaluation of perfusion in colorectal anastomosis is still a field of study and progress for the development of new modalities that allow reducing the ratio of dehiscence or anastomotic leakage (al) in said surgery. our objective with this work is to highlight the utility of indocyanine green (icg) in the said evaluation after colo-rectal surgery. methods: we present a series of cases of colorectal surgery (benign and malignant disease) intervened in the period between and . the population sample has been homogenized according to age criteria, risk factors and comorbidity. a retrospective database has been developed with the spss v. software for the evaluation of the results obtained. the primary outcome measure was al rate with at least month of follow-up. results: a significant reduction in the incidence of al was observed in patients who underwent colo-rectal surgery (p = . ). low al rates were shown in rectal cancer surgery (p = . ). there was no significant decrease in the al rate when colorectal procedures for benign and malignant disease were combined. conclusions: the use of the image by fluorescence with indocyanine green is a safe, reproducible and relatively simple method with which to evaluate the perfusion of the colorectal anastomosis as well as reduce the rate of anastomotic leak in the postoperative period. large well-designed randomized control trials are needed to provide evidence for its routine use in colorectal surgery. introduction: currently colonoscopy is the gold standard investigation for colonic evaluation. although caecal intubation is one of its quality indicators, it is not attained in up to % of cases. this remains a significant concern. limited data are available on the follow-up of patients with incomplete colonoscopy. aims: to assess colonoscopy completion rate, the reasons for incomplete colonoscopy, and the methods used to complete colonic evaluations after incomplete colonoscopy. methods: we performed a retrospective study of incomplete colonoscopies in our unit over a one year period ( ) these results compare favorably with published data. few statistically significant differences between groups suggest varying modalities of treatment broadly result in similar qol. this data highlights a need for well-delivered support programmes for specific issues, for example stoma care and sexual dysfunction. future studies will need to include a baseline questionnaire to truly measure the impact of surgery and measure quality in an increasingly elderly and comorbid population. splenic flexure cancer (sfc), comprising the tumours raised in the distal transverse colon and proximal descendingcolon, accountfor to %of all surgically treated colorectal cancers.in cme forsfc, dissection of both the transverse and descending mesocolon must be considered. however, the use of laparoscopic surgery as a curative treatment for sfc, has never been investigated in adequate controlled trials, because of difficulty in deciding on the appropriate operative procedure, as well as technical difficulties with laparoscopic lymph node dissection. the aim of this multicenter study is to evaluate the oncologic effectiveness of laparoscopic segmental resection with cme with for cancer located at the splenic flexure. we performed a retrospective analysis of all cases of sfc treated with a laparoscopic segmental resection with cme in five different institution. intra and post operative were evaluated. patientes were evaluated, the mean operative time was . ± . min. a total of ( . %) conversions occurred, due to splenic artery lesion, one for difficult adesyolisis and three due to locally advanced tumour. recurrence was observed in ( . %) patients. there was a significant association between disease stage and recurrence (p \ . ) with a higher proportion of stage iv patients in the recurrence group ( . % vs . %). at days follow-up no mortalitywere recorded.during a median follow-up of months (range - ), deaths occurred (all of them for disease progression). keplan mayer curves showed a compareble suvival with other colo-rectal cancer. in conclusion, laparoscopic segmental resection with cme and cvl seems to be an oncologically safe and effective procedure for treatment of sfc. it may be regarded as the standard surgical method for elective management of this disease. in the future, more tailored patient-and tumor-specific segmental resection might be achieved with the use of routine lymph node road mapping. it is very important to establish a minimum number of lymph nodes to analyse for a correct staging. it has been established as . the treatment of colorectal cancer is essentially surgical. the review of the medical literature indicates that laparoscopic colorectal surgery is a safe procedure that has not found significant differences in the survival rate from open surgery. aim: the aim of our study is to compare the outcomes of laparoscopic and open resection for colorectal cancer surgery evaluating lymph node assessment. methods: the patients were collected in our hospital during the period from / / to / / and the number of lymph nodes obtained in lymphadenectomy has been studied comparing the laparoscopic and laparotomy approaches. results: interventions were performed. were laparotomic, were laparoscopic and converted laparoscopic (fig ) . the average number of nodes found in these interventions was , . nowadays, the recommendations to obtain a proper lymphadenectomy is to find more than lymph nodes. analysing our procedures, surgeries had obtained a good lymphadenectomy. according to the approach, , % of the interventions ( ) are laparotomy, , % ( ) are laparoscopic procedures and , % ( ) are by reconverted laparotomy (figure ). the average number of lymph nodes isolated was similar. laparotomy approach found , nodes while , nodes were found in laparoscopy. converted laparoscopy found , ( figure ). conclusion: the treatment of colorectal cancer is essentially surgical. today, there are a lot of studies that support that laparoscopic surgery has a survival rate similar to laparotomy surgery. according to our study, the data collected indicates that the number of isolated lymph nodes in both approaches is very similar. to sump up, laparoscopic colorectal surgery is safe and has demonstrated oncological adequacy comparable to open approach and better short-term outcomes due to a less invasive approach. background: laparoscopic low anterior resection highlights the advantages of laparoscopic surgery (better surgical field, less bloodloss, less postoperativepain, better cosmeticresult). defunctioning ileostomy prevents anastomotic leakage in low rectal cancers, butincreases morbidity, degrades thequality of life and requires a second surgery for its closure. method: in the last months we performed laparoscopic low anterior resections for rectal cancer, whithout performing any protectiveileostomy, afterchecking the anastomosis intraoperatively( men, women. average age: years). the typical placement of trocars included one supraumbilical mm trocar, two right sided mm trocars in the midclavicular line, one mm in the left midclavicular line and one mm trocar in the suprapubic midline which is also used for specimen removal, after a cm transverse extension of the incision. we present themain stages of the procedure (dissection andmesorectal excision, division of the rectum with linear stapler using the 'chinese hat-parnex' technique, creation of an end-to-end intracorporeal anastomosisusing circular stapler under direct laparoscopic vision). results: no major postoperative complication was observed. the mean operative time was min ( - ) and free surgical margins were achieved. in one case a conversion to open surgery occured. the average length of hospital stay was days ( - ). conclusions: the laparoscopic approach facilitates access to the middle and lower rectum, total mesorectal excision and avoidance of ileostomy if possible. it is a demanding operation with extended learning curve, and requires adequate experience in laparoscopic surgery and colorectal surgical oncology. background: in colorectal cancer, local excision is an attractive treatment option, but additional resection is considered when lymph node metastasis(lnm) is expected at high rate. in lower rectal cancer, advanced surgery techniques are required, so it is often difficult to make judgments. the aim of the current study is to assess the reliability of laparoscopic surgery for submucosally invasive rectal adenocarcinoma (pt ) analyzing short-term outcomes and long-term survival. method: this cohort study analyzed patients who underwent laparoscopic rectal resection for submucosally invasive rectal adenocarcinoma (pt ). conversion rate and functional and oncologic outcomes were analyzed. data on long-term results and survival were evaluated. result: surgical procedure was low anterior resection / intersphincteric resection / abdominoperineal resection: / / , and conversion to open surgery was needed for ( . %) patients. sphincter-preserving procedures were performed in ( . %) patients. there were no perioperative mortalities and positive resection margin. the mean length of hospital stay was . days. complications beyond clavien-dindo grade iii occurred in ( . %) patients,the anastomotic leakage rate was . % ( / ). the positive lymph node metastasis rate was . % ( / ). high tumor budding (p = . ), lymphatic invasion (p \ . ), and mucinous /poor histological differentiation (p = . ) were significantly associated with lymph node metastasis on univariate analysis. on multivariate analysis, only lymphatic invasion was associated with lymph node metastasis (p \ . ).the median follow-up time was months (range, - months), recurrence free survival rates was . % ( / ). conculusion: the outcomes of this study suggest that laparoscopic surgery can be used for safe and radical resection of submucosally invasive rectal adenocarcinoma (pt )?and the absence of lymphatic invasion, budding, and mucinous /poor histological differentiation are each associated with low risk of lnm. risk stratification models integrating these factors need to be investigated further. conclusions: this study highlights the complex nature of sarcopenia, as well as its common incidence. minimally invasive surgery had a higher incidence of sarcopenia than that of open surgery when both were performed within an enhanced recovery setting. despite colorectal patients being a typically well-nourished cohort at low risk of complications, there may well be benefit from interventional strategies such as perioperative immunonutrition or pre-habilitation to reduce the incidence of this poor prognostic indicator. backgrounds: urinary dysfunction is frequently observed after rectal resection and justifies urinary drainage. the concept of enhanced recovery after surgery (eras) has been widely spread from the early s. however, the optimal duration of postoperative urinary drainage is unknown. aims: the aim of this study was to comprehend short-term outcome of early removal of urinary catheter after robotic rectal surgery (rrs). patients and methods: (patients) the data of consecutive patients who underwent rrs at two hospitals between april and november were retrospectively reviewed. the main indication of rrs was the patients who need rectal mobilization with autonomic nerve preservation regardless of benign or malignant disease. perioperative management: none of the patients received epidural anesthesia for postoperative analgesia. our basic principle was to remove urinary catheter on postoperative day (pod) . after removal of urinary catheter, trans-urethral catheterization (tuc) was performed in the following situations: ) no autonomous urination over h after removal ) the decrease in urine volume (\ ml/ hr) ) the appearance of subjective symptoms like abdominal distension. when tuc was required even once, residual urine volume was measured with ultrasonic examination device since then. results: twenty seven male and female were included. the median age of patients and bmi were years old and . kg/m , respectively. the surgical procedures included anterior resection (n = ), intersphincteric resection (n = ), abdominoperineal resection (n = ), hartmann's procedure (n = ), and total coloproctectomy (n = ). only one patient received lateral pelvic lymph node dissection. urinary catheter was removed on pod in cases ( . %), on pod in cases ( . %). although tuc was needed in three cases ( . %) immediately after removal, tuc was no longer needed within three days in all three patients. late dysuria was observed in two cases ( . %), and bladder overdistension was suspected in these two cases. conclusions: our study showed that urinary catheter could be safely removed on pod after rrs. however, careful follow-up observation to avoid bladder overdistension is essential after removal. introduction: intersphincterian low rectal resection is a valid alternative to lower rectal cancers located at about - cm from the anus. methods: we present cases from our personal experience for tumors localized - cm from the anus. of them required preoperative radiochemotherapy. in cases, abdominal surgery was performed laparoscopic, having the surgical specimen extracted transanal. lone star device was used for the perineal procedure in all cases. cases required a manually, separate wires anastomosis; the others cases benefited from mechanical anastomosis performed endoanal with - mm circular stapler. we performed complete mesorectum excision in all cases, ligation at the origin of inferior mesenteric artery, complete mobilization of left splenic flexure and lateral protective ileostomy. all pacients underwent inspection rectoscopy before transit reintegration, and cases were reintegrated over a period of - weeks, except for cases which developed a colo-anal fistula, that closed under conservative treatment over a period of - months. results: there were no postoperative anal incontinence. in one case, a relative anal stenosis occured, which required endoscopic dilation. there was case of tumor recurrence and required abdominoperineal resection. conclusion: literature data sustain a - / ratio for very low rectal resection versus rectum amputation. the limit resection under the tumor is accepted as . cm. very good functional results by considering oncological principles, is a sustainable argument for choosing this kind of procedure as an alternative of rectum amputation. in the few studies conducted on crcs, the reported rate of sln micro-metastases is up to - %. the aim of this ongoing prospective study is to assess the predictability of the ex-vivo nirf sln mapping and of the research of micrometastases in nnd crc patients to propose adjuvant chemotherapy. materials and methods: fifty-eight patients undergoing standard oncological crc laparoscopic resection have been prospectively enrolled in two centre. as previously described by the authors, the intact surgical specimen was extracted and opened longitudinally and ml of indocyanine green (icg; mg/ml) was injected submucosally at four corners around the tumor in order to identify the lymphatic pathway and the slns. each sln presenting as negative at conventional histological analysis, was further investigated with ultrastaging techniques including serial sectioning and additional immunohistochemistry, in order to detect the presence of micrometastases. results: thirty patients were n ? , and were nnd. overall, a total of lymph nodes were retrieved. a total of sln were identified (mean . per case) and of those were nnd. after ultrastaging investigations, micrometastatic cases were found in nnd patients. the patients were so upstaged to n . sln located deeper in the mesenteric and mesorectal fat could easily be identified by nirf (even after nchrt). conclusions: in our preliminary series, the ex-vivo nirf sln mapping rightly predicts the status of loco-regional nodes, as confirmed by the histological investigations. the micrometastases' identification let selected patients to undergo the adjuvant treatment with the aim to reduce the risk of recurrence. ( / ) in lateral node positive group and . % ( / ) in lateral node negative group. four of local recurrence were lateral lymph node recurrence. two patients recurred the other lateral side of previous lpl, then they were laparoscopically resected and no recurrence ( , months). two patients recurred the same side after lpl were not curable because of liver metastasis and extensive invasion to the common iliac vessels. conclusion: selective lpl for rectal cancer was safe and good local control for lateral lymph node positive patients. also curable local recurrence resection was possible for non-treated lateral lymph node recurrence. intestinal malrotation is an embriologic anomaly generally discovered in the first months of life due to bowel obstruction. adult presentation is rare and its association with colon cancer is far more rare. we report a case of a years old man affected by asymptomatic intestinal malrotation incidentally found during an abdominal computed tomography (ct) performed for retroperitoneal colonic perforation in a patient with an endoscopically diagnosed aenocarcinoma of the caecum and a large polyp of the descending colon. preoperative vascular anatomic study allowed us to plan a laparoscopic approach safely also with adequate lymphoadenectomy. the abdominal cavity was entered throught a right flank mm optical trocar on the transverse umbilical line. three additional mm trocars were placed in right iliac fossa, right and left hypocondrium respectively. exploratory laparoscopy confirmed midgut malrotation and a fresh flogistic area at the descending colon perforation site. caecum and ascending colon were on midline and attached due to adhesions to sacral promontory. ileocolic artery (ica), middle colic artery (mca) and ima were selectively ligated but not at their origins due to aberrant anatomy. laparoscopic subtotal colectomy with intracorporeal stapled ileosigmoid anastomosis were carried out (endogia mm, double layer / polyglicolic acid suturing of the breech). the anisoperistaltic nature of the anastomosis is due to the disposition of the mesenterium which did not allow an isoperistalting orientation of the two resected stumps. the specimen was extracted throught a pfannestiel incision. the postoperative course was complicated by intestinal obstruction conservatively treated with slow bowel function's restoration. the patient was discharged from the hospital in th postoperative day. unexpectedly specimen histology revealed two villous adenomas with high grade dysplasia. lymphnodes were retrieved from the specimen (ptisn ). to date our case is the only fully laparoscopic colonic resection reported in literature in malrotation as well as the first intracorporeal stapled ileo-sigmoid anastomosis for such disease. the median hospital stay was days. in-hospital mortality was nil. the overall morbidity was %. the median length of follow-up was months. conclusions: our preliminary results suggest that robotic-assisted surgery for colorectal cancer can be carried out safely and according to oncological principles. robotic surgery is advantageous for both surgeons (in that it facilitates dissection in a narrow pelvis) and patients (in that it affords a very good quality of life via the preservation of sexual and urinary function in the vast majority of patients and it has low morbidity and good midterm oncological outcomes). in rectal cancer surgery, the robotic approach is a promising alternative and is expected to overcome the low penetration rate of laparoscopy in this field. aims: postoperative inflammation have been reported as one of the independent prognostic factors in several types of malignancies.the aim of this study is to clarify the impact of laparoscopic approach on postoperative inflammatory status after surgery for colorectal cancer, and to analyze the association between postoperative inflammation and prognosis in patients with colorectal cancer. methods: a total of patients with stage l-lll colorectal cancer (crc) who underwent curative surgery were retrospectively analyzed. the maximum crp value measured between the times of surgical resection and discharge was defined as 'max crp'. the optimal cut-off value of max crp that best predicts rfs was determined to be mg/dl by the minimum p-value approach. methods: trainees working in this firm were responsible for data collection. patients who underwent emergency surgery during the calendar year of had the following details collected-the presence or absence of a complication in the -day post-operative period, the type of complication and description of complication along with the grade of the complication (see fig. .) . patients who underwent intermediate to major surgery were followed up at outpatients and were specifically asked for the occurrence of complications from the point of discharge up until the outpatient appointment. with one centralised national hospital-the people who were discharged and subsequently experienced considerable or major complications invariably represented back to hospital via the a&e department. results: a total of emergency surgeries were performed by this surgical firm in , % of these being done laparoscopically. of these cases- patients experienced post-operative complications within the first days after their procedure. this equated to a complication rate of . %. the most common complications were abdominal pain, nausea & vomiting, and wound infection. there were complications for each of these categories. post-operative bleeding occurred in cases with fistulas or leak of an anastomosis occurring in cases. death of a patient occurred in instances once as a result of post-operative bleeding from the site of anastomosis after a whipple's procedure, the nd occurred subsequent to post-operative bleeding from a peptic ulcer and in the rd case occurred in an instance of faecal peritonitis as a result of anastomotic failure after a roux-en-y bypass for a patient with pancreatic malignancy. conclusion: the davien-clindo classification proved to be simple, efficient and useful in analysing post-operative outcomes. the results indicate that despite the emergency setting & elderly cohort of patients-minimally invasive surgery proved to be a safe and viable option. conclusions: in this prospective study, we observed greater rates of detection of adenomas among endoscopists. screening colonoscopy on symptomatic and/or high risk group for crc is valuable in early detection and the prevention of crc. large sample size and long period of screening colonoscopy was needed. limitation of our study was the small sample size and no use of high detention endoscopy. results: the mean intraoperative blood loss volume was significantly less in the lap group than in the open group ( vs. ml, respectively, p \ . ). the mean operative time was not significantly different between the lap group and the open group ( vs min, respectively, p = . ). the incidence of severe postoperative complication (grade or higher in the clavien-dindo classification) was lower in the lap group ( / ( %) vs / ( %), respectively). the mean postoperative hospital stay was significantly shorter in the lap group than that in the open group ( vs. days, respectively, p = . ). conclusions: lap-tpe can be a safe and feasible procedure. background: amyloid light chain (al) amyloidosis is a rare protein deposition disorder with an incidence ranging between - cases per million people. it can present insidiously with localized or multisystem symptoms and usually occurs later in life. prognosis is poor as al typically presents at an advanced stage. intestinal pseudo-obstruction is a rarely reported complication of al amyloidosis. here we report a case of al amyloidosis which was identified during surgery for intestinal pseudo-obstruction. case presentation: a year old male presented to the emergency department with a month history of abdominal pain and distension, as well as marked swelling of his lower limbs. this had worsened in the previous weeks and he had developed intermittent diarrhoea. ct showed ileitis with marked dilation of the proximal small bowel. laparatomy revealed small bowel that was grossly distended that rapidly developed multiple petechiae and subsequent haematomas upon handling. two days later a repeat laparotomy was performed and . m of ishaemic small bowel was resected. histology showed amyloid deposition with positive congo red staining. subsequent cardiac events led to an echo being performed that showed concentric left ventricular hypertrophy attributed to amyloid deposition within the myocardium. free serum light chain ratio was sent and confirmed the diagnosis of al amyloidosis. he has recently been started on a treatment regimen consisting of cyclophosphamide and dexamethasone. discussion: systemic al amyloidosis frequently involves the gastrointestinal tract, typically presenting with chronic diarrhoea and associated malabsorption. only case presenting with pseudo-obstruction has been reported in the literature. al amyloidosis presents insidiously with non-specific symptoms depending on which organs are affected. treatment aims to prevent further deposition of protein within the organs. prognosis is determined by the organs that are affected and the extent of protein deposition within them. cardiac involvement holds the worst prognosis ultimately causing sudden cardiac death. the mainstays of management are early identification and treatment implementation to prevent protein build up and subsequent organ failure. conclusion: a diagnosis of amyloidosis should be considered in patients with intestinal pseudo-obstruction to expedite the diagnosis of al amyloidosis and improve survival. aim: in the management of locally advanced rectal cancer (larc), the achievement of a complete total mesorectal excision (tme) with clear resection margins was demonstrated to be the main predictor of overall and disease-free survival. predicting surgical difficulty in larc patients may be of particular importance to choose the best surgical approach. this study proposes a mri-based score to identify preoperatively larc patients with a high risk of having a difficult surgery. methods: this is a retrospective study based on the european mri and rectal cancer surgery (eumarcs) database, including patients with mid-low larc who were treated with neoadjuvant chemoradiation therapy and laparoscopic tme with primary anastomosis. data on pre-treatment and restaging through magnetic resonance imaging were available for all patients. surgical difficulty was defined as high or low grade taking in to account operative (e.g. duration of surgery), and postoperative factors (e.g. hospital stay). score accuracy was evaluated by estimating sensitivity, specificity and area under the receiver operating characteristics curve (aroc). results: seventeen ( . %) of larc patients were graded as high surgical difficulty. the eumarcs score was developed using the following significant predictors of surgical difficulty: bmi [ , interspinous distance \ . mm, ymrtstage = t b, and male sex. the score ranged from to . the cut-off score to best differentiate patients with a high probability of difficult surgery was = points. this cut-off value showed the best balance in sensitivity and specificity. the eumarcs score demonstrated high accuracy (aroc: . ) conclusions: the eumarcs score was found to be sensitive and specific in predicting surgical difficulty in larc patients who were candidate for laparoscopic tme. the score has the advantage of considering patient and cancer related characteristics that can be all assessed preoperatively and it can be useful in the decision making process. this score has not yet been externally validated. background: recently published two non-inferiority randomised control trials has raised questions on laparoscopic surgery for rectal cancer, showing lower quality pathological specimens to those achieved using an open technique. locally advanced rectal cancers add to the level of difficulty for laparoscopy approach. our study was aimed to assess feasibility of laparoscopic rectal surgery, comparing short term outcomes, quality of surgical specimen, morbidity and mortality, between propensity score match groups of locally advanced and early rectal cancers. methods: prospectively acquired data from consecutive patients undergoing laparoscopic surgery for rectal cancer at the minimally invasive colorectal unit in united kingdom between and . locally advanced rectal tumours were identified as t b or t with pre-operative mri scans. all the patients were operated by the same team and the procedures were performed laparoscopically. : propensity score matching was performed to create a perfect match in terms of tumour height. results: total of laparoscopic rectal resections were performed during the study period, out of which patients had locally advanced (la) disease and were propensity-score matched for tumour height with non-locally advanced (nla) patients. median operative time was higher for the la surgery group ( min vs min p = . ). however, conversion to open surgery (p = . ), readmission (p = . ), re-operation (p = . ), clinical anastomotic leak (p = . ) and -day mortality rates (p = . ) were all equivalent between the two groups. r resection was achieved in % of la group as compare to % of nla group (p = . ). conclusion: this study demonstrate that standardised approach to laparoscopy is safe and feasible in locally advanced rectal cancers. comparable post-operative short-term clinical and pathological outcomes were seen between la and nla groups. aims: the application of colorectal cancer screening programs, has showed a decrease in recurrence and mortality. for this reason, these programs are being implemented at a national level in the different spanish regions, as has happened in our community.to present the initial short-term results on the morbidity of the immediate postoperative period to days of colon cancer, mortality and hospital stay after the implementation of a screening program in our center. methods: a retrospective study was performed. patients aged between and years were included in the study, diagnosed with colon cancer. they underwent minimally invasive surgery, in most cases, with any type of colonic resection, from january to december . all patients were diagnosed, conventionally or through a screening program, the latter according to the plan implemented in our community. the sample was divided into two groups of patients according to the way of being diagnosed (group si screening = patients, group no screening = patients) and they were compared according different variables: dependent factors of the patient, factor of type colon cancer, factors of colon cancer resection and follow-up. results: both groups were comparable in all study variables. regarding the variables included in the follow-up, no statistically significant differences were found in terms of postoperative mortality-clavien-dindo v. however we found differences statistically significant in postoperative morbidity (p = . ) and in its classification according to clavien dindo i-iv (p = . ). the complications analyzed independently, such as anastomotic dehiscence (p = . ) or postoperative ileus (p = . ), have also presented significant differences, unlike surgical wound infection (p = . ). conclusion: at our center, the application of the screening program has not influenced in the initial stage of colon cancer or its surgical approach. however, we have found a lower overall morbidity rate and minor complications, justified by a lower incidence of anastomotic dehiscence and postoperative ileus. background: colorectal carcinoma is one of the most common malignancies. surgery is the only definitive method to achieve cure for this illness and can be performed via an open or a laparoscopic approach. the pros and cons of each approach have been discussed extensively, with the oncologic efficiency of the laparoscopic approach being one of the leading topics. objective: the aim of this study was to establish oncological non-inferiority of the laparoscopic approach to colorectal cancer. primary outcome measure was defined as number of harvested lymph nodes. secondary outcome measures were medium-term disease free and overall survival as well as length of hospital stay, time to oral feeding and short-and long-term complication rate. methods: this was a single center retrospective chart review. all consecutive patients who underwent colon or rectal resection due to colorectal carcinoma at hadassah medical center between the years - were included. patients who were operated on for recurrent disease or who had metastatic disease at the time of surgery were excluded. patients were divided into three groups according to the surgical approach: laparoscopic, open or converted. medium-term oncological outcomes were the same for all groups. time to oral feeding, length of hospital stay, short-and long-term complication rate were all significantly improved in the laparoscopic group. conclusions: we were unable to prove non-inferiority of the laparoscopic approach regarding the number of harvested lymph nodes. however, all surgical approaches yielded a high number of harvested lymph nodes which is most probably oncologically sufficient, as reflected by the non-existent difference in medium-term oncological follow up. this study supports previous studies showing the superiority of the laparoscopic approach regarding short term recovery and overall complications rates. aims: two non-inferiority randomised control trials have questioned the utility of laparoscopic surgery for rectal cancer by failing to prove that pathological markers of high quality surgery are equivalent to those achieved by open technique. we intend to present short and long-term postoperative outcomes from the largest single surgeon series of consecutive patients undergoing laparoscopic tme for rectal cancer. we describe the standardised laparoscopic technique developed by the principal surgeon, and the short-term outcomes from three surgeons who were trained in and subsequently adopted the same approach. methods: prospectively acquired data from consecutive patients undergoing surgery for rectal cancer by the principal surgeon (ap) at the minimally invasive colorectal unit in portsmouth between and were analysed along with data acquired between and from surgeons (tq,nf,ah) at three further international centres. end-points were overall and diseasefree survival at years, and early post-operative clinical and pathological outcomes. results: consecutive patients underwent laparoscopic tme surgery by the principal surgeon (ap). at years overall survival was . % (dukes' a = . %; b = . %; c = . %); disease-free survival was . % (dukes' a = . %; b = . %; c = . %). post-operative length of stay, lymph node harvest, mean operating time, rate of conversion, incomplete resection, major morbidity and day mortality were not significantly different between the principal surgeon and those he had trained when subsequently in independent practices. conclusion: laparoscopic tme produces excellent long-term survival outcomes for patients with rectal cancer. a standardised approach has the potential to improve outcomes by setting bench-marks for surgical quality, and providing a step-by-step method for surgical training. results: analysis of association of tumor location (sigmoid, right or left colon), operation time, blood loss, extraction site, type of surgical sutures used for wound closure with postoperative complications or specimen quality either did not show significant correlation or could not be conducted due to data nature. unexpectedly, a significant difference was demonstrated between two surgical teams in terms of hernias. majority of cases- ( . %) were performed by surgeon (s ), surgeon (s ) operated on ( . %) patients, nevertheless minilaparotomy closure was usually performed by junior members of the team. conversion rate was . % for s and . % for s (p = . ). operation time and blood loss were smaller in s group compared to s ( . ± . min vs . ± . min, p = . and . ± . ml vs . ± . ml, p = . respectively). specimen quality and early postoperative complications did not differ. postoperative hernia rate was . % for s and . % for s (p = , ). both surgeons used the same specimen extraction sites and materials for wound closure. hernias were more frequent after vertical minilaparotomy- % ( of patients), and in converted patients , % ( of patients), compared to , % ( of ) in transverse minilaparotomy group. there was no association of hernias and wound infections. conclusions: our study demonstrates, that besides consultant dependent surgical surrogates, steps which are often performed by other members of surgical team (such as wound closure) may contribute to complication rate as well. more thorough supervision of wound closure may be needed. aims: laparoscopic complete mesocolic excision (cme) right hemicolectomy is considered a demanding procedure and it is actually adopted in few centers from the west. the aim of the present study is to analyze the safety of laparoscopic cme right hemicolectomy and to compare its short-term results with standard right hemicolectomy in a single western center. methods: prospectively collected data from patients who underwent laparoscopic cme right hemicolectomy between june and november were retrospectively analyzed (cme group) and compared with data from patients submitted to standard laparoscopic right hemicolectomy between april and november (s group). results: no differences were observed between the cme and the standard right hemicolectomy groups in terms of clinical characteristics. in the cme group, . % of patients were = years old, . % of patients were asa class , . % of patients had = comorbidities, . % of patients had bmi [ and . % of patients had = previous abdominal surgeries. no differences were observed in terms of duration of surgery ( ± min vs. ± min; p = . ) and intraoperative complications ( . % vs. . %; p = . ) between cme and s groups; mean blood loss was lower in the cme group ( . ± . ml vs . ± . ml, p = . ). the percentage of overall ( . % vs. . %; p = . ) and severe (clavien-dindo = ) complications ( . % vs. . %; p = . ), redo surgery ( . % vs. . %; p = . ) and readmission ( . % vs. . %; p = . ) was comparable between cme group and s group. a significant difference was observed in the length of specimen ( ± mm vs. ± mm; p \ . ) as well as in the length of proximal ( ± mm vs. ± mm; p = . ) and distal margins ( ± mm vs. ± mm; p = . ) in favor of the cme group. the number of lymph nodes harvested was slightly higher in the cme group ( . ± . vs. . ± . ; p = . ) as it was for the percentage of cases with less than retrieved lymph nodes ( . % vs. . %; p = . ), although these differences did not reach statistical significance. conclusions: this study represents one of the few western experiences demonstrating the safety of laparoscopic cme right hemicolectomy. cme technique showed good short-term results and better quality specimens when compared with the standard procedure. aim and background: peritoneal dissemination of colorectal cancer (pc) makes the complete resection of cancer lesions impossible. in such cases, multidisciplinary therapy is essential with mainly chemotherapy. preoperative diagnosis of pc is usually uncertain by ct or mri image. for diagnosis of pc needs surgical materials with laparotomy. but the laparotomy and resection of pc with general anesthesia tends to make impossible for immediate chemotherapy. less invasive diagnosis of pc is necessary and expected.endocytoscopy (ec) makes the histological diagnosis with precise images gained by high magnification (x ). as a preliminary examination, ec diagnosis for resected specimens of pc were evaluated. methods: two cases of pc diagnosed in operation were evaluated. under general anesthesia, laparotomy was conducted. peritoneal dissemination lesions obviously diagnosed as pc were resected. immediately the lesions were stained by methylene blue solution for to s. ec observation was done according ec classification ) and ecv classification ). results: in two cases, ec observation was successfully done. images of dilated surface microvessels of a nonhomogeneous caliber or arrangement were observed in nbi ec corresponding to ec-v . histopathological diagnosis of resected specimens was metastatic colorectal carcinoma in peritoneum in both cases. conclusions: histological diagnosis for pc is gained by ec with resected specimen. as the result of this investigation, ec examination via camera port in laparoscopic operation might be possible for diagnosis for pc of colorectal cancer in vivo. aims: the aim of this presentation is to demonstrate and analyze surgical complications, arising during laparoscopic colorectal resections for cancer and to analyze the reasons of adverse events. methods: we demonstrate videos from our surgeries, where different types of complications occurred and share our classification of types of mistakes, that may lead to intraoperative complications and ways to prevent them. results: we divide mistakes in laparoscopic colorectal resections into two large groups-'false strategy' and 'dangerous techniques'. the first includes poor diagnosis, too extensive or insufficient extent of surgery and improper enthusiasm in using platforms. prevention of first type mistakes is in thorough training and peer-review of each consultant practice. second type of mistakes includes two subtypes : 'faulty habits'-use of unsafe techniques (blind port insertion, poor vascular exposure prior to clipping, not obtaining 'critical views', unsafe use of energy and stapling devices etc.) and 'failure in a certain case'-when despite correct general approach a complication occurred (misinterpretation of fascial layers or vessels). prevention of 'faulty habits' lies in supervised training in high volume colorectal departments including dedicated surgical devices training. to avoid 'failure in a certain case' standardization of surgical procedure is essential, as the most efficient way to prevent this type of mistake is 'pattern recognition'ability of a surgeon to compare the picture he sees during a procedure with a 'standard' view, he used to have during previously performed standard surgeries-this is apparentely impossible when every procedure is done differently. regular reviews of own surgeries recording and other surgeons' procedures may also fascilitate pattern formation. in case a complication occurres we use the four step course of action: preservation of the view, temporary control, decision on conversion, permanent control. conclusion: as popularity of laparoscopic colorectal resections is growing rapidly the number of intraoperative compliactions is increasing as well. we demonstrate videos of complications and our approach to classification of possible mistakes. systematic aproach to reasons, underlying certain mistakes helps to produce a strategy to reduce intraoperative complication rate. introduction: the drains placement inside the abdominal cavity has traditionally been carried out to evacuate hematic remains or postoperative collections. there is no scientific evidence of the prophylactic use of drainage in elective colorectal cancer (ccr) surgery to avoid anastomotic complications or other complications. however, it is traditionally used. when the anastomotic leak is produced, it is generally agreed that drainage system should be used for therapeutic purposes. aims: the aim of this study is to evaluate the effectiveness of the use of prophylactic drainage in elective surgery of ccr. we would check if they avoided the appearance of complications, and if they are useful when the anastomotic leak appears. methods and results: we analyzed the data collected in our hospital from / / to / / . we studied the number and type of interventions in which prophylactic drainages were placed, the appearance of anastomotic complications and if these drains were effective. interventions were performed during this period of time. % of these procedures had used prophylactic drainage ( interventions). this percentage was up to % in patients who have performed a left colon surgery as a sigmoidectomy or rectal procedure. during this period, there were cases of anastomotic leakage. in all of them had been placed drainage but only of them were effective. conclusions: we have seen that prophylactic drainage is a common practice independently of the location of the anastomosis. the last multimodal rehabilitation guidelines recommended the nonuse of drains systematically above the peritoneal reflection with a high level of scientific evidence. they cause discomfort to the patient and delay early mobilization. however, it may be useful to use drains in the first h of a pelvic floor procedure. there is not enough evidence to show sistematic drainage after colorectal anastomosis prevents complications of the anastomosis or other complications. aims: colonic cancers of the splenic flexure is uncommon and associated with poor prognosis. several studies were published aimed to identify the optimal surgical option for the best oncological outcomes. however, whether an extended colectomy or a segmental resection is required is still controversial. the aim of this study is to analyse the outcome of the two different approaches through the experience of a single centre. materials and methods: retrospective data of consecutive patients with diagnosis of colonic cancer situated at the splenic flexure of our department between and were analysed. based on type of surgical procedure, patients were enrolled in arm a (segmental resection) and arm b (extended resection). arm a patients were treated with segmental resections with a wide mobilisation of the transverse and descending colon and ligation of the left colic artery, sparing the middle colic artery and the inferior mesenteric artery. functional lateral to lateral anastomosis was performed extracorporeally. arm b patients were treated with more extended colectomies, both associated with central vascular ligation. results: out of patients included, were allocated in arm a and in arm b. patients' population of the arms was homogeneous as concerns demographic characteristics and stage of the disease. operative time was comparable ( , min vs min, p = , ). the length of the specimen was significantly shorter in arms a ( , vs , , p = , ) . the number of harvested lymphnodes did not differ between the two groups ( , vs p = , )postoperative short term complications was comparable in both arms ( vs , p = , ). no postoperative mortality was observed. overall -year survival and disease free survival rates were similar in arm a and b ( . % vs . %, p = , and , % vs , %, p = , ). hospital stay was similar in the two groups (p = , ). conclusions: despite a shorter length of surgical specimen after limited resections, postoperative complications, lymph node harvest, and survival were comparable in both.in our opinion the extracorporeal anastomosis is functional to both the achievement of a cleaner operative field and a better control of the resection margins. incidence of neuroendocrine tumours in the rectal area has increased in recent years.before the onset of minimally invasive colorectal surgery, these lesion had to be treated by a more radical technique when not suitable for endoscopic resection.selection of the cases is mandatory in order to achieve good results not only surgical, but also oncological. we present our series of neuroendocrine tumoirs treated by tamis approach, including technical aspects, deffect closure techniques and data regarding pathological findings.all cases were low grade carcinoid tumours. resection with free margin was obtained in all cases. defect closure was performed in all cases. the tumours were settled , and cm form the anal verge. postoperative course was uneventful, ann no adyuvant therapy was needed.tamis apporach for rectal neuroendocrine tumours is a safe and feasible technique. proper selection of the cases is mandatory in order to achieve good results. surg endosc ( ) aim: to assess the safety and efficacy of single layer of barbed vs double layer 'hybrid' (interrupted and running) suture for the closure of anastomotic stapler access enterotomy after laparoscopic right colectomy with intracorporeal anastomosis. methods: from april to november , laparoscopic right colectomy with intracorporeal anastomosis were performed in our surgical department. all patients in both groups were perioperatively managed using an eras pathway. seventy-two patients had the enterotomy closed with a single layer running suture of filbloc tm (assut europe). these patients were matched with patients who underwent intracorporeal right colectomy with enterotomy closed with a 'hybrid' double layer technique (first layer interrupted stitches in maxon tm - (covidien), second layer using a running suture in pds tm - (ethicon). intraoperative variables, anastomotic leak rate, morbidity and mortality rates were analyzed. results: the two groups were homogeneous with respect to demographics, body mass index (bmi), american surgical association score (asa) as well as for tumor stage. in the barbed group, median operating time was . min vs . min in the hybrid group (p = . ). anastomotic leak occurred in ( . %) patients in the hybrid vs ( . %) patients in the barbed group (p = . ). all patients required a reoperation. intraoperative findings show in ( . %) cases in the hybrid group a leak at the enterotomy closure, while an intact staler access was observed in both patients in the barbed group. no difference was observed with respect to noninfectious complications between the two groups (p = . ). patients in the hybrid group experienced a longer hospital stay when compared to the barbed group (p = . ). a re-admission occurred in the hybrid due an intraabdominal collection, while no re-admission was observed in the barbed group. no patient died in the postoperative period. aims: lymph node status is one of the key prognostic factors in patients with colorectal cancer, and remains the most important selection criteria for adjuvant chemotherapy. it is believed that at least % of node negative patients will suffer disease recurrence within the first years after surgery. this may be due to understaging lymph node status. sentinel lymph node mapping is widely used for staging of breast cancer and melanoma, with injection of colloid tc and isosulfan blue (ib). however, indocyanine green (icg) fluorescence guidance is a new technical approach to this issue, with promising results as it is not influenced by body mass index or lymphatic invasion. intraoperative fluorescence icg navigation also aims for detection of aberrant lymphatic drainage outside the planned resection. the icg lymphography has the advantage of offering a good visualization of the lymphatic channels but there are problems to identify the lymphatic nodes. our objective with this study is to rate the use of the intraoperative lymphogram in cases of elective colorectal surgery to evaluate if there were changes in the surgical attitude regarding the performance of lymphadenectomy. methods: indocyanine green was injected into the submucosal layer around the tumor at points with?a -gauge localized injection before lymph node dissection and the lymph flow was observed at , and min after injection, using a near-infrared camera system. in addition, a complete mesocolic excision with central vascular ligation guided the region where the lymph flow was observed to be fluorescent. the following table summarizes the procedures carried out as well as the lymphadenectomy performed before and after the use of icg. in brief, after the application of intraoperative icg it was observed that in % of patients additional lymph nodes were obtained after the expansion of the surgical plan, moreover % affected lymph nodes were spotted after the expansion of the surgical plan. conclusions: intraoperative real-time visualization of the lymph flow using indocyanine green fluorescence imaging during laparoscopic colon cancer surgery is feasible and a helpful technique for lymph node mapping which may lead to intraoperative changes in lymphadenectomy. tamis resection of rectal tumours has proven to be a sefe and feasible technique, specially for lesion located in the mid and low rectum.when the tumour is located in the upper rectum, and specially near the colorectal junction, tamis resection may be more difficult, not only due to technical aspects, but also due to the risk of a free perforation, specially when a full thickness resection is performed.we present our results of tamis resections of lesions located around the colorectal junction. four resections where performed with the aim of an endostpaler in order to achieve full resection without the risk of a free colonic perforation.in cases, an abdominal combined laparoscopic exploration was made, in order to help and assure proper resection of the lesion as well as avoiding intraoperative complications.distance from the anal verge ranged from to cm.postoperative course was uneventful in all cases, and a complete specimen resection was obtained in all cases.tamis resection of tumours located in the rectosigmoid junction may be a safe and feasible technique in selected patients. methods: between january to april , patients with diagnosis of right colon adenocarcinomas underwent right hemicolectomies. the data was analysed for patients demographic, histology, type of surgical approach, intraoperative details (length of surgical procedure, blood loss, blood transfusion, conversion rate) and short-term post-operative outcomes including complications. introduction: postoperative ischemic colitis is a life-threatening vascular gastrointestinal condition, that mainly occurs after cardiovascular surgery. we present a surprising case following a laparoscopic rectum resection. case report: a -year-old diabetic patient with upper rectal adenocarcinoma undergoing laparoscopic anterior rectal resection (partial mesorectum excision) and mechanical anastomosis following chemotherapy / radiotherapy. after h postsurgery he presented abdominal pain, distension and fever. on adominal computed tomography (ct) scan (contrast enema) no anastomotic leakage (al) finfings were revealed. neither digital palpation nor proctosigmoidoscopy ( th day) showed al signs. the patient clinical situation improve with conservative treatment (antibiotics, digestive rest …), c-reactive protein levels decreased and the blood cultures were negative. on the th day he was discharged presenting semiliquid stools. eight days later he needed hospital readmission: air and feculent/purulent discharge from the previos abdominal drainage orifice. ct scan: no evidence of dehiscense found although rectum and sigmoid colon distention and an image of a 'large fecaloma' were observable. on the th day of hospitalization he expulsed a large malodorous segment of tissue with necrotic asppearance (image) through the anus with surprising histologic features: 'complete-thickness necrotic colonic wall'. further rectosigmoidoscopy: complete anastomosis, signs of ischemic colitis proximate to the anastomosis and a fistulous orifice. surprisingly, the patient progressed favorably, being discharged the th day for ambulatory control with a low debit enterocutaneous fistula. histopathological diagnosis: ypt n m . follow up: the fistula discharge quantity increased maintaining diarrheal stools through anus along with persistent anemia and malnutrition. a exploratory laparotomy was schedule. fistulous tract towards a small stenotic segment of colon inmediately proximal to the colorectal anastomosis was identify and resected. finally a terminal colostomy was performed. subsequent postoperative without incidents. currently the patient is asymptomatic. comments: it seems indisputable that a colon segment, proximal to the anastomosis, was necrosed and expelled through a colorectal anastomosis. the mechanism seems inexplicable to us. it is even more disconcerting that there was no disruption of the anastomosis. objectives: fluorescence-guided surgery has emerged as a new imaging modality to improve the detection of liver and lymph node metastasis in colorectal cancer. in right-sided colon cancer, the standard lymphadenectomy should reach the ileocolic vessels and the right branch of the middle colic vessels. the purpose of this study is to perform an objective estimation of lymphatic drainage and metastatic lymphonodes in right-sided colon carcinoma through indocyanine green (icg) lymphography. methods: patients with right-sided colon adenocarcinoma were included, excluding those in stage iv, t and those who underwent urgent surgery. cc of icg peritumoral were injected using a peripheral intravenous catheter at the beginning of the intervention. the lymphatic drainage mapping of the tumor was identified. lymphadenectomy of the ileocolic vessels and right branch of the middle colic vessels was performed extending it to the left branch and origin of middle colic vessels if it was shown in the mapping. results: patients were included. the average age was . in patients the tumor was located in the ascending colon and in patients in the hepatic angle. in patients, the mapping showed lymphatic drainage to ileocolic vessels and right branch of the middle colic vessels. in patients ( %) it showed drainage to the left branch and origin of the middle colic artery, therefore extended lymphadenectomy was performed at that level. in patients, the postoperative period was uneventful. patient presented infection of the surgical wound and another patient developed a cm perianastomotic collection treated with percutaneous drainage. the anatomopathological report showed nodal metastasis in of the patients ( %) in whom lymphatic drainage was observed in the territory of the middle colic vessels with icg. these patients presented the tumor in the hepatic angle. therefore, of the patients with right-sided colon carcinoma ( %) presented nodal metastasis in the territory of the middle colic vessels. conclusions: fluorescent lymphography may improve the results of lymphadenectomy in colon cancer. in patients with tumors of the hepatic angle, lymphadenectomy extended to the left branch and origin of middle colic vessels, could be an adequate alternative. introduction: over the last decade, the common principles of surgical treatment in colon surgery are central vascular ligation (cvl) and complete mesocolic excision (cme). however, the superior mesenteric vessels anatomy, while performing the right colectomy is characterized by wide variability, which can lead to complications, especially during minimally invasive surgical intervention. objective. the purpose of this study is describing vascular variations around the superior mesenteric artery and vein-middle colic, right colic and ileocolic vessels, henle trunk in the laparoscopic right colectomy. materials and methods: the study was held in the 'dobrobut' clinic and o. o. bohomolets national medical university, department of general surgery (kyiv, ukraine) during the - period. patients were included to the study, females ( . %), males ( . %) in the average age of , ± , years. all the patients underwent the laparoscopic right colectomy (cme ? cvl) with d lymph node dissection. recorded video materials from each laparoscopic right colectomy were analyzed during the study. results: ileocolic vessels were the most stable. there were typical anatomical position in all cases. . % of cases, ileocolic vein was identified anteriorly to the ileocolic artery, while . % being posteriorly. right colic vein was absent in . % of cases. right colic vein drainage was to henle trunk and inferior mesenteric vein in . % and . % respectively. the right colic artery was present in % of patients, it's origin was superior mesenteric artery in . % and . % the middle colic artery. the middle colic vein was present and drained to superior mesenteric vein in % of cases. same as the middle colic artery with the superior mesenteric artery origin. henle trunk was present in . %, gastro-pancreato-colic trunk in . % of cases gastro-pancreatic trunk in . %, gastro-colic in . %. conclusions: knowing the options of surgical vessels anatomy, while performing the right colectomy, altogether with surgeons preparation, using the ct-scan data can reduce the risk of iatrogenic damage and complications risks. introduction: the enhanced recovery after surgery (eras) protocol was designed to accelerate convalescence, reduce morbidity and shorten the length of hospital stay (los). one of its major interventions is balanced perioperative fluid therapy. the impact of this single intervention on short-term outcomes is widely discuss. aim: the aim of this study was to assess the impact of perioperative fluid therapy on short-term outcomes. material and methods: the analysis included consecutive prospectively registered patients operated laparoscopically for colorectal cancer between november and january . patients were divided into two groups: balanced (= ml) or unbalanced ([ ml) perioperative fluid therapy. all patients were treated according to eras protocol. study outcomes were: recovery parameters, morbidity rate, los, -day readmission rate. results: group consisted of and group of patients. there were no statistically significant differences between the groups in terms of demographic and operative parameters. morbidity was lower in group ( . % vs . %, p = . ). patients in group were discharged home earlier than in group ( vs days, p \ . ). moreover, we observed differences in recovery parameters between the groups: tolerance of an oral diet on the st postoperative day ( % vs. %, p = . ) and patient mobilization on the day of surgery ( % vs. %, p = . ). -day readmission rate was lower in group ( . % vs. %, p = . ). conclusion: a balanced perioperative fluid therapy on the day of surgery may be associated with faster convalescence, lower morbidity rate, shorter los and lower -day readmission rate. methods: a retrospective analysis was performed including patients who underwent lcs or ocs for cancer treated as emergency in a single centre between and . patients who underwent palliative surgery were excluded. lcs were : propensity score-matched based on pposum and stage of disease with ocs. short-term outcomes included oncological quality, length of hospital stay (los) and postoperative mortality. for long-term outcomes, -year overall and disease free survival (os and dfs) rates were analyzed. results: during the study period, a total of emergency colorectal resections were performed. of them, % (n = ) were coloniccancers. lcs were matched to an equal number of ocs.median age was ( ) years and % were females. median follow-up was ( ) months. the majority of resections were right hemicolectomies ( %), followed by sigmoid resections ( %) and subtotal colectomies ( %). operative time ( ( ) background: total mesorectal excision (tme) offers the best reported rates for local recurrence and survival in patients with rectal cancer. our series from a single high-volume center, assessed the feasibility, safety and long-term oncologic adequacy of laparoscopic total mesorectal excision methods: we reviewed the prospective database of consecutive unselected patients undergoing laparoscopic tme for rectal cancer between and at the department of general surgery, onze-lieve-vrouwziekenhuis hospital (olv), campus aalst, belgium. the objective of the present study was to evaluate the effectiveness of laparoscopic tme, with an emphasis on perioperative variables and long-term oncological outcomes. results: pts with mid and distal rectal cancer up to cm from the anal verge had laparoscopic tme resection. patients ( . %) underwent a sphincter-preserving surgery and the remaining patients ( . %) had an abdominoperineal resection. end-to-end anastomoses: pts ( %), j-colonic pouch: pts ( % introduction: the rica clinical pathway (intensified recovery in abdominal surgery), also called surgical multimodal rehabilitation, is the application of a series of perioperative measures and strategies in those patients who are going to undergo a surgical procedure with the objective of reducing secondary stress to the surgical intervention. in this way, we achieve a better recovery of the patient and significantly reduce complications and morbidity. objective: s to analyze, through our database of patients undergoing crc, the percentage of postoperative ileuses and the following quality indicators: the postsurgical hospital stay, the anastomotic leak, and the infection of the surgical site. to check if the implantation of the rica pathway has meant an improvement in our postoperative hospital stay and with that, a lower sanitary cost. methods and results: we analyzed the data collected from those patients who underwent ccr in our hospital between / / and / / , during which time we implemented the rica clinical pathway. the average hospital stay was days. of the patients, . % presented anastomotic leak, . % infection of the surgical wound and . % paralytic ileus. we have verified how the average hospital stay increases with the appearance of anastomotic leak ( . days), infection of the surgical wound ( . days) and paralytic ileus ( . days). when we divided this -month period into two halves to see the impact of the implantation of the clinical pathway, we obtained the following results: the post-surgical hospital stay in the period from / / to / / was . . the stay from / / to / / was . . the implantation of the rica clinical pathway is providing us with important advantages in our clinical practice, with greater postoperative comfort and an improvement in our quality indicators, such as the decrease in the average hospital stay of our patients. on the other hand, after starting its implementation we have encountered the resistance to change clinical habits and the one that requires a multidisciplinary participation, so adherence to this is being progressive, and requires periodic audits to reinforce and consolidate our achievements, and identify our points of improvement. however, tem has not yet achieved widespread use. recently, transanal minimally invasive surgery (tamis) using single-port surgery devices has been reported. initially facilitated by existing single-port surgery devices, two platforms for transanal access, the gelpoint Ò path (applied medical, rancho santa margarita, ca, usa) and the sils tm port. the gelpoint Ò path is the only platform to be specifically designed for tamis y tatme. objetive: in the present study, usesa gelpoint Ò path was performed in patients with lower rectal neplasms. results: complete full-thickness excision was performed in all cases of tamis and free margins over rectal cancer. on two cases no neoplasm was visualizad. the patient characteristics, operative techniques and operative outcomes were evaluated. the mean age of the patients was . years (range - ). the mean operating time were min (range - ). patients was selsted for tatme, for tamis and two patients for evaluation and biopsy if was necesary. additional transabdominal rectal resection was not performed, and adjuvant chemoradiotherapy was performed in all cases. tamis using a gelpoint Ò path was revealed to be easy and safe to perform. although only a small number of cases were treated, and the operation was demonstrated to be sufficiently feasible. conclusion: gelpoint path is a good tool for colorectal surgery in tatme, tamis and evalluation of anastomoses or de novo lesions introduction: several improvements in rectal cancer treatment, in the last decades, resulted in a markedly increased survival. nevertheless, surgery remains the prevalent treatment and to % of operated patients experience some kind of functional abnormalities. as nowadays we acknowledge the importance to focus not only on survival rates but also on quality of life, we craved for a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, we performed a thorough translation with cultural adaptation of the patient reported outcome tool, low anterior resection syndrome (lars) score, to the portuguese language (lars-pt) and population. methods: according to the current international recommendations, we designed this study encompassing three main phases: (i) cultural and linguistic validation to european portuguese; (ii) feasibility and reliability tests of the version obtained in the previous phase; and (iii) validity tests to produce a final version. the questionnaire was completed by patients from six portuguese colorectal cancer units, and completed it twice. results: the portuguese version of lars score showed high construct validity. regarding the test-retest, the global intraclass correlation showed very strong test-retest reliability. looking at all five items, only items and presented a moderate correlation. lars score was able to discriminate symptoms showing worse quality of life in patients submitted to preoperative radio and chemotherapy. conclusion: lars questionnaire has been properly translated into european portuguese, demonstrating high construct validity and reliability. this is a precise, reproducible, simple, clear and user-friendly tool for evaluating bowel function in rectal cancer patients after sphincter saving operation. therefore, his sistematic use should be implemented. oesophagectomy is the mainstay of curative treatment for oesophageal cancer and post-oesophagectomy diaphragmatic hernia (podh) represents a potentially life-threatening surgical complication characterized by an underestimated occurrence rate and unknown related risk factors. this study analyses the experience of two tertiary designated centers in order to evaluate key elements concerning development and treatment of podh. a cohort of consecutive patients affected by a clinically resectable oesophageal cancer (any t, any n and m ) underwent ivor-lewis oesophagectomy between march and april according to three different approaches: totally open incision procedure (oilo), hybrid (hilo) and totally mininvasive to esophagectomy (milo). all population was retrospectively observed in the context of a postoperative calendarised follow-up in order to record the incidence and postrepair results of podh. patients underwent ivor-lewis oesophagectomy for cancer and ( . %) developed podh within a median follow-up period of months ( - ). surgical repair was generally applied by the mean of laparoscopic cruroplasty ( %) with a conversion rate of %. postoperative morbidity did not include early recurrences but exclusively cardio-pulmonary complications ( patients) with one case of respiratory failure leading to death. the discharge was reached after a median hospital stay of days ( - ) while recurrences ( %) occurred over a median followup period of . months. a wide univariate analysis identified statistically significant associations between podh occurrence and the administration of preoperative chemoradiotherapy, the complete pathological response (cpr) and a lymph node harvest (lnh) larger than stations (p-value of . , . and . respectively). the strong influence of an extended lnh was confirmed by the multivariable analyses ( . ) along with cpr which should however be considered as longer survival-related bias. the minimally invasive surgery and the neoadjuvant chemoradiotherapy represent a considerable part of multimodal treatment for oesophageal cancer presenting a not statistically significant association with podh development while a lnh including more than nodes resulted to be an independent risk factor mirroring the extent of surgical demolition in oesophagectomy. l. barbulescu aim: to asses the safety and effectiveness of robotic total meso-rectal excision vs laparoscopic total meso-rectal excision and to analyse the primary outcomes. methods: the operative, post-operative and oncological outcomes were evaluated to assess the effectiveness of both techniques of tme. in our center were performed robotic rectal resections and laparoscopic resections from january to present. results: the rtme was associated with longer operation time, early bowel movements, lower risk of conversion and shorter hospitalization. the statistical equivalence was seen between rtme and ltme for non-oncological variables like blood loss, morbidity and reintervention risk. the oncological variables such as number of harvested nodes and positive circumferential resection margin risk were also comparable in both groups. the length of distal resection margins was similar in both groups. conclusion: rtme in patients with rectal cancer was associated with a lower rate of conversion and less incidence of urinary retention. the operative time in rtme was significantly longer than in ltme. the initial oncological and function outcomes of rtme seem to be equivalent with ltme. c. athanasiou aims: two randomized controlled trials failed to show non-inferiority of the laparoscopic total mesorectal excision (ltme) compared to open. ltme becomes particularly challenging in low rectal cancers and in narrow pelves. many surgeons report that robotic tme (rtme) may be beneficial in that setting. our aim was to systematically review the literature and compare the pathologic outcomes of open, laparoscopic and robotic tme for rectal cancer methods: medline, embase, scopus, cochrane library and web of knowledge databases were searched for randomized controlled trials (rct) reporting patholologic outcomes of open, laparoscopic or robotic tme with no language restriction. our primary outcome was quality of tme on macroscopic assessment of the specimen. secondary outcomes included positive circumferential resection margin, distance to radial margin, number of lymph nodes and positive radial margin. the included studies were quality assessed and the jadad score was reported. the grade approach was used to rate the certainty of each network estimate. results: fourteen rcts were included in our study. seven rcts compared the otme to the ltme, six compared the ltme and rtme and one study the otme to the rtme. no statistical significant difference was found in quality of tme when the the ltme was compared to the otme or = . ( . , , ) or the rtme or = . ( . , . ) . no difference was found in pcrm for the laparoscopic or = . ( . , . ) or the robotic approach or = . ( . , . ) when compared to open. distance to radial margin and number or lymph nodes didn't differ between the groups. conclusions: no significant advantage on pathologic specimen quality has been found with the robotic approach. the ltme doesn't seem to compromise the quality of the specimen. h. samura , j. arakaki , k. sugata , y. hori , y. nagamine , f. kohagura , h. motonari , s. kameyama , t. ishimine division of digestive and general surgery, urasoe general hospital, okinawa, japan; department of surgery, urasoe general hospital, okinawa, japan colorectal cancer often invade adjacent organs and it is known that prognosis improves with resection of the involved organ. we report our experience of invaded adjacent organ resection, which include seminal vesicle, uterine and bilateral appendages, posterior wall of the vagina and bladder wall. method: although the range of resection is predicted by image study preoperatively, at the time of operation, it was decided by palpation with a forceps. each operation is evaluated by operation time, blood loss, blood transfusion volume, postoperative complication, postoperative hospital stay, and short term prognosis. result: resection cases of seminal vesicle, posterior vaginal wall, uterine and bilateral appendages and bladder wall were , , and , respectively. the results are shown in the order of seminal vesicle / vaginal posterior wall / uterine / bladder. median age was , , and years old. the median operation time was , , , min, the median blood loss was , , , ml, and only one case of uterine and bilateral appendages resection required the blood transfusion. the average postoperative hospital stay was , , , days. nine cases have postoperative complication, that include delayed wound healing, anastomotic leakage and rectovesical fistula, postoperative ileus, chyle ascites and neurogenic blodder. all of those were improved with conservative treatment. the mean hospital stay in complication cases was days ( - ) and ( - ) days without complications. the median observation period was days ( - ), and there was no local recurrence. all of the case of stage iv were dead. there was no local recurrence and all patient without stage iv are alive, it seems that the resection range was sufficient. conclusion: even with adjacent organ invasion colorectal cancer, it was possible to determine the resection line by palpation with laparoscopic forceps manipulation, and possible to resect margin free of cancer. laparoscopic low rectal resection with/without diverting ileostomy p. ihnát, m. tesar, p. ostruszka, p. gunková, p. vávra background: the construction of diverting ileostomy (di) is recommended to avoid septic complications of anastomotic leakage. the aim of our study was to assess the benefits and risks of di constructed during laparoscopic low anterior resection (lar). methods: retrospective clinical cohort study was conducted in university hospital ostrava, czech republic. all patients undergoing laparoscopic lar with tme because of rectal cancer within a -year study period were assessed for study eligibility. results: a total of patients ( patients without di, patients with di) after laparoscopic lar were enrolled into the study and underwent analysis. both study subgroups were comparable in terms of demographic and clinical features. postoperative -day morbidity was significantly lower in patients without di ( . % vs. . %, p = . ). anastomotic leakage frequency was higher in patients without di ( . % vs. . %, p = . ); surgical intervention was necessary in . % of patients without di. stoma-related complications were noted in . % of patients with di; some patients had more than one complication. surgical intervention because of stoma-related complications was needed in patients ( . %). distinctive complications of di laparoscopic construction (small bowel obstruction due to di semi-rotation around its longitudinal axis) was noted in patients ( . %). mean stoma period (interval between lar and di reversal) was more than months in our study; only . % of patients were reversed without delay (= months). postoperative morbidity after di reversal was . %; re-laparotomy was needed in . % of patients. conclusions: despite benefits of di in protecting low rectal anastomosis, ileostomy construction remains fraught with many stoma-related complications and long stoma periods associated with significantly decreased quality of life. aims: single port laparoscopic is a minimally invasive surgical technique that joint the cosmetic advantages with the well recognized benefits of the standard laparoscopic approach [ ] . we describe a laparoscopic single port hartmann reversal in a patient by the use of the umbilical colostomy site for surgical access [ ] . methods: a years old patient was submitted to a laparoscopic single port hartmann procedure with an trans-umbilical colostomy for a recurrent sigmoid volvulus that was treated at the beginning by endoscopic de-rotation. after three months the patient was reevaluated for a hartmann reversal with a laparoscopic single port technique. after routine skin preparation and laparoscopic setup, the colostomy is mobilized from its mucocutaneous border, and the anvil of a circular stapler is secured to the distal lumen. by the use of a gelpoint system with trocars, the intra-abdominal adhesiolisis in performed. the splenic flexure is mobilized to achieve a sufficient mobilization of the left colon that allows the fashion of a tension free anastomosis. the rectal stump is mobilized to the mid rectum, starting from the posterior mesorectal fascia around to the anterior rectal wall. a tension-free colorectal anastomosis is secured with a standard circular mm stapling device inserted transanally. the colostomy wound is closed. the operative time was min. results: the postoperative course was uneventful, the patient was discharged at forth postoperative day, oral intake started on postoperative day three. conclusions: single port laparoscopic hartmann reversal thought the umbilical stoma site is a minimally invasive surgical option that is safe in selected patients and offer the best cosmetic results. [ the progressive evolution of surgical techniques and oncologic protocols on rectal cancer disease facilitates surgeons to challenge the skills for anus preservation in low rectal cancer surgery. the laparoscopic surgery is already one of the best ways to reach the pelvic floor and to try procedures, which were previously difficult to apply through open surgery. the anastomotic leakage has particularly high occurrence if the anastomosis is performed in the anal or distal rectum area. it is evident that although the fecal diversion does not decrease post operatory mortality, it significantly reduce the risk of anastomotic leak and the risk of a second major surgery when the leak occur. diverting stomas are low-risk procedures from a technical point of view, but they potentially expose the patients to postoperative morbidity, impacting the patients' quality of life. it is not easy to decide whether the fecal diversion is needed or not. this decision must be made on a case to case basis, trying to apply the stomas only when they are really needed. we report our initial experience by living a transmesenterial cotton loop around the pre terminal ileum which extremities are turned out usually through the lateral trocar wound in laparoscopy or by applying a dedicated mini incision in open surgery. the purpose is to perform (in case of suspected fistula), a mini invasive diverting procedure, by widening the loop wound and by pulling up the ileum in a lateral loop ileostomy. we applied this procedure to consecutive patients with low colorectal anastomosis and in two of them we performed a lateral loop ileostomy with good results. we believe this can be an alternative that needs to be standardized. purpose: sarcoidosis is a chronic, multisystem inflammatory disorder with unknown aetiology characterised by noncaseating granulomas within involved organs. gallbladder involvement in sarcoidosis is extremely rare and literature review revealed only reported cases to date. in this paper, we present a case of gallbladder associated sarcoidosis. method: a -year-old lady was known to the clinic for regular surveillance of liver steatosis and incidental gallbladder polyps. the largest polyp was mm at presentation in and has grown to mm in . in view of worsening symptoms of biliary colic and growing polyps, a laparoscopic cholecystectomy was performed. results: laparoscopic cholecystectomy was unremarkable and specimens of the gallbladder and lymph nodes were sent for histology. histological examination revealed chronic cholecystitis with polypoid cholesterolosis of the gallbladder and noncaseating granulomata within a lymph node, which strongly suggest sarcoidosis. conclusion: in conclusion, we report a case of incidental finding of gallbladder sarcoidosis over the course of treatment of biliary colic and symptomatic gallbladder polyps. therefore, the definitive treatment for patients with symptomatic gallbladder sarcoidosis is a cholecystectomy. the surgical management of cholelithiasis can be associated with significant morbidities. despite the relatively low incidence of bile duct injuries during laparoscopic cholecystectomy, the total number is large due to the high frequency of the operation. the subtotal cholecystectomy with its variants is a well known bailout strategy to the surgical community. however, there is no agreement on when and how to perform these procedures. indeed, the majority of surgeons will adopt these solutions when there is a struggle to identify the critical view of safety. this struggle results increases the risk of injuries. we hypothesize that a primary intent gall bladder lithotomy and disconnection (glad) when the dissection of the gb pedicle is anticipated difficult dissection is a safe and feasible strategic option. methods: out patients elevtively admitted to aberdeen univesity hospital with gall stone disease between march and november , consecutive patients were operated with glad procedure based on intraoperative criteria. the primary outcome was the operative time. secondary outcomes were length of hospital stay, the criteria to do this procedure will be explained, the outcomes will be listed. indocyanine green is a molecule that becomes fluorescent when excited struck with light of a specific wavelength in the infrared spectrum (nir-infrared), allowing the visualization of anatomical structures in which it has accumulated. the aim of the study is the application of icg enhanced fluorescence in laparoscopic cholecystectomy in order to identify the anatomy of the biliary tract, to reduce the risk of iatrogenic lesions and the conversion rate. the study involves laparoscopic cholecystectomy for cholecystitis and gallstones of main biliary tract. the evening before the surgery, a vial of icg ( mg) diluted in ml of saline solution was intravenous injected. during the procedure, after opening the calot triangle, switching to the nir mode on the camera, the anatomy of the biliary tract and in particular of the main biliary tract is visualized. the cystic duct and cystic artery are isolated, their section between clips is cut and the cholecystectomy is performed. from january patients were enrolled: cases of acute cholecystitis, cases of gallstones of main biliary tract, undergoing preoperative ercp. in cases of cholecystitis, the angiography allowed the visualization of the main biliary tract. in one case, an abnormal course of the cystic duct was identified. in two cases of gallstones of common bile duct, it favoured the visualization of the biliary tract anatomy. all cases were completed with laparoscopic technique. there were no intra-and post-operative complications. icg-enhanced fluorescence is a safe, effective, cheap and rapid tool that can also be applied in small hospitals with no need for training. its use does not extend the time of surgery and allows the visualization of the anatomy of the biliary tract, especially in situations where it can be altered by reducing the conversion rate and potentially the risk of iatrogenic lesions of the main biliary tract. case presentation: patient is a year old female with no significant past medical or surgical history presented to the emergency department with a day history of worsening sharp right upper quadrant pain with associated nausea, vomiting, and po intolerance. the pain started a few months prior, however it was self-limited with diet modifications. an ultrasound demonstrated a contracted gallbladder with a mm gallbladder wall. white blood cell count was within normal limits and total bilirubin was slightly elevated to . mg/dl. no palpable mass was noted on physical exam. an mr cholangiopancreatography was performed which demonstrated a dilated gallbladder measuring . x . cm, a severely thickened gallbladder with a small intramural collection and multiple gallstones. the patient proceeded with a laparoscopic cholecystectomy. intraoperatively, the omentum was densely adhered to the gallbladder and needle decompression of the gallbladder was unsuccessful due to the wall thickness. the gallbladder was subsequently removed without any complications. patient's remaining hospital course was uncomplicated. surgical pathology returned demonstrating acute on chronic cholecystitis. discussion: cholecystomegaly or 'giant gallbladder' disease is a rare pathology encountered in the surgical world. there have been few reported cases, most of which occurred in the elderly ([ years). kuznetsov et al. defined an enlarged gallbladder to have a volume of - cc and a giant gallbladder as exceeding cc (the average weight of the liver). the etiology remains unknown, however certain factors exist to allow the gallbladder to reach this size without life-threatening sequela. preoperative imaging, such as mr cholangiopancreatography, is important to differentiate biliary pathology and delineate anatomy. removal of the gallbladder is recommended to prevent the development of complications like cholangitis or bowel obstruction. the cause of cholecystomegaly still remains uncertain and warrants further research. the management and treatment remains similar to acute cholecystitis. aims: mini-laparoscopic cholecysectomy (mlc) is considered to be the best variant of minimizing surgical trauma and improving cosmesis in laparoscopic cholecystectomy. the most challenging techniqual step of mlc is clipping the cystic duct. it may be impossible or unsafe when diameter of cystic duct exceeds mm, which is common in severe chronic colecystitis or acure cholecystits. there is very limited data in the literature about the use of mlc in acute cholecystits. the aim of study was to access the first results of new technique of mlc. methods: five women with the mean age of years ( - ) underwent mlc. the st -mm troacar was inserted in the umbilicus and used for the camera and removal of the gallbladder. the nd -mm troacar was inserted in subxyphoidal area and used for the main working instruments, including medium-large polymer clip-applier (hem-o-lok type). the rd and th -mm troacars were placed in right subcostal area and used for mini-graspers (karl storz). in initial procedures we used conventional -mm clip-applier with adopted medium-large titanium clips. to improve safety, we aplied -mm hem-o-lok type clip-applier for the last patient with acute cholecystitis. in this case the diameter of cystic duct was , mm. the clipping was performed successfully. the -mm drain was placed via subcostal troacar incision. also, in this case we applied original technique of removal of the bladder using wound retraction instrument (karl storz). results: in all the cases there were no intra-or postoperative complications. the mean duration of procedures was min ( - min). the postoperative stay was days in every patient. the patients estimated their pain on postop day as 'almost absent' and cosmethic results mo postop as 'exellent'. conclusions: . new technique of mlc alowed to perform the clipping of cystic duct safely, which is essential in acute calculous cholecystitis. was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as cholelithiasis included in the study and were planned either for mini-laparoscopic cholecystectomy and conventional laparoscopic cholecystectomy were explored for outcome while the patients with empyema gallbladder, gangrene, mucocele gallbladder and adhesions were excluded from the study. results: during one year study period, total five hundred patients were diagnosed as cholelithiasis with means age . ± . (sd). of five hundred, ( . %) were underwent for mini-laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), excellent cosmetic results, mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), minor oozing ( . %), port size hernia ( . %). remaining ( . %) were underwent for conventional laparoscopic cholecystectomy with ( . %) were males and ( . %) were females. the outcome were measured as postoperative pain (vas) . ± . , size of wound (umbilical mm, epigastrium mm and subcostal mm), mean ± sd for hospital stay (hrs) and operative time (minutes) was . ± . and . ± . , early return to work ( . %), port size hernia ( . %) along with zero ( %) mortality. conclusion: it has been concluded that mini-laparoscopic cholecystectomy is superior and feasible than conventional laparoscopic cholecystectomy and has decreased early postoperative incisional pain, avoided late incisional discomfort and safe procedure with nearly scarless wounds with superior cosmetic effect especially for young female patients. objective: to determine the outcome of immediate versus late laparoscopic cholecystectomy in acute cholecystitis at tertiary care hospital hyderabad / jamshoro sindh pakistan patients and methods: the descriptive case series study of one year ( - ) was conducted in department of surgery lumhs jamshoro. all the patients having age = year of age, either gender presented with history of abdominal pain, nausea and vomiting and were diagnosed as acute cholecystitis (cholillthiasis) included in the study and were planned for laparoscopic cholecystectomy and were explored for outcome as immediate (within h) and late components ([ weeks). the frequency and percentage was calculated for categorical variables and mean ± sd was calculated for numerical variables. as this was descriptive case series so there was no any statistical test of significance was applied. results: during one year study period, total one hundred patients were diagnosed as acute cholecystitis with means age . ± . (sd). of one hundred, % were females and % were males. the immediate outcome reported as tissue fragile %, pancreatitis %, slipage of ligature of cystic duct %, empyema gallbladder %, mucocele % and gangrenous gallbladder % while the late outcome reported as adhesions %, cholecystoduodenal fistula and mirizzi syndrome % and %, gallstone ileus %, perforated gallbladder % and cholidochiolithiasis % while the mean ± sd for hospital stay (days) in immediate as . ± . while in late outcome (days) during acute cholecystitis . ± . and after surgery ( weeks later) as . ± . respectively. conclusion: it has been concluded that early lc for acute cholecystitis with cholelithiasis is safe, low cost and feasible intervention and offering the additional benefit of shorter hospital stay and reduce the economical burden. surg endosc ( ) :s -s general surgery, chang gung memorial hospital kaohsiung division, kaohsiung, taiwan background: the treatment of common bile duct (cbd) stones is challenging while unclear hepatic hilum anatomy especial experience of previous laparotomy. a minimally-invasive approach choledocholithotomy is feasible, but can be difficult and converted for the unclear anatomy of the biliary tree. near-infrared (nir) cholangiography by systemic administration of indocyanine green (icg) can enhance the visualization of the biliary tree anatomy but is limited by the high intensity of background fluorescence signal coming from the liver. nir fluorescence cholecysto-cholangiography by direct biliary tree administration of the icg can enhance the biliary tree without background noise signal. we created the nir cholangiography via different route according to patient situation : systemic circulation or biliary tree injection to see the feasibility of those application. material and method: ten patients who suffered from obstructive jaundice due to cbd stone and patients received percutaneous biliary tree drainage as first treatment and patients received endoscopic biliary tree drainage. those patients received laparoscopic choledocholithotomy as definite treatment after acute infection phase. patients received biliary tree icg injection via drain tube and patients by systemic injection. visualization and fluorescence patterns around cbd was recorded. results: in our series, one patient received previous gastrectomy and patients had previous biliary tree surgery. background: laparoscopic cholecystectomy (lc) has become the gold standard for the treatment of gallstone disease. multiple studies have confirmed its safety, lc at index admission is still not widely practiced in ireland. we present our experience in performing index cholecystectomy at cuh after the start of acute care surgery program in may . aim: the aim of this study is to determine the safety of laparoscopic cholecystectomy at index admission, complications,re-admissions, and los. methods: electronic records, theatre records and imaging reports were searched to enroll all patients who underwent lc for gallstone disease at index admission from may to october . patient demographics, indication for surgery, postoperative complications, readmission and conversion rate were recorded.in addition timings of mrcp and ercp, imaging findings, and los were also noted. results: a total of patients underwent lc during the study period. median age was years ( - ). male to female ratio was : . . ( %) patients had acute cholecystitis, ( %)had acute biliary pancreatitis, ( . %)biliary colic and ( . %) had cholecystitis with signs of cbd obstruction. ( . %)patients had obstructive jaundice and one with adenomyomatosis. patients ( %) had preop mrcp while ( %) underwent pre-op ercp. all except patients undergoing ercp had preprocedure mrcp. patients had pre-op cholangiograms. in terms of complications, ( . %) patients had bile leak and one( . %) had re-operation. one patient had the post-op hematoma which was drained percutaneously, one patient had procedure abandoned because of bradycardia upon induction of anesthesia. there was no common bile duct injury, no conversion to open and no days mortality was reported. the average length of hospital stay has been days. ( to days). conclusions: laparoscopic cholecystectomy at index admission for cholecystitis, choledocholithiasis, and biliary pancreatitis, has been a safe and feasible treatment option in our hospital. a safe practice can be ensured by adherence to a care pathway and a multidisciplinary, consultant-led service. index cholecystectomy service can be provided safely across the country to prevent diseaserelated morbidity and multiple re-admissions in patients awaiting interval surgery. when to use the two-stage surgery to treat choledocholithiasis: the size aims: the treatment of choledocholithiasis has been provided by various of studies worldwide. the most common accepted minimal invasive treatment was two-stage treatment using endoscopic retrograde cholangiopancreatography before or after laparoscopic cholecystectomy(ercp ? lc), and one-stage treatment with laparoscopic exploration of the common bile duct(lcbde). in fact, despite several large studies have been published in recent years, the debate for the ideal treatment of choledocholithiasis is way from being concluded. we aim to find the proper treatment option for the patients with variable sizes of choledocholithiasis. methods: we retrospectively analyzed patients who underwent treatments for cholidocholithiasis in our institute between january , and july , . the patients who received either ercp and lc in the same admission, and the patient who received lcbde, irrespective of trans-cystic(ltcbde), or choledochotomy(lcd), were included. the data was analyzed with chi-square test and mann-whitney u test. results: the stone size of the ercp ? lc group is significantly smaller than the lcbde group. we further analyzed the ercp failure case, and the group of stone size [= . mm has a significantly higher rate of procedure failure. the failure rate is increasing with the stone size. conclusions: both the treatment of lcbde and ercp ? lc have similar safety and success rate, and the rate of residual stone was also similar in both group. however, the failure rate for ercp is significantly increased when stone size is larger than . mm in this study. aims: the xanthogranulomatous cholecystitis (xc) is a rare entity that can cause doubt in the choice of surgical treatment, because of differential diagnosis with gallbladder carcinoma (gc). methods: a -year-old patient presented acute abdominal pain in the right upper quadrant, nausea and low-grade fever with signs of peritonitis. he had elevated pcr, leukocytosis with neutrophilia. abdominal ultrasound showed an acute xanthogranulomatous cholecystitis. a laparoscopic cholecystectomy was decided but it was converted to open surgery due to the difficulty in the dissection, with fundus embedded in the hepatic bed and intraoperative finding of hilar adenopathic conglomerate .the postoperative period was torpid, with abdominal pain, jaundice, elevated bilirubin and enzymes of cholestasis. postoperative abdominal tomography showed injury in the iv segment of the liver suggestive of neoplasia. metastatic adenopathic conglomerate at the hepatic hilum caused extrinsic biliary obstruction with hepatic failure later so an internal-external drain was placed in the bile duct. the patient was died a week later. the pathological anatomy reported a stage four of gc. results: xc is a rare, non-neoplastic, inflammatory and destructive entity of the gallbladder wall, considered a variant of chronic lithiasic cholecystitis. it may be due to extravasation of bile or ulceration of the mucosa, causing an inflammatory reaction and fibrosis, with xanthomatous cells. the prevalence is to % in the resected gallbladders. it is more frequent in - years oldfemales. its clinical presentation does not have specific characteristics that differ from cholelithiasis, except for the weight loss. radiologically it is characterized by nodular thickening and increased attenuation of the vesicular wall with signs of cholecystitis, indistinguishable from a vc. the xanthogranulomatous inflammatory foci infiltrate the hepatic parenchyma, having an invasive behavior; hence, it mimics a neoplastic disease. the confusion in diagnostic and the risk of gc (up to %) makes treatment contentious. conclusions: the xc can simulate an advanced gc that sometimes makes us wonder if we should perform a radical surgical treatment; when presented in an emergency situation, our therapeutic decision can focus on solving the acute problem and be conditioned by the patient's general condition. single port transumbilical laparoscopic surgery (sptls) is a techinque that has been around for about years. although the enthusiasm for this type of surgery seems to have diminished in recent years it is expected to rise considering the recent development of sophisticated devices for its execution . we report retrospectively our year experience with procedures performed by sptls technique. in a private practice setting in mexico city. procedures include cholecystectomy ( ), appendectomy ( ), inguinal hernia tapp and tep ( ), hiatus and esophageal ( ) , sleeve gastrectomy ( ), colon ( ), gyn ( ) . different access platforms were employed. we explain our selection criteria for the application of the technique and describe the evolution of the instruments employed during the past years, from laparoscopic conventional to curved and bendable; regular scopes to extra long telescopes with different angles. or time, top bleeding, conversion rate, the need to employ an extra trocar, complications, pathology reports, scheduled or urgent kind of surgery and length of hospital stay were recorded from the beggining; patients variables such as bmi, asa status, tep risk, satisfaction with the procedure and other were recorded . we describe the evolution of our technique, and our learning curve with cholecystectomies. we compare our group of sptls transvaginal assisted laparoscopic hysterectomy (tvalh) patients vs tvalh multiport patients. we explain the feasibilty, and efficiency of the procedure in our hands compared to other series. background: in japan, the severity of acute cholecystitis(ac) is assessed by the severity classification of the tokyo guidelines (tg ). the value of c-reactive protein (crp) is not included in the severity classification criteria. the first line treatment, according to tg , for mild (grade i) to moderate (grade ) ac is laparoscopic cholecystectomy, but laparoscopic surgery may not be feasible in some cases due to adhesion or local inflammation of the gall bladder. aim: the aim of this study is to assess the effect of crp on the open conversion rate in laparoscopic cholecystectomy for acute cholecystitis.method: we conducted a retrospective study. patients who were diagnosed with ac and treated with emergent laparoscopic cholecystectomy between june and may in our institution are included. we set the cutoff value for crp at mg/dl and compared the open conversion rate. secondary endpoints are amount of bleeding, operation time, post-operative course (peak in body temperature and inflammatory markers) and the frequency of complications according to the clavien-dindo classification. results: out of patients had a crp value greater than or equal to mg/dl. the median crp values for the crp \ group and crp = group were . and . , respectively. the open conversion rate of the crp = group was significantly higher than that of the crp \ group ( / , / , p = . ). the most common reason for these conversions was local adhesion ( / ) . there were no differences in the amount of bleeding, operation time, post-operative course, and frequency of complications with clavien-dindo grade ii or higher. background: reports about clinical value of fluorescent cholangiography using indocyanine green (icg) during single-incision laparoscopic cholecystectomy (silc) were increasing. we report clinical value and pitfalls of fluorescent cholangiography during silc for the patients with the infraportal type of the right posterior bile duct. methods: our silc procedure utilized the sils-port with an additional -mm forceps through the umbilical incision. before silc, ml of icg ( . mg) was administrated by intravenous injection. for fluorescent cholangiography, icg fluorescent laparoscope system was used. results: we performed fluorescent cholangiography during silc in patients with the infraportal type of the right posterior bile duct. all procedures were completed successfully. the interval from the injection of icg to the first obtained fluorescent cholangiography before the dissection of calot's triangle ranged from to min. detectability of infraportal type of the right posterior bile duct before dissection in claot's triangle was . % (n = ) and that during dissection in calot's triangle was . % (n = ). the infraportal type of the right posterior bile duct could be identified under fluorescent cholangiography only when it joined into the common hepatic duct. conclusions: utilization of fluorescent cholangiography can lead silc to safe even for the patients with the infraportal type of the right posterior bile duct. its benefit is emphasized when the infraportal type of the right posterior bile duct joins into the common hepatic duct. aims: due to the development of laparoscopic surgery and the progress made in surgical treatment ofhydrocephalus, surgeons may come across patients with ventriculoperitoneal (vp) shunt, as candidates for laparoscopic procedures. according to this fact, we report a case of an unusual complication of laparoscopy surgery that can appear in this kind of patients. methods: we present a case of a -year-old man with medical history of normotensive hydrocephalus with vp shunt, that came to the emergency room complaining of abdominal pain and fever since two days. blood test showed an elevation of infection parameters and inflammatory markers, and the ultrasound study revealed an emphysematous cholecystitis. therefore, we decide to carry out an emergency laparoscopic cholecistectomy. the patient did not present any adverse event during the surgery or the immediate postoperative period, being discharged the third postoperative day and evaluated ambulatory one month after the surgery with no complications. two months after surgery, the patient returned to the emergency room presenting alteration in consciousness and fever. results: during the study of the pacient, an abdominal ct was performed, showing a complete section of the vp shunt in the subcutaneus space of the upper abdominal wall and intraperitoneal migration of the remaining catheter. the patient was transferred to neurosurgery to carry out an emergent replacement of the ventriculoperitoneal shunt. after surgery and intravenous antibiotic treatment, the patient evolved favourably and was discharged a few days later. conclusions: the rate of serious complications associated with a laparoscopic approach is overall low and up to % of them occur during the abdominal access for camera or port placement and may not be recognized until postoperative period. vp shunts should not be a contraindication for laparoscopic surgery. however, laparoscopy approach must be carry out with good anesthetic and monitoring facilities and taking several previous considerations, such as verifying the proper functioning of the vp shunt, identifying the path of the catheter within the abdominal wall to avoid inadvertent damage to the catheter during trocar placement and ensuring that the intraperitoneal portion of the catheter is not twisted or obstructed prior to decompression of the abdomen. surg endosc ( ) introduction: since advantages of robotic surgery is being more emphasized, robotic cholecystectomy (rc) cases are increasing. ajou group had introduced a method called which technique places the trocars transversally on the bikini line and it makes cosmesis and pain beneficial. however, rc with low incision port has several limitations. therefore, we changed port placement which may be a one of safe tehniques for rc. method: this study retrospectively reviewed data for patients who received rc with port changing method (rcpc, n = ) and rc with low incision port (rcli, n = ) from february -february and surgical variables were analyzed. results: patients in both groups had similar demographic features and indications for surgery. the rcpc group required no conversions to conventional robotic surgery and no additional operation, whereas the rcli group had one incisional hernia ( . %) and two bowel perforation ( . %) cases. length of stay ( . ± . vs. . ± . days, respectively; p = . ) did not significantly differ between the rcpc and scli groups. however, the rcpc group had shorter operative time ( . ± . vs. . ± . min; p = . ) than the rcli group, although the parameters mentioned above were not statistically significant. conclusion: robotic cholecystectomy with bikini line incision has some limitations even though it has cosmetic benefits. whereas robotic surgery with changing port method is one of safe and feasible procedures for performing robotic cholecystectomy. also nothing more to say that it gains cosmesis effect and escapes complications. mini surgery, odessa medical university, odessa, ukraine the aim of the study was to optimize the diagnostic and therapeutic tactics for yatrogenic injuries of the extrahepatic bile ducts. methods: patients were examined. typical manifestations were jaundice, cholangitis, biliary peritonitis, external biliary fistula, subhepatic abscess.cholecystectomy main cause of damage.a visual, manual and x-ray examination of the hepato-choledochus and cholangioscopy were performed. ultrasound, endoscopic retrograde cholangiopancreatography, fistuloholangiography or percutaneous transhepatic cholangiography play a leading role in diagnosing. the results: high damage to the bile duct was detected in . % of patients, low-in . %.percutaneous transhepatic drainage under ultrasound control was performed in . % of patients.emergency laparotomy, sanation of the abdominal cavity and external drainage of the bile ducts were performed with bile peritonitis. recovery operations produced . % of patients. reconstructive interventions were performed in . % of patients after - weeks after the first stage. the covery operations were successful in . % of patients. . % of the sick had complications in the form of biloma. a scar stricture formed in . % of patients after - months. patient underwent recanalization of the stricture zone with a dilatation balloon through interchangeable transhepatic drainage. balloon dilatation was performed retrogradely through the large duodenal papilla in patients. deaths in the postoperative period was not observed. conclusions: the surgical team should be strengthened by an experienced surgeon when intraoperative diagnosis of yatrogenic damage to the bile ducts.the operation should be completed by external drainage of the bile duct and the abdominal cavity in the absence of an experienced specialist.recovery operations are shown only with lateral injury of the ducts.the patient must be sent to a specialized institution for radical surgical treatment after stabilization of his general condition. aim of the study sub-hepatic bile collections, biloma and hematoma are rare complications and we present our experience in treatment this complications. material and methods: from laparoscopic cholecystectomy performed in our clinic, three patients (two women and one men) to whom it was performed laparoscopic cholecystectomy, came back two weeks later after they were released from the hospital because of epigastric discomfort, fever and nausea. results: clinical examination after rehospitalization showed tenderness in the epigastrium and right subcostal region. in all patients were measured high levels of leukocytosis and crp . an ultrasound examination of the abdomen revealed a large hypoechoic collection in the sub hepatic space, after the abdominal ct scan was performed, the density of the collection did not indicate the presence of blood in two patients. percutaneous drainage of the collection in both patients was realized under us guidance and - fr catheter was inserted in the sub hepatic region. in the first patient cc of bile-stained liquid, and in the second patient cc of biliary liquid was drained. in a third patient h after surgery signs of significant hypotension and limited tenderness at the right subcostal region occurred. a complete blood count (cbc) showed a decrease in the level of haemoglobin to . g%. ultrasound examination revealed a fluid collection in the sub hepatic space, which is also confirmed by computed tomography. laparotomy was performed and the large sub hepatic hematoma was evacuated. after that the fr abdominal drain was inserted into the sub hepatic space. the postoperative course of all three patients was not complicated. conclusion: sub hepatic biloma and hematoma are rare complications of laparoscopic cholecystectomy, while early diagnosis followed by percutaneous drainage or open laparotomy is the only way to resolve these complications. ( ), hemoperitoneum . % ( ) . the average number of days of hospitalization was . days. there was no mortality at days. conclusion: in the emergency setting the rendezvous technique has an adequate success rate of cannulation and clearence of the bile duct, an acceptable surgical time, few complications, these being more frequent in those patients with inflammation of the gallbladder and without associated mortality at days. there is a need for controlled randomized studies with a greater number of patients recruited and follow-up to determine the usefulness of this technique. intraoperative cholangiography could serve as a fundamental solution to avoid the bile duct injury during laparoscopic cholecystectomy. however, it is difficult to identify the cystic duct to which the contrast catheter should be inserted in cases with high degrees of adhesion around the calot's triangle. in these cases, it is not possible to conduct cholangiography from the cystic duct. for these types of cases, intraoperative cholecystography may serve as an option. however, since the bladder is a bag-like organ that expands when liquids are entered, directly inserting a contrast dye into the bladder would make the bladder itself expand, which makes it impossible for to maintain enough pressure in the contrast dye to flow into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct. also, since it is difficult to control leakage of the contrast dye from the catheter insertion site, it is not possible to obtain enough images to sufficiently understand the anatomical characteristics of the bile duct in many cases. therefore, cholecystography is not generally recognized as a method to be used during surgery. in our facility, we insert the contrast catheter through the bladder after stretching the gallbladder neck as much as possible, hold the gallbladder neck with a removable intestinal clamp, and then apply the contrast dye to the bile duct. through this method, it is possible to insert enough contrast dye into the cystic duct, extrahepatic bile duct, and intrahepatic bile duct to understand the anatomical characteristics of the bile duct, allowing us to obtain appropriate images of the biliary tract. because this method uses equipment that is highly versatile, we believe that it is inexpensive and convenient. during this presentation, we will also conduct a case presentation of the methods of bladder contrasting that we utilize in our facility during laparoscopic cholecystectomy. introduction: retrieval of a thick walled gallbladder during a difficult laparoscopic cholecystectomy (lc) for an acute or chronic calculous cholecystitis can be exasperating. it increases operative time and often necessitates enlargement of mm port to deliver the specimen. the 'in-situ cholecystotomy', which we wish to call the 'delhi maneuver' is very helpful in improving the ergonomics of specimen retrieval, saves time and conserves cosmesis. patients & methods: one hundred and ten patients of acute or chronic calculous cholecystitis were placed randomly in groups. a disposable transparent plastic bag was used in all cases to retrieve the gallbladder specimen through the - mm port using a rampley's sponge holding forceps. retrieval was done using conventional technique in patients (group b). the delhi maneuver was used in the remaining patients (group a). it involved cutting the gall bladder inside the plastic bag in a certain fashion, delivering the gallstones in the bag, and removal of gallbladder preceding the stones. the retrieval time, number of insertions of sponge holder, any rupture of plastic bag as well as the number of cases needing port enlargement were noted. results: the average time taken by delhi maneuver (group a) was min as compared to was min by conventional method (group b). the number of insertions of sponge holder ranged from - in group a (mean ) and - in group b (mean ). four patients needed port enlargement in group a ( %) while patients needed enlargement in group b ( . %). there were incidences of bag rupture in group a ( %) and in group b ( %). the delhi maneuver improved the ease and speed of specimen extraction at laparoscopic cholecystectomy for thick walled gallbladders. it also decreased the need for port enlargement for specimen retrieval. the bile duct injuries are a very complex desease to confront, the inciian managment is to clasificate the injury and to identifie the mechamism of the injury. it's important for the optimal heal of the patient to have a multidisciplinary approach including internal medicine, surgery, endoscopy and interventional radiology specialists. the laparoscopic cholecystectomy responsible for %- % of them.this is a retrospective study on the incidence, classification and management of bile duct injuries in a private sector hospital in monterrey nl. mexico. in this study, bile duct injuries were identified in years of experience in a single center. were categorized using the strasberg classification. variables were evaluated such as type of injury, mechanism of injury, hospital stay, if the surgery was scheduled or of emergency, the moment in which the surgeon evidenced the injury, the way in which the surgeon became aware of the injury performed. the type of management that was given to this lesion was also studied and the days of intrahospital stay and the number of reinterventions or procedures performed were compared.the average age of the patients was years, patients belonged to the female sex, although there were lesions of all kinds in this work, there was a greater incidence in strasberg type a lesions, which represented % of the lesions. the most common diagnosis presented was cholecystolithiasis. in surgeries the evidence and repair of the bile duct was in the same intervention aims: bile leak is a rare but recognised complication after laparoscopic cholecystectomy. this usually occurs after a difficult procedure complicated by adhesions, unusual anatomy or if the surgeon is inexperienced or unfamiliar with the anatomy. this video aims to demonstrate the laparoscopic diagnosis and treatment of this complication particularly for surgical trainees. methods: we report a case of significant bile leak occurring soon after a straightforward laparoscopic cholecystectomy due to very short cystic duct (cd). the procedure was carried out uneventfully but the cd was clipped flush with the bile duct. the patient was discharged on the day of surgery feeling well but readmitted with abdominal pain h later. results: after readmission the patient underwent a ct demonstrating only a small amount of fluid suggestive of a small collection. she was treated conservatively but suddenly deteriorated and a repeat ct confirmed significant intraperitoneal fluid. a diagnostic laparoscopy was carried out urgently confirming a cd stump bile leak where the clips had sloughed off causing the leak. two litres of bile was aspirated with copious irrigation and a latex t-tube inserted into the cbd. patient made a full and rapid recovery. conclusions: this is a rare complication and learning opportunities for trainees are therefore infrequent. this video demonstrates a successful laparoscopic approach to management of postoperative bile leak showing t-tube insertion technique and highlighting the need for careful cd closure techniques during laparoscopic cholecystectomy when the duct is very short. about - % of bile duct stones could not be extracted using conventional endoscopic techniques (baloon, sphincterotomy). there is lower success rate in elderly patients; among the biggest challenges are intrahepatic stones, size of stone is large, etc. aims: to present the case of a recurrent intrahepatic lithiasis and its management using spyglass choledochoscopy.to expose, other cases and the main outcome and complications of other difficult cases of bile duct stones that solvedusing this choledochoscope vs. the traditional one and the beneffits. we present a case of years old male who presented with cholangitis caused by an intrahepatic stone that required multiple sessions of endoscopic retrograde cholangiopancreatography with spyglass for clearance. one year later, he presented again with cholangitis, that required another session of spyglass lithotripsy and cholecistectomy. conclusions: besides ercp, there are different approaches to treat difficult bile duct stones, as transhepatic percutaneous drainage, surgical techniques, or other endoscopic techniques (doubleballoon, enteroscopy). ercp and sphincterotomy are the first step of endoscopic treatment with more than % of success rate, and a low mortality and morbility rate; other steps include some lithotripsy techniques, or the use of biliary stent as a bridge before definite treatment. spyglass is a visualization & intervention system used when common ercp has been unsuccessful, and it is first line for better and direct image of biliary ducts, with °range of motion, with multiple advantages like the concomitant use of lithotripsy devices. aims: the number of elderly people has increased, because of the strong association between age and gallstone disease, both prevalence and incidence of this disease are increasing. this presentation aims to review our current management options of octogenerian patients with acute cholecystitis. methods: we retrospectively analyzed octogenerian patients who were admitted to the our hospital with the diagnosis of acute cholecystitis between january and october . the patients were initially allocated to four different treatment groups as follows: immediate surgery, delayed surgery, medical treatment and cholecystostomy. differences in the outcomes between the treatment groups were evaluated. results: there were males ( . %) and females ( . %) with a mean age of . years (range - years). the patients had different co-morbid diseases, especially hypertension ( , . %) cardiovascular disease ( , . %) and diabetes mellitus ( methods: a retrospective observational study where were analyzed patients older than years who underwent urgent surgery for ac who fulfilled an indication for surgery according to tokyo guidelines . the type of cholecystitis, stay and postoperative complications, the type of intervention, the conversion rate, the need for reoperation and re-admissions in patients older than years were analyzed and compared with those of patients operated on for cholecystitis younger than years. outcomes: a total of patients were registered, older than years ( %) and younger ( %). in cases, cholecystitis were complicated ( . %), cases older than years ( . %) and in cases younger than years ( . %). the approach was laparoscopic in % of the cases older than years, with a conversion rate of . %, not finding statistically significant differences with younger than years ( % laparoscopies with . % of conversions). % of patients older than years had some type of postoperative complication, not finding statistically significant differences in patient younger than years ( %); being the most frequent complication the intrabdomintal abscess ( . % of patients [ years, and . % of those \ years = '' span = '' [ being not statistically significant with % ci. any patient older than years required re-entry after discharge, compared to patients younger than years who were re-entered, not being statistically significant; and any patient older than years required reintervention, while it was necessary to reoperate patients younger than years ( %), being not statistically significant. mortality was very low, finding case in older than years ( . %) and case in younger ( . %), not obtaining statistically significant differences. the postoperative stay in patients younger than years of age has a median of days and in older than years a median of days, not finding statistically significant differences with % ci conclusions: laparoscopic cholecystectomy is safe and effective in the treatment of elderly patients with (ac), there being no differences with younger patients. introduction: significant bile leak is an uncommon but serious complication of laparoscopic cholecystectomy. our study aims to evaluate the efficacy of relaparoscopy in treating symptomatic bile leak and biloma formation. material and methods: patients presenting with postoperative bile leak after different operations on extrahepatic biliary tree from january to december were reviewed retrospectively (in total, , laparoscopic surgical interventions were performed for the period under study). the sites of bile leaks were the cystic duct stump in thirty seven patients, the bile ducts of luschka in fifty two, liver beds in cases after hepatectomy, in had small injury of cbd, and seven patients with tubular stenosis of the common bile duct. results: three main approaches of mini-invasive treatment of bile leakage was used: ( ) percutaneous puncture with or without drain under ct-scan or ultrasound guidance in patients; ( ) endoscopic management in patients (in patients ( . %) were managed with ercp alone and fifteen ( . %) were treated with a percutaneous intervention followed by ercp. endobiliary stent placement was performed after es in patients and without es in twenty seven patients ( ) relaparoscopy has been performed in patients, in cases of biliary peritonitis. conclusions: relaparoscopy was the ultimate method of treating postoperative complications of laparoscopic surgery in . % of patients. in general, this method, as well as laparoscopic intervention, is highly effective in the diagnosis and correction of postoperative complications, with minimal surgical trauma for the patient, with great therapeutic effect and subsequent rapid social rehabilitation of patients. introduction: laparoscopic operations have already become routine, even for pancreatoduodenectomy for periampular cancer. for unresectable cases, endoscopic bibliary stenting or hepaticojejunostomy are usually used. these methods are quite expensive and may be accompanied by complications. materials and methods: laparoscopic cholecystogastroanastomosis was performed in patients with unresectable periampullary cancer. there were females and men and average age was , . the indications for surgery in all patients was unresectable periampullary cancer and biliary hypertension with preserved patency of the cystic duct. the level of bilirubinemia ranged from to lmol/l (the average level was , lmol/l). we used -port technique. optical trocar was placed in the right iliac region, one mm above the navel and one mm in the right hypochondrium after punction gallblaber and aspiration of bile, we cut the apex of the gallbladder and gastric antrum up to . cm and performed cholecystogastroanastomosis with barbed-suture v-loc. results: we had not conversion to open surgery. the average operation time was min. postoperative stay was average days and on median follow-up of month. post-operatively, there were no major morbidity and nil mortality. we had cases of leakage of bile through drainage for up to - days, which spontaneously stopped. all patients showed a decrease in the level of bilirubinemia. patients were later radical operated (pancreatoduodenectomy), while they did not have such phenomena as cholangitis, pancreatitis, inflammation of the hepatoduodenal ligament elements, which we often observe after endoscopic biliary stenting. conclusions: laparoscopic cholecystogastroanastomosis is safe, effective and feasible for patients with periampular cancer and obstructive jaundice. aims: surgeons with the expertise and resources to perform laparoscopic common bile duct exploration often prefer the 'one stage approach' over endoscopic retrograde cholangio-pancreatography (ercp) for the management of common bile duct (cbd) stones. this case series aims to evaluate the effectiveness of lcbde in a single benign upper gastrointestinal (gi) unit. methods: all patients with suspected and confirmed pre-operatively cbd stones who underwent a lcbde between january and october were included. lcbde was performed on the basis of pre-operative suspicion of cbd stone confirmed by intra-operative imaging. results: patients with confirmed choledocolithiasis had lcbde during this time period. the indications for lcbde were deranged liver function tests, dilated cbd or confirmed stones on preoperative imaging. median age was (range - ), % of whom were female. % of patients had confirmed cbd stones pre-op. % of cases were performed as emergencies and conversion rate to open was . %. choledocotomy was performed in % of cases. in % of these t-tube was left in situ. transcystic approach was used in the remaining %. despite positive intraoperative imaging no stones were found on cbd exploration in cases ( %). in patients stones were unable to be cleared with lcbde. the overall morbidity was %. % of patients had gallstone related complications. overall mortality was % (due to bile leak). / patients required re-intervention with re-look laparoscopy (n = ) or ercp (n = ). patients re-presented within months with cbd stones. overall median length of stay was days. conclusions: our case series demonstrates that lcbde is an effective and safe treatment for choledocolithiasis in both the elective and emergency settings. complication rates are comparable with therapeutic ercp ( % specific complications) followed by laparascopic cholecystectomy ( % day morbidity). the variability in anatomic location of subvesical bile ducts puts them in danger during hepato-biliary operations. its prevalence varies between % and %. the origin and drainage of these ducts were limited mainly to the right lobe of the liver, but great variation could be seen. some authors think of them as small bile ducts that drain directly into the body of the gallbladder; others consider them to be networks of miniscule bile ducts between the liver capsule and the gallbladder. recent studies suggest that clinically relevant bile leaks complicate approximately . - . % of cholecystectomies. injury to a subvesical duct is one of the most common causes of cholecystectomy associated bile leak and occurs as often as major bile duct injuries and leaks from the cystic duct stump. indeed, recent studies suggest that about % of clinically relevant bile leaks are caused by inadvertent injury to a subvesical bile duct. there are four types of subvesical bile ducts, including ( ) superficial variations of segmental and sectorial bile ducts, ( ) superficial or intercommunicating accessory bile ducts, ( ) hepaticocholecystic ducts, and ( ) aberrant bile ducts.we present a case of year old patient who developed a coleperitoneum after a routine daycase colecystectomy due to the inadvertent injury of a hepatocholecystic duct. a superior comprehension of ductal anatomy is essential in preventing and managing operative injury to the subvesical ducts, although some times is unavoidable. nowadays, the diagnosis of liver cancer is primarily radiological, as recommended by the principal international societies. in doubtful cases or due to the clinician needs, diagnostic evaluations can eventually be completed with a liver biopsy. the goal is to perform the examination, or the examinations, that guarantee the most elevated sensibility and specificity levels being as little invasive as possible. nevertheless, even using the best radiological tools, the diagnosis is not certain, due both to device limitations and radiology experience. recently, various diagnostic algorithms have been proposed, relating with contrast enhancement characteristics, different radiological techniques, blood examinations and cross evaluations from different radiologists. one of the most recent algorithm purposed is liver imaging reporting and data system (li-rads), that evaluates ct and mri imaging to classify hepatic lesions in different diagnostic categories, in order to perform a better and more precise diagnosis of hcc or other liver benign or malignant lesion. through a retrospective study, we evaluated and compared preoperative imaging and post-operative histological reports. results reveal that li-rads routine use increases hcc diagnosis up to %. background: we previously developed a modified difficulty scoring system (dss-ihd) of laparoscopic liver resection (llr) for patients with intrahepatic duct (ihd) stone. we validated dss-ihd in patients who underwent llr for hepatolithiasis. methods: dss-ihd was based on the extent of liver resection ( to ), stone location ( to ),atrophy of liver parenchyma ( to ), ductal stricture \ cm from the bifurcation ( to ), and combined choledochoscopic examination for remnant ihd ( to ). results: the dss-ihd ranged from to and divided to -level groups of low group (score * ; n = ), intermediate group ( objective: improving the surgical treatment of patients with cholangiogenic abscesses of the liver through the application of minimally invasive technologies. material and method: in the presented study presented results of treatment of patients with biliary liver abscesses. surgical interventions for hepatic abscesses were performed simultaneously with the elimination of the primary pathological process of the biliary system, which caused the occurrence of cholangitis, or in the near future (up to days) after biliary drainage drainage. among patients with biliary liver abscesses, treated with minimally invasive methods, revealed abscesses of the right hepatic lobe, -abscesses of the left hepatic lobe, -abscesses and right and left hepatic lobes. single abscesses were detected in patients, and in -two or more abscesses. in terms of liver abscesses, more than cm were detected in patients, more than cm in patients. drainage of the biliary tract was carried out endoscopically transpapillary and (if the endoscopic approach was unsuccessful) with transcutaneous transhepatic approach. results: drainage under ultrasound guidance was performed on patients with solitary and patients with two or more cholangiogenic abscesses of the liver. laparoscopic interventions were performed on patients. among the patients operated on using minimally invasive technologies, occurred complications ( . %). patient died due to the development of biliary sepsis ( . %). conclusion: percutaneous drainage of liver abscesses under ultrasound control is appropriate not only for single abscesses, but also for their larger number, which has many advantages over other interventions. it was proved possibility of simultaneous drainage of liver abscess and bile duct. percutaneous drainage of the liver abscess, drainage of the biliary tract and laparoscopic surgical intervention are complementary aspects in the treatment of liver abscesses of biliary origin. after laparoscopy residual calculus can be removed endoscopically in more favorable conditions after stabilization of the patient's condition is achieved and the infection-associated disorders are eliminated. in case of localization of abscesses in the marginal segments of the liver, laparoscopic atypical resection of the liver with an abscess is most desirable. general surgery, rambam medical center, haifa, israel background: recently robotic surgery has emerged as one of the most promising surgical advances. despite its worldwide acceptance in many different surgical specialties, the use of robotic assistance in the field of hepatobiliary (hbp) surgery remains relatively unexplored. our study presents single institution's initial experience of robotic assisted surgery for treatment of benign hepatobiliary pathologies. methods: a retrospective analysis of a prospectively maintained database on clinical outcomes was performed for consecutive patients that underwent robotic assisted surgery for benign hbp disease at rambam medical center during - . results: there were robotic assisted surgical procedures performed for benign hbp pathologies during the study period. there were anatomical robotic liver resections for symptomatic hemangiomas, cases of giant liver cyst, robotic assisted surgery for type i choledochal cyst, case of benign (iatrogenic) common bile duct (cbd) stricture, cases of robotic (cbd) exploration due to large intra choledochal stones and cases of cholecystectomy for cholelithiasis. the median postoperative hospital stays for all procedures were . days (range - days). general morbidity (minor) was %. there was no mortality in our series. conclusion: robotic surgery is feasible and can be safely performed in patients with different benign hbp pathologies. further evaluation with clinical trials is required to validate it's real benefits. most liver cysts are asymptomatic and tend to have a benign clinical course. however, symptomatic or complicated liver cysts sometimes require surgical intervention. needle aspiration is safe and can be the lease invasive procedure, this procedure is however associated with a high failure rate and rapid recurrence. surgical approach is the crucial and provides definitive treatment for such cysts. thirteen cases were nominated from shonan kamakura general hospital between january and december . mean age and body mass index (bmi) were . and . , respectively. all patients have had any complaint such as upper abdominal pain, dyspnea, and fever. two cases were clinically diagnosed as the infectious cyst and serum crp was elevated before surgery. additional cholecystectomy was planned for one case of chronic cholecystitis with gallbladder stones. all cases were prompted the reduced port surgery (rps) and cases were performed rps with trans-vaginal approach (hybrid notes) and case was chosen in single port surgery. cyst unroofing was performed for all cases. mean operation time and blood loss of all cases were . min. and . ml, respectively. no surgical complication has been occurred in all cases, an infectious cyst case was however required additional drainage for infectious control after surgery. although statistic difference was not shown, fewer blood loss and shorter hospital stay was seen in non-infectious cases, compared to laparotomy cases. mean hospital stay after surgery of whole cases, non-infectious cases, infectious cases was . , . , . days, respectively . no recurrence of any symptom was shown in any cases in observation period ( - days) . laparoscopic unroofing is the definitive treatment for the complicated or symptomatic liver cyst. however, for the infectious cyst, infection control such as intensive drainage and/or administration of antibiotic before surgery may be needed to avoid additional treatment, leading to longer hospital stay. laparoscopic unroofing of liver cyst can be the first choice for symptomatic or complicated liver cyst. also, reduced port surgery can be nominated to achieve less invasiveness. introducction: laparoscopic liver resection (llr) has been increasing since it was first reported in . three international expert consensus conferences on llr surgery were held in louisville, ky, usa, in , morioka, japan in and southampton, uk, in . while most initial minimally invasive liver resections were typically done for benign lesions in anterior o left segments, llr is currently being applied for major anatomic resections, malignancy, cirrhosis and liver donor hepatectomy. clinical case report: this is a -year-old male patient with a history of hta and liver cirrhosis due to hepatitis b virus. hepatocarcinoma is diagnosed in liver segment vi with a size of cm . in the digestive study the patient presents a child a stage, meld \ , without signs of portal hypertension. complete analytical with normal afp and cea . markers. after presentation of the patient in a multidisciplinary committee and being a stadium according to the early bclc classification, laparoscopic surgery with segment vi resection was decided. discussion: laparoscopic liver resection is becoming widely accepted for the treatment of hepatocellular carcinoma. liver resection is a first-line option in very early and early-stage disease. many meta-analysis have shown that llr is better than open liver resection in terms of short-term outcomes for patients with child-pugh a cirrhosis, solitary tumors, and minor resections. in the long-term setting, the results demonstrate that a minimally invasive approach is comparable to an open approach in terms of overall. in conclusion, the current evidence conclude than llrs for hcc are safe and may be considered a standard practice in specific settings. results: there were women ( %) and men ( %). the age of patients ranged from to years. the patients underwent complex examination including abdominal ultrasound, esophagogastroduodenoscopy, and some of them underwent ct (computed tomography). all patients in the first stage were performed antegrade external drainage of biliary tracts with x-rays of the biliary tracts, and specifying the level and extent of the block.total miniinvasive interventions were hold. two patients in connection with the uncoupling of equity ducts were performed antegrade bilobar stenting with preliminary split external bile release.there were complications after carried out interventions in cases, which were associated with dislocation of holangiostomic drainage in patients ( . %); with acute cholecystitis in patient ( . %); with hydrothorax in patients ( . %); perihepatic biloma in case ( . %). patient ( . %) had a recurrence of obstructive jaundice due to germination of endobiliary stent in the late period after stenting. lethal outcome appeared in patient. conclusions: ultrasound examination allows us to determine the level of obstruction of the biliary tract, to substantiate the tactical position in the application of mini-invasive technologies. antegrade miniinvasive technologies in the treatment of tumor lesions of the proximal bile ducts allow timely and effectively stop biliary hypertension and to determine further treatment strategy. acknowledgements this study was supported by the russian science foundation under project ? - - . background: repeat hepatectomy is an effective treatment, with long-term surgical outcomes for recurrent hcc and colorectal liver metastasis(crlm). however, the efficacy of a minimally invasive surgical approach for recurrent liver tumor is not yet confirmed. the purpose of this study is to examine the efficacy of laparoscopic repeat hepatectomy(lrh) compared with open repeat hepatectomy(orh) for recurrent liver tumor. we retrospectively analyzed the clinicopathological features and short-term surgical outcomes between lrh and orh. methods: from to , patients with liver cancer underwent repeat hepatectomy. of those patients, patients underwent partial hepatectomy, patients were undergone laparoscopically, and patients underwent open hepatectomy. we compared the clinicopathological and surgical parameters in the lrh group with those in the orh group. results: there were no significant differences in patients' gender, age, viral infection status, child-pugh classification, tumor size, tumor number, and tumor location in the two groups. the operative times were similar, but blood loss was significantly lower in lrh group ( vs. ml, p \ . ). the postoperative hospital stay was significantly shorter in the lrh group ( . vs. . days, p = . ). postoperative complications(cd = a) were observed only in the orh group, with a complication rate of . %. conclusions: we demonstrate that lrh reduces blood loss and postoperative complications compared with orh. lrh might be a feasible and effective procedure for the selected patients. background: the liver is the most common site of metastatic disease with up - % of all cancers having the potentiality for sending liver metastasis during the disease. consequently, increasing value for surgical resection of hepatic deposits of different types of cancers, the need for accurate evaluation of the extent of hepatic metastasis was established for choosing the most suitable patients for surgery and in planning the extent of hepatic resection. the aim of this work is to evaluate the role of intra-operative ultrasound in the detection of hepatic deposits in intra-abdominal malignancies with special emphasis on its accuracy, sensitivity, specificity. patients and method: this study was carried out on thirty patients who were admitted to the gastrointestinal surgery unit, main alexandria university hospital with intra-abdominal malignancies for whom elective open surgical intervention was recommended in the period from st of september till the th of march . results: in the present study consisted of males ( . %) and females ( . %). their mean age at admission was . ± . years. six of the included patients ( %) were found to have hepatic lesions by using ious including the four cases ( . %) already detected by preoperative imaging. two cases ( . %) were newly discovered in the operative room by using ious. conclusion: the current study has proved that ious demonstrates superior lesion detection over the various non-invasive preoperative imaging modalities causing significant impact on change of the planned surgical strategy laparoscopic approach to the liver has become an integral part of surgery. two consecutive international consensus meeting recommends major hepatectomy has been on the expert hands. tumors located in the right posterior section are considered to be difficult for laparoscopic resection. patients and methods: since , until , cnuhh has been performing laparoscopic hepatectomies including major hepatectomies. among major ones, there are rh, lh, rps, ch, and as. we analyze data on patient demographics, tumor characteristics, operative date, and posterior outcome retrospectively. results: during - , laparoscopic rps were performed. the diagnosis were hcc in and crlm in patients. median operative time was min, and median blood loss was ml. no blood transfusion was occurred. median tumor size was mm, and median resection margin was . mm. six of the patients ( %) were cirrhotic on pathology. there was no conversion and was no postoperative mortality. median hospital stay was . days. conclusion: laparoscopic rps is known challenging procecedure. strict preoperative planning and operative procedure is mandatory. even though it should be performed by the experienced hands both on hepatic surgery and laparoscopic skill, it can be an good option for treatment of the tumor locating over right posterior section. purpose: previously we developed a new sponge (named endoractor) as an organ retraction device in laparoscopic surgery in and have reported that it is useful in various surgical procedures including rectal surgery we confirmed that it is also useful in laparoscopic radiofrequency ablation of the liver in terms of pulling and protecting organ, so we report it materials and methods: a case is an -year-old female with liver cirrhosis. she had primary hepatocellular carcinoma in s lesion with a diameter of . cm very close to the inferior vena cava and middle hepatic vein root and in s lesion with a diameter of . cm we thought she could not put up with hepatic resection because of her poor hepatic reserve capacity. and we could not expect treatment effect by embolization therapy since contrast effect was poor. so we decided to select ablation therapy in the puncture and ablation of the s tumor, since there was concern about the thermal damage of the middle hepatic vein and the cooling effect by the inferior vena cava, we would dissect the right coronary mesentery sufficiently and pull the liver apart from the inferior vena cava and the middle hepatic vein as much as possible using our endoractor also, in the puncture and ablation of the s tumor, it was feared that the stomach would be thermally damaged, so we would place endoclactor between the liver and the stomach to protect the stomach results: when ablating the s tumor, we could pull the liver securely without slipping, so we did not cause thermal damage to the middle hepatic vein. and there was no cooling effect by the inferior vena cava, so we could obtain sufficient cautery margin. in ablation of s tumor, we were able to puncture by stabilizing the lateral segment of the liver on our endoractor, and avoid thermal damage of the stomach conclusion: it seems possible to perform safe and reliable puncture and ablation by using our endoractor as well in laparoscopic radiofrequency ablation surg endosc ( ) surgical reinterventions in patients with complicated hepatic hydatid cysts usually occur as a result of diagnostic or technical failures during the initial procedure. according to recent studies, the most common complication after liver hydatid cyst surgery is local sepsis at the residual cavity and long-term biliary leak. we report the case of a -year-old male with a history of liver hydatid disease four years before the current episode, admitted in our surgical department for intense upper right quadrant pain. abdominal ultrasonography, ct and mri scans revealed three cysts in the gastrosplenic ligament, in liver segments vii-viii, and ii-iii respectively, sized between and cm. the intraoperative aspect during laparoscopy was strongly suggestive for liver hydatid disease. laparoscopic fenestration with tunneling for the hepatic cyst in segment viii, partial cystectomy in the left liver lobe and ideal cystectomy in the gastrosplenic ligament were performed. postoperatively, the patient displayed a constant biliary drainage output of - ml from the cavity remnant in the segment viii. conservative therapy for external biliary fistula and concomitant treatment with albendazole for months were initiated. evolution was slowly favorable with decreased biliary drainage to ml two months after surgery and complete symptom resolution five months after hospital discharge. aims: this study aimed to evaluate the effectiveness of fluorescence imaging with indocyanine green (icg) during laparoscopic deroofing of hepatic cysts. methods: this was a single-center, case-control study. we included patients who underwent laparoscopic deroofing between november and october . imaging with and without icg fluorescence was performed in (icg group) and (non-icg group) patients, respectively. icg was intravenously administered between min and . h before surgery. we performed a standard laparoscopic procedure. we detected a thin bile duct on the hepatic cyst on using intraoperative icg fluorescence imaging. we adjusted the resection line of the cyst wall and ligated the bile duct at the point at which it crossed the resection line. data on age, sex, cyst size, resected cyst size, operative time, estimated blood loss, post-operative hospital stay, complications, and recurrence were compared between the groups. results: the mean cyst size was ± . and ± . mm, the mean resected cyst size was ± . and ± . mm, and the mean operative time was . ± . and ± . min in the icg and non-icg groups, respectively. using icg fluorescence imaging, the bile duct was detected on the cyst wall in patients ( %). all surgeries were completed laparoscopically, and no post-operative complications occurred in either group. recurrence of the hepatic cyst occurred in one patient ( %) of the non-icg group. conclusions: fluorescence imaging with icg is used widely in hepatobiliary surgery for intraoperative identification of biliary and vascular anatomies. this method does not require complicated techniques or instruments. icg fluorescence imaging may facilitate the prevention of intra-or post-operative complications, such as biliary leakage, in laparoscopic surgery. in this study, icg fluorescence imaging was found to be effective in detecting the bile duct on the cyst wall intraoperatively, allowing for wider resection of the cyst and avoiding inadvertent injury. our study suggests that wider resection of the cyst wall might prevent recurrence of hepatic and that icg fluorescence imaging could ensure procedural safety. abdominal ct showed: large hepatic cyst ( x , x cm size), with no malignity signs, that occupies practically the whole right liver, causing subsegmentary atelectasis of the middle lobe, superior and inferior cava vein compression, and displacement of right kidney, pancreas and right atrial. due to breath involvement, a percutaneous drainage is performed achieving clinical improvement and reduction of the size of the injury. the patient was released but a cyst superinfection occurred; once this problem was solved, the drainage was removed. results: in light of the complication, surgical treatment was decided, which confirmed the large cyst located in right posterior hepatic segments with tight diaphragmatic adhesions. we carried out the cyst evacuation and a wide laparoscopic resection of the cyst walls, until the posterior area of the cava vein, combining supra and infrahepatic access. the patient was released on the sixth postoperative day and continues asymptomatic. conclusions: simple cysts can be approached in a no surgical way (punction-aspiration with/ without sclerosing products injections) or in a surgical way (cyst wall fenestrations, cystectomy or liver resections). a conservative treatment will obtain symptomatic relief but with a high risk of recurring. recurrence is the main drawback of unroofing. cystectomy is the better option but may be too complicated depending on the cyst's location. to our patient, we carried out a wide laparoscopic unroofing (even though its posterior localization) to minimize recurrence possibilities. in conclusion, laparoscopic resection of the cyst wall is a simple and effective approach in symptomatic or complicated cases. background: single-incision laparoscopic surgery or laparoendoscopic single-site surgery is emerging as an alternative to conventional multiple-incision laparoscopic surgery. it has a potential benefit of less postoperative pain and faster recovery compared with conventional multiple-incision laparoscopic surgery. single-incision laparoscopic hepatectomy (silh) has been reported in only a few small series and the majority were minor resections. case report: a y/o male patient is a case of chronic viral hepatitis b and early cirrhosis of liver. two atypical hepatocellular carcinomas (up to . cm in diameter) located at the junctions of segments & and segments & were impressed by liver magnetic resonance imaging (mri). we performed single-incision laparoscopic anatomical hepatic resection of the right posterior section via a -cm transverse incision on the right middle abdominal wall. inflow control was carried out with an extra-glissonian approach before parenchymal transection. the glissonean pedicles of segments and were divided by linear staplers respectively as well as a major branch of the right hepatic vein in segment . the operative time was min and the estimated blood loss was ml. the pathologic examination revealed two foci of hepatocyte dysplasia with a safe margin of cm. the patient was discharged eight days after the surgery uneventfully. conclusion: single-incision laparoscopic anatomical right posterior sectionectomy is feasible and safe by experienced laparoscopic surgeons. it provides a fast recovery but needs a long operative time. the mortality in the patient with liver cirrhosis is very high. the aim of this work was to decrease mortality and morbidity by using endoscopic local heamostasis and laparoscopic operations, in the patients with bleeding from cirrhosis by variceal bleeding. methods and material: we observed patients with cirrhosis complicated by variceal bleeding during years. there were patients with child phue a, ones with child phue b, ones with child phue c. all the patients were performed prolonged endoscopic heamostasis with conservative therapy. the main methods that we used were the ligation in cases, sealing in cases, sclerotherapy in cases. in cases we couldn't stop the bleeding with band ligation method and introduce the danis stents into esophagus and stopped the bleeding successfully. to prevent the re-bleeding we performed the laparoscopic dissection the abdominal part of esophagus with suturing the venous vessels, coagulations and dissection of short gastric vessels between stomach and spleen, clipping the left gastric artery and vein in the patients. in patients we performed laparoscopical suturing the variceal veins by introducing the laparoscopic trocars into the stomach. in cases with varices vien of stomach, with non-effective local endoscopic heamostasis we performed laparoscopic resection the fundal part of stomach. results: endoscopic local heamostasis were successful (in %) in cases. the relapse of bleeding were in patients. patients died. there was no mortality after laparoscopic operations. there were cases for trocar wounds infection, cases of subphrenic abscess. goals: the advance of laparoscopic surgery also includes the more complex procedures of abdominal surgery such as those affecting the liver and pancreas. there are multiple indications that laparoscopy has in hepatobiliopancreatic surgery, both in benign and malignant pathologies. material and methods: we present the video of a -year-old male patient with a history of right hemicolectomy due to disease-free intestinal lymphoma who, in the control analysis by his attending physician, detects the elevation of tumor markers. an extension study was started showing a hepatic lesion in the caudate lobe with a pathological anatomy suggestive of hepatocarcinoma and an adenopathy suspicious for malignancy adjacent to the right renal vein. the clinical case is presented in a multidisciplinary tumor committee and it is decided to perform surgery. a laparoscopic caudate lobe resection was performed, previously performing intraoperative ultrasound and a lymphadenectomy of the portal territory, vena cava and exeresis of adenopathy of the right renal vein. introduction: major vascular complications during laparoscopic surgery occur approximately in one in cases, but mortality rate can reach - %. most major vascular injuries lead to conversion to laparotomy but successful laparoscopic repair is also possible. simulation training improves laparoscopic performance and possibly reduces surgeons mental strain. materials & methods: during two editions of advanced laparoscopic training course participants had a task to control a major vessel damage (damage). before the task an educational video explaining the methods of obtaining haemostasis was shown. the algorithm of the 'damage' task was as follows: without previous preparation a cm injury of a major vessel was done with l-hook electrocautery. after the injury participants were free to control the damage the way they wanted. heart rate of the participants was measured with an ear electrode. measurements were carried out times-before the injury, immediately after, and afterwards obtaining vessel control. after participants were interviewed for their feelings after the 'damage' task. results: there were vessel injuries in animals. one animal died during the 'damage' task min after desuflation due to relapse of bleeding. there was no conversion to open procedure. temporary vessel control was obtained with different methods. all participants used vicryl . or pds ii . suture for final hemostatic purposes. heart rate of the participants before injury were - ± . bpm, immediately after the injury it rose to - ± . bpm, and after obtaining vessel control were in the range - ± . bpm. a statistically significant difference was found between the ratio of the first and second hr measurement (p = . , t = - . ), and second compared to the third (p = . , t = . ) measurement. participants judged their experience on a -point scale ( - was not helpful at all; -was extremely educative). the educational value of the task received points in cases and points in one case. conclusion: participants feel stress during major vessel bleeding even in animal model, and this stress can result in a serious intraoperative mental strain and significantly increase heart rate. participants found the 'damage' task very useful for their daily practice. the aim of study was to improve the results of treatment of patients with hepatic echinococcal cysts by using of argon plasma coagulation. methods: the analysis of treatment results of patients was put into the basis of this study. it was ( . %) men and ( . %) women in total. an average age of them was . ± . years. the main difference between groups was a way of liver parenchyma coagulation in order to make reliable hemostasis. in main group the final stage of surgical intervention on liver was argon plasma coagulation. it was performed to ( . %) patients. alternatively, monopolar coagulation was performed to ( . %) patients (comparison group). results: in main group in the . % cases pericystectomy was conducted. the resecting surgeries was performed to . % cases. in comparison group was conducted in . % cases. in early postoperative period in main group the complications were observed in . % of cases. the same parameter was . % in comparison group. it led to relaparomies. the forming of external biliary fistulas was observed in ( . %) patients in main group and in ( . %) patients in comparison group. however, all the fistulas have closed spontaneously on th- th day in both groups. hernias of abdominal wall and peritoneal adhesions that manifested by intestinal obstruction of different degree were considered as complications of late postoperative period. these values were % and . % in main group versus % and . % in comparison group, respectively. the resection of hepatic echinococcal cysts with further application of argon plasma coagulation on the cyst bed was accompanied by complications quantity decrease in patients that underwent surgery in early as well as in late postoperative period. in this case more positive dynamics of functional liver values improvements was observed. aims: indocyanine green (icg) fluorescence imaging has been reported as a reliable and safe navigation tool in laparoscopic hepatectomy. however, the factors affecting the sensitivity of tumor detection with icg fluorescence imaging is relatively unclear. the aim of the present study is to analyze the factors of successful icg fluorescence in laparoscopic hepatectomy. methods: this is a retrospective single-center study. this study population consisted of laparoscopic hepatectomies from january to november undertaken at kurashiki central hospital. we excluded patients whose tumors were located more than mm from the liver surface, those who did not receive icg fluorescence imaging, and those who were not injected with icg dye ( . mg/kg) intravenously within days of surgery. the pinpoint endoscopic fluorescence imaging system was used to detect the tumor location. we evaluated the relationship between successful fluorescence and the timing of injecting icg before operation, tumor size, icg r , liver damage and bmi. results: following exclusion, patients were eligible for analysis. among the tumors resected, icg fluorescence imaging detected tumors ( . %), including hepatocellular carcinomas and liver metastases. icg fluorescence imaging detected all tumors in the patients injected with icg to days before hepatectomies . icg fluorescence imaging detected all tumors which were more than mm in diameter. there was no relationship between indocyanine green fluorescence with icg r , liver damage and bmi. conclusions: the injection of icg to days before operation and a tumor size of more than mm can be factors in successful fluorescence in laparoscopic hepatectomy. introduction: cysts in the liver have a wide variety of aetiologies. it is important to characterize the cystic lesion before treating it. the simple cyst has a low prevalence and is more frequent in women. fenestration is a useful option for the treatment of simple cysts in selected patients. case presentation: a -year-old woman was referred to our hospital with a one-year history of intermittent, right upper quadrant pain, with no other associated symptoms. computed tomography and magnetic resonance imaging showed a large cyst ( , x , cm) in the right of the liver. the cyst presented lobulated morphology, smooth edges and well delimited. there were other smaller cysts in the left lobe. hepatic function in blood analysis was normal. biomarkers, tumor markers and hepatitis virus markers were negative. outpatient follow-up and symptomatic treatment of pain was decided. after six months of follow-up, the pain persisted, so surgical treatment was proposed. a laparoscopic fenestration was performed, widely resecting the free wall of the cyst. there was no evidence of a connection to the bile duct. there were no complications. on days she was discharged. discussion: some giant hepatics cysts become symptomatic due to mass effect. persistence of pain is an indication of surgical treatment. laparoscopic fenestration is an alternative for the management of simple hepatic cysts. aim: laparoscopic liver resection for malignant pathology such as colorectal cancer metastases has been a matter of discussion for several groups in the last years. it has been proposed as a safe and feasible treatment but subjects like short and long term outcomes and oncologic results have not been adequately assessed. methods: we performed an observacional retrospective study of patients undergoing laparoscopic liver resection for colorectal metastases in our center. from november to november a total of patients underwent laparoscopic liver resection. data for resection margin, hepatic and extrahepatic recurrence and both disease free survival and overal survival were collected. patients were discussed in a multidisciplinary group with oncologist, radioterapic oncologist and surgeons. the surgical procedures were perfomed by the same team in all the cases to minimize bias. results: a total of patients ( . %) were non resectable at the time of surgery.the mean overall survival was months with a maximum of months. we got a mean of disease free survival in our patients of . months. the hepatic recurrence was %, most of them in high risk patients, and from this group . % underwent a new liver resection. major complications took place in patients ( . %) two biliar leaks, one bowel perforation, two hepatic failure, one evisceration and three respiratory insufficiency needing urgent surgery in three of the cases. mean hospital stay was . days. a mean of days of this stay were in an intensive care unit. conclusions: laparoscopic liver resection for colorectal liver metastases could be a feasible technique when perfomed by trained surgeons. it improves the postoperatory recovery with a reduction of hospital stay and less postoperatory pain without increasing the development of major complications or mortality in the first days after surgery. we got good oncological results that have been improving with the experience acquisition of the surgical team. aged to underwent surgery for cirrhosis with massive refractory ascites child c ( - ), without obvious signs of hepatic encephalopathy. major etiological factors were: viral hepatitis c ( patients ( . %)), b ( patients ( . %)), b ? d ( patients ( . %)), toxicity ( patients ( , %) ). to prevent possible bleeding at the first stage, endoscopic filling of esophageal varices with fibrin glue was performed in patients ( . %). after testing the effectiveness of varices filling, in the following - days decompression surgery of thoracic lymphatic duct was performed under local anesthesia to improve lymphatic drainage from liver and abdominal organs. simultaneously, laparoscopic sanitation of abdominal cavity was performed, with complete evacuation of ascites fluid, rinsing and drainage. fractional post-surgery rinsing was repeated daily for - days towards removing peritoneum edema and improving its absorptive properties. results evaluation was performed , and months after surgery, based on criteria of liver reserves and ascites volume. results: post-surgery mortality from liver failure was . % ( patients) . other patients died of the same cause the following - months. annual survival rate was . %. complete ascites regression over - months after surgery was noted in patients ( . %), significant regression and stabilization in ( . %), moderate regression with need for periodic decompressive laparocentesis in cases. in all patients, functional liver reserves and life quality significantly improved. conclusions: the use of the given technique of refractory ascites correction, in patients with depleted liver cirrhosis, by laparoscopic sanitation with post-surgery fractional rinsing of abdominal cavity, with simultaneous decompression of thoracic lymphatic duct showed very high efficiency and deserves establishment as a clinical practice. t. urade, hepato-biliary-pancreatic surgery, kobe university, kobe, japan aim: anatomical liver resections guided by a demarcation line after portal staining or inflow clamping of the target territory were established as essential methods for the curative treatment of hepatocellular carcinoma (hcc) and then subsequently applied to other malignancies. however, laparoscopic anatomical liver resection (lalr) is much more difficult to reproduce these procedures and to confirm demarcation of the hepatic segment visually on the monitor. recently, laparoscopic fluorescence imaging system has been used as a tool for real-time intraoperative navigation in llr. the aim of this study is to demonstrate how to perform lalr using indocyanine green (icg) fluorescence imaging. methods: three patients underwent pure lalr using icg fluorescence imaging. the following operative procedures were performed: partial liver resection for hcc, segmentectomy for liver metastasis and right anterior sectionectomy for hcc. in all patients, preoperative d simulation images from dynamic ct were reconstructed using a d workstation to decide on cutting points of the glissonean branches. after mobilization of the liver, intraoperative ultrasonography was performed to identify the location of the tumor and glissonean pedicles corresponding to the tumor-bearing hepatic region. we dissected or transected the hepatic parenchyma to encircle the glissonean pedicles. after clamping or closure of them, . mg of icg was injected intravenously to identify the boundaries of the hepatic segments under near-infrared light. parenchymal transection was started according to the demarcation on the liver surface. the lateral aspect of the parenchymal transection was carried out based on the demarcation between non-fluorescing and fluorescing liver parenchyma as far as possible. results: in all the cases, demarcation lines on the liver surface could be visualized clearly after injection of icg. in addition, boundaries of cone units, segments and sections could be recognized to some extent because the tumor-bearing hepatic region became non-fluorescing parenchyma during parenchymal transection. these procedures were completed successfully, and the postoperative courses were almost uneventful. aim: sintrahepatic cholangiocarcinoma is the second most common primary liver cancer after hepatocellular carcinoma (hcc). although the laparoscopic approach of these tumours is not frequent due to its complexity, it is performed increasingly by hepatic surgeons.traditionally, the abdominal surgery in cirrhotic patients has been reserved to selected cases secondary to the high rate of complications. the advance on the treatment of the hcc on liver cirrhosis and the higher safety when performed by laparoscopic approach has encourage some surgeons to extend surgery to child b-c or portal hypertension patients. methods: we present a male of years old, diagnosed in of liver cirrhosis accompanied with portal hypertension. on mri in was found a solid lesion of mm located on segment ii hepatic. biopsy confirmed the diagnostic of intrahepatic cholangiocarcinoma. after a liver function evaluation (child c, meld ), an hepatic chemoembolization was performed. sequentially ct scans indicated a complete radiologic response. after years of follow up, mri showed a recurrence of mm between segment ii and iii of the liver.on multidisciplinary committee liver resection was decided due to suitable liver function and low aggressiveness of the tumour. a laparoscopic left lobe liver resection was performed. sonastarÒ and ligasure tm were used to perform the liver transection and endo gia tm for portal and hepatic veins sections. the surgery develop was complicated due to trend to bleeding that finally was achieve through cauterization. results: early after the surgery, the patient presented a haematic debt through the drain of cc accompanied of hypotension, therefore an emergent surgery was indicated. an exploratory laparoscopy was performed finding hemoperitoneum and diffuse bleeding of the liver surface that was controlled. the patient had a proper recovery and was discharged on the th day post-surgery. the analysis of the specimen showed a . cm cholangiocarcinoma with a . cm margin of resection. conclusion: there is an augmented risk of complications on liver resection of cirrhotic patients with portal hypertension. the laparoscopic approach allows to reduce potential complications, despite bleeding continuous to jeopardize this surgery, this option could be proposed on selected patients. introduction: accessory spleen itself is found in approximately % to % of the population. most ( %) are located near the splenic hilum but intrapancreatic accessory spleens (ipas) are the second most frequent location ( . %) of accessory spleens. in adults, ipas are clinically silent. they may become clinically important because of their radiographic similar appearence of cancer. intrapancreatic accessory spleen is a rare cause of pancreatic pseudotumors and is located in the pancreatic tail in approximately % to %. ipas can be difficult to differentiate radiologically from hypervascular pancreatic tumors such as pancreatic endocrine neoplasms because theycan share a similar enhancement pattern. as a result, most of the reported cases of ipas have been diagnosedonly after distal pancreatectomy was completed. material and methods: we present the case of a -year-old male patient with a history of large vessel vasculitis followed-up for rheumatology, which showed a pancreatic nodule in a control ct so he was referred to digestive for study. an echoendoscopy was performed. it showed, at the level of the tail, in the third distal, a lesion of x mm, hypoechoic, with rounded morphology and well-defined edges that can not be biopsied given the absence of adequate window for the realization of fine needle aspiration biopsy (fnab). based on these radiographic findings, the differential diagnosis included a pancreatic endocrine tumor. due to the high suspicion of malignancy and the absence of biopsy, he was referred to general surgery for scheduled surgery. a laparoscopic corporocaudal pancreatectomy was performed without incidents and the definitive histology showed an intrapancreatic accessory spleen in the pancreatic tail that excluded the presence of cancer. conclusion: intrapanceratic accesory spleen is a challenging diagnosis to make and it should be included in the differential diagnosis of pancreatic neoplasm. its early identification precludes surgical resection. however, the preoperative diagnosis of ipasmay be difficult, and distal pancreatectomy is a safe and relatively simple operation, most of the reported cases of ipas being diagnosed correctly only after surgery there are various options for treating pps. this paper describes our tailored and methodological approach to laparoscopic drainage of pancreatic pseudocysts based on an anatomical classification. methods: we adopted the laparoscopic approach in patients who had pps requiring surgical drainage. the laparoscopic method had been decided according to preoperative computed tomography (ct) and intraoperative findings. the results shown represent median (range). the aim of this work was to decrease mortality and morbidity in patients with combined trauma. methods and material: for years patients were brought to our clinic with combined trauma. everybody was performed ct and ultrasound examination. patients were performed open laparatomic operation due to massive liver rupture, spleen rupture and massive trauma of bowels, pancreas and kidney with massive bleeding. in circumstances we didn't found the trauma of the abdominal organs and the massive abdominal bleeding after ct observation. those patients were cured conservatively. in circumstances with combined trauma after ct examination we performed laparoscopic operation. in circumstances from the patients, who we started laparoscopic operation in, we conversed to laparotomy, due to massive liver rupture, and trauma spleen and hollow organs. in those circumstances we performed urgent laparotomies with suture ligation of bleeding points, suturing of liver and hollow organs and drainage of abdomen cavity. results: we performed laparoscopic operation in patients. in circumstances with trauma of liver we performed laparoscopic electro coagulation and argon-plasma coagulation. in circumstances with trauma of liver we performed electro coagulation with packing the omenture to its surface. in circumstances with trauma of spleen we performed argon plasma coagulation and used fibrin glue. after laparotomic operations mortality were in circumstances, morbidity were in patients. after laparoscopic operation mortality were in circumstances of severe combined trauma with multiple abdominal trauma and morbidity in patients. conclusion: laparoscopic operations in patients with combined trauma decrease mortality and morbility. aims: in laparoscopic distal pancreatectomy, getting away liver and stomach from the surface of the pancreas is sometimes difficult. when we separate the pancreatic body from the retroperitoneum, we must not injure the pancreas to prevent breaking a tumor. when we cut the dorsal side of the spleen from the retroperitoneum, we rarely cut into the spleen accidentally. based on our experiences, we gradually explored a set of procedural operation steps to resolve these problems. our three-step maneuver simplifies the procedure and improves the efficiency and safety of laparoscopic distal pancreatectomy. methods: as the first step, to get away the liver we sutured the round ligament of liver and crus of the diaphragm using - pds and the both ends were tugged form the outside of the body through both side of the xiphoid process. and the stomach was hung from the outside using two nylon thread like a bridge, so we could see the surface of the pancreas body with a good view. the second step was a rolling up maneuver of the pancreas. when we separate the pancreatic body and tail from the retroperitoneum, we rolled the pancreas with gauze for use in laparoscopic surgery and lifted the gauze up in only one assistant's forceps. then we could find the correct line for dissection clearly. the last step was a hanging maneuver of the spleen. when we cut the dorsal side of the spleen from the retroperitoneum, we hanged the hilum of spleen with cotton tape. with this technique we could find easily the correct line to dissect. results: the operation time was h and min and the estimated blood loss was a little. we did not injure the tumor or spleen in this operation. the patient recovered uneventfully after short hospitalization. conclusion: our three-step maneuver can be effective to perform laparoscopic distal pancreatectomy. about - % of patients with pancreatic collections will develop walled off necrosis, with an associated - % mortality. there are multiple options for intervention and drainage, usually the outcomes after endoscopic drainage are related with the nature of the collections. aims: to evaluate and present the rol of endoscopy in pseudocyst and walled off necrosis treatment, and favorable outcomes. methods and results: we present a case of a years old male, who presented biliary pancreatitis treated with cholecystectomy and transoperative cholangiogram weeks ago. he continued with persistent abdominal pain; his ct scan showed a big walled off necrosis; he was taken to surgery for an endoscopy-assisted laparoscopic cystogastrostomy with necrosectomy, he was discharged days po. conclusions: the step-up management of walled off necrosis has proven to be a better option than conventional surgical or endoscopical techniques alone; by reducing complications and mortality vs conventional necrosectomy. the use of endoscopic treatments reduce the pro-inflamatory response. drainage of walled off necrosis can be done by a transpapilar or transmural endoscopic apporach each one with its own advantages. some authors avoid the use of endoscopy in walled off necrosis because of a higher rate of complications, re-interventions and a greater lenght hospital stay. in our experience, we have achieved excellent results with this combined technique. nearest and long- patients underwent chemotherapy after electroporation procedure. day mortality was . % (n = ) in electroporation group. it was found that erreversible electroporation improved local recurrence-free survival ( and months, respectively, p = . ) and distant recurrence free survival ( and months, respectively, p = . ) . overall survival was and months, respectively (p = . ). conclusion: irreversible electroporation of locally advanced pancreatic cancer is safe. four month chemotherapy followed by surgical procedure is associated with good local response and better overall survival compared with chemotherapy alone. these data will be validated in further multicenter study. introduction: pancreatic pseudocysts are the most frequent complication of acute or chronic pancreatitis. usually asymptomatic, they can be managed conservative or, in case of complications, by several methods, endoscopic, percutaneous or by surgery. material and method: we present the case of a years old patient known with an episode of acute pancreatitis five years ago, who was hospitalised now for an upper gastrointestinal bleeding with hematemesis. the upper endoscopy showed a subcardial bulking with an erosion of the posterior gastric wall, with signs of recent bleeding, managed by clipping. patient work-up showed a cm pancreatic pseudocyst at endoscopic ultrasound. taking into consideration the history of the patient, the size and the complication of the cyst, the patient was proposed for a drainage intervention. results: a minimally invasive approach was decided. using ultrasonography guidance, a posterior gastrotomy was performed with the cystotome, establishing the comunication with the pancreatic pseudocyst. dilatation of the path with mm cre baloon, with partial evacuation of turbid liquid. the drainage consisted in pigtail fr plastic stents. the patient was discharged the following day in a good health condition.the endoscopic ultrasound control at weeks showed complete resolution of the pancreatic cyst and was followed by stent removal. the endoscopic drainage of the pancreatic pseudocyst represents the first treatment option as an alternative to the surgical intervention, being minimally invasive, with low risk and fast recovery. clinical case report: a -year-old man was admitted to the hospital with a diagnosis of severe acute pancreatitis and multi-organ failure. during the first month patient has in uci and non invasive procedures were attempted: enteral feeding by a nasoduodenal tube was started and antibiotics were administered to control sepsis. on day , percutaneous drainage was performed for large retroperitoneal abscess. on days, endoscopic transgastric necrosectomy was performed and the left collection was resolved. due to the multi-organ failure persistence and the evidence of size increase of the right retroperitoneal collection, a vard was decided.the right collection was accessed following the previously pigtail catheter. a mm trocar was placed to create retro-pneumoperitoneum with a pressure between - mmhg. a trocar of mmhg was placed, purulent content was aspirated and a debridement was performed. irrigation and aspirate was performed with normal saline and povidone-iodine solution. drainage was used to perform washes with physiological saline and urokinase.on days, the ct confirmed collection resolution. on days he was discharged. after months, the patient is in good clinical condition. discussion: drainage of the retroperitoneal abscesses via laparotomy is highly invasive and risky. vard enables radical necrosectomy and drainage less invasively. in this patient, the complete resolution of the right collection is obtained with retroperitoneal debridement without complications. we conclude that careful retroperitoneal necrosectomy is a valid alternative for the management of right collections. aims: in this study we analyze laparoscopic approach for hepatocellular carcinoma in order to clarify iwe can take advantage in some outcomes as complications, postoperative recovery or long-term survival outcomes. methods: a retrospective case consecutive study has been taken analyzing: age, sex, body max index, comorbidity, surgical extension and tumor size. the outcomes analyzed were: operation time, intraoperative blood loss, blood transfusion, postoperative morbidity and mortality, intensive care stay, hospital stay, tumor size, r resection, conversion rate, early reintervention, disease-free survival rate, overall survival rate results: in this study patients were analyzed males and females with ages between and years (mean age ) and diverse comorbidities: arterial high pressure ( / ; %), diabetes ( / ; , %) ; dislipemy ( / ; , %) , hepatophaty measured as liver cirrhosis ( / ; , %). all of them underwent laparoscopic liver surgery, in cases non-anatomical resection was performed while in the other a segmentectomy was performed. in cases the laparoscopic was strict, in and assistance incision was needed. operative time was - min (mean: min). blood loss mean was , g/dl and only intraoperative transfusion were needed. massive blood loss was reported in case. postoperative medical complications were observed: hepatic failure and renal insufficiency and in case we observed a postoperative hemorrhage that needed an urgent reintervention. the mean of intensive care stay was day and hospital stay was . days. about oncological outcomes r resection was achieve in / ( %), r in / ( %). at years / cases were free disease, dead by progression of disease and dead by other causes. aim: the purpose of this study is to analyze our initial experience with laparoscopic duodenopancreatic resection. introduction: laparoscopic procedures have advanced to represent the new gold standard in many surgical fields. laparoscopic pancreatoduodenectomy and laparoscopic distal pancreatectomy(ldp) are advocated to improved perioperative outcomes, including decreased blood loss, shorter length of stay, reduced postoperative pain and expedited time to functional recovery. however, the indication to minimally invasive approach for pancreatic surgery is often benign or low grade malignances. material and method. the steps of ldp procedures are similar to the open procedure. we perform destructive part of procedure totally laparoscopically and we prefer to do reconstructive part of procedure using hand-assisted techniques. for the period - , we have been perform pd, ( %) we have done with laparoscopic approach. ( %) of patients were operated totally laparoscopic and ( %) of patients were operated by handassisted techniques. results: a significantly higher conversion rate was encountered when lc was done - weeks after es, as compared to week after ercp. it is estimated that pancreatitis after ercp affects roughly three to percent of patients and many endoscopists quote a post-ercp pancreatitis rate of - %. however, - % is probably a more realistic answer for the majority of ercp endoscopists. wise endoscopists inform their patients that there is a spectrum of post ercp pancreatitis severity, from mild ([ % of cases) to severe ( - % of cases). in mild forms, pancreatitis after ercp may resolve itself. conclusion: endoscopic retrograde cholangiopancreatography is a procedure used to diagnose and treat disorders involving the pancreatic and bile ducts. acute pancreatitis is the most common and feared complication of endoscopic retrograde cholangiopancreatography. the assumption is that the duration of the laparoscopic method is longer, but on the other hand the patient have better wound healing and fewer possibility of developing postoperative hernia . the postoperative period is much more simple due to the significantly shorter hospitalization and the faster recovery, and according to patients the level of pain is much smaller as well. however the oncology results are the same. introduction: spiegel hernias are a rare, representing only between . % and % of all abdominal wall hernias. due to its location, below the spiegel line, its diagnosis requires a high index of suspicion. the physical examination only detects % of the spiegel hernias and, in many occasions, imaging tests are necessary for the diagnosis. goals: our objective is to describe the case of an urgent laparoscopic repair of a case of high grade bowel obstruction secondary to a spiegel hernia. material and methods: we present the case of a -year-old male patient with no medical history that comes to the emergency department of our center due to an eight hour evolution of abdominal discomfort associated with nausea without vomiting or other symptoms. the patient was afebrile and hemodynamically stable at all time. on physical examination, the abdomen is soft and depressible, painful on the left flank where a tumor compatible with spiegel's hernia is palpable. in the blood count there is no leukocytosis nor alteration of inflammatory parameters. an abdominal computed tomography (ct) scan was requested from the emergency department which demonstrated a high-grade small bowel obstruction caused by an entrapped loop of distal jejunum conditioned by a left-sided spiegel hernia. given the situation, an informed consent was obtained, and the patient was taken to the operating room for emergency laparoscopic repair. we performed a laparoscopic hernioplasty with ventralpatch mesh between oblique major and transverse and primary closure of defect in continuous suture. after this, the evolution of the patient is favorable, with good oral tolerance and re-establishment of intestinal transit, being able to be discharged h after surgery. the spiegel hernia is a rare entity that requires a high index of suspicion for its diagnosis. despite the limited evidence published in the literature on the laparoscopic repair of incarcerated spiegel hernias, the studies published so far suggest that the laparoscopic repair is a valid alternative to the classic approach when it is performed by a well-trained laparoscopic surgeon. introduction: repair of lateral abdominal wall hernias (both primary and incisional) can be challenging due to the complexity of anatomy, issues with fixation and the low incidence of such cases. a good understanding of abdominal wall and retroperitoneal anatomy, coupled with proficient laparoscopic technique is essential for successful repair via the minimally invasive approach. methods: a retrospective review of a prospectively maintained database was performed to identify patients with lateral abdominal wall hernias who underwent laparoscopic repair from january to july . results: patients with hernias were identified ( primary, incisional). mean patient age was (range - ) and mean bmi was . kg/m (range . - . ). according to ehs classification, the incisional hernia defects were located at subcostal (l , n = ), flank (l , n = ), iliac (l , n = ) and lumbar (l , n = ) regions. background: it is commonly admitted that laparoscopic surgery has the advantage of abdominal wall preservation. however, the increased use of laparoscopy has resulted in certain complications specifically associated with the laparoscopic approach, such as trocar-site incisional hernia. until today, it is not finally clarified 'patient-dependent' factors contributing to the occurrence of postoperative hernia after laparoscopic abdominal surgery. methods: between and , patients were operated due to trocar-site incisional hernia in one surgical centre. 'the patient-depending' factors which caused postoperative trocar site incisional hernia data was collected and retrospectivily analysed. results: port site incisional hernia occurred in % ( patients) after the use of trocars with mm or larger diameter. the presence of metabolic syndrome was the decisive factor in the development of postoperative incisional hernia in % ( patients). in % ( patients) the postoperative hernia occurred on the background of a long cough symptoms caused by chronic obstructive pulmonary diseases. the cause of postoperative hernia in % ( patients) of patients was the condition of lifting a one-time severity or heavy physical work. in % ( patients) of postoperative patients hernia developed due to prolonged constipation of chronic inflammatory colon diseases. conclusions: thus, when the aponeurosis of the trocars is adequately closed, the reason of the occurrence of postoperative hernias was caused by patient-dependent factors which increase intra-abdominal pressure. for this method, small midline incision cm in length - cm away from hernia orifice was carried out initially. dissection of intraperitoneal adhesion was carried out by sils with sils device. subsequently after closure of initial laparotomy unilateral anterior rectus sheath was incised from the same incision and dissection of retro-rectus space up to preperitoneal space was done under laparoscopic vision. dissecting the other side was carried out by same fashion. initial dissection of linea alba could be done by open surgery from initial incision. further dissection of linea alba, retro-rectus space, and hernia orifice was carried out by sils. defect closure of anterior and posterior rectus sheath using barbed suture was also done by sils and self-grip mesh was inserted. additional trocar to assist retro-rectus dissection, defect closure, and decompression of intraperitoneal cavity was inserted as required. aims: the laparo-endocsopic approach of inguinal hernia contiue to bring many clarifications concerning inter-parieto-peritoneal space of this region through in vivo exploration, obtained by magnification by means of specific optic intrumentation. our study aimed to revalue the in vivo fascias, to establish their embryological correspondences and to reunite the variable nomenclature existing in the classical anatomy of this region. these observations find their applicability in tapp and tep hernia procedures, as the old anatomical descriptions are no longer operative. methods: we have tried to identify the structures that delimit the anatomical regions of retzius and bogros in recording of tapp procedures performed on men, on the right side, for small indirect hernias on patintes with clear view of the structures. additional, a review of literature on this subject has been performed through a search in the detabases according to the following keywords: bogros space, retzius space, preperitoneal approach, urogenital fascia. results: retzius and bogros are the medial and lateral compartments of the inter-parietalperitoneal space, located between the transversal fascia and the parietal peritoneum. these narrow, virtual spaces are best highlighted today with the help of insufflation techniques during laparo-endoscopic procedures. a competent and careful dissection confirms a 'deep and superficial' stratification, highlighting embryonic relics derived from the uro-genital fascia: urinaryprevesical fascia and spermatic fascia. in addition, the real retzius space is located previously and the real bogros space is located behind this strcuture. the confluence area of the two spaces is a critical point of laparo-endoscopic dissection, its non-recognition may 'wander' the dissection. conclusions: literature data in this topic reflects a certain terminological confusion using general terms such as 'preperitoneal tissue' or 'arreolar tissue' to denote what we consider to be the urogenital fascia or its prologations. the data obtained were synthesized in several drawings and diagrams very useful in training surgeons to use tapp / tep techniques. aim: spigelian hernia containing epiploic appendage is really rare entity. in this paper, we present a very rare case of spigelian hernia involving epiploic appendage performed laparoscopic hernia repair. case report: a -year-old woman presented to the emergency department with sudden onset abdominal pain in the left lower quadrant. on physical examination, she had a small, palpable tender mass in the left lower abdominal quadrant. temperature and white blood cell count were normal. an inflamed epiploic appendage with an oval shape, a fatty core, and a central thin hyperdense line in the hernia sac was detected on abdominal computed tomography. its intraabdominal relationship with the normal wall of the sigmoid colon was well appreciated (figure a, b) . diagnostic laparoscopy was performed. (figure ) adhesions between the sac and epiploic appendage are released using sharp dissection. a peritoneal flap is then created (figure ). laparoscopic tapp repair was used without closing the defect (figure ) . the patient was discharged on th days uneventfully. aims: morgagni's hernia is an in infrequent, congenital, anterior or retrosternal diaphragmatic defect. the right side is the most frequently affected, up to % of cases. it represents between and % of congenital diaphragmatic hernias. in childhood, they usually attend asymptomatically or with respiratory symptoms. up to % are diagnosed in adulthood, incidentally or after gastrointestinal obstruction debut. the treatment is surgery, which can be by laparoscopic or open approach.we present a case of laparoscopic approach with intra-abdominal mesh placement of giant morgagni's hernia diagnosed in senile age. methods: -year-old woman with a history of advanced alzheimer's dementia, partially dependent in daily life activities and institutionalized who consulted for intermittent episodes of oral diet intolerance associated with vomits of one month of evolution. abdominal examination was anodine. chest radiograph revealed a right lower lung field mass with fluid collected. thoracoabdominal scan showed small bilateral pleural effusion and large, right anterolateral morgagni's hernia, which contains dilated segment of transverse colon and greater omentum . results: laparoscopic approach was performed. hernia was reduced and hernia sac was removed. the defect was repaired with a dual-component (absorbable and non absorbable) mesh anchored with intracorporeal suture. patient recovered and was discharged days after surgery. conclusion: laparoscopic approach for morgagni's hernia reapir is secure and offers the advantages of less post-opertive pain, faster recovery and short postopatory stay. introduction: recently, laparoscopic operations for ileus are increasing. we have undergone laparoscopic operation to adhesive ileus with umbilicar incision at the beginning. the umbilicar incision at the beginning makes it possible to secure the laparoscopic field by peeling the adhesion under direct view, and makes it easy to repair damage to the intestinal tract. surgical procedure: at first, the umbilicus - cm incision was made and peeled the adhesion as much as possible under direct vision. secondly, ez access was set and inserted one mm port, therefore laparoscopic operation was performed with or pieces of mm ports. when the repair or resection of small intestinal due to damage is necessary, it is pulled out through the ez access. objective: to investigate the possibility of problems of laparoscopic ileus operation to adhesion ileus by umbilicar incision at the beginning. introduction: small bowel obstruction (sbo) during pregnancy is a rare condition with an incidence of . - . % and in around % of cases it is most caused by adhesions from previous abdominal surgery. other diagnosis, such as, hernias, malignancy, volvulus or intussusception are extremely rare. when sbo occurs in pregnancy, it carries a significant risk to mother and fetus. its diagnosis of can be difficult to make as symptoms are often attributed mistakenly to the pregnancy. goals: a case report of congenital bowel obstruction during the second trimester of pregnancy handled by laparoscopy. material and methods: we report the case of a year old woman with a history of chronic lung disease, pregnant because in vitro fertilization ( ? weeks) who attended the emergency department with abdominal pain and bloating accompanied by nausea and vomiting for two days. on physical examination she showed a distended, soft, depressible and painful abdomen without peritonism. laboratory tests were normal. a nasogastric tube was placed with generous output fecaloid intestinal contents. abdominal ultrasound by expert radiologists in abdomen showed a moderate amount of free abdominal fluid with normal uterus moderate and sbo to the ileum because of intestinal adhesion. this results were confirmed with an magnetic resonance imaging (mri). results: the patient was operated by laparoscopic approach with three trocars. the main problem was discovered. we founded a congenital adhesion which conditionated the obstructive syndrome. postoperative recovery was uneventful and the patient was discharged h after surgery. conclusion: the non-obstetrical acute abdomen in pregnant patient is a reality that occurs in one of every pregnancies. its diagnosis in more difficult than in nonpregnant patients requiring or high index of suspicion. the laparoscopic approach of acute abdomen during pregnancy is a valid and safe option, even in the early hours after diagnosis of bowel obstruction when it is performed by a well-trained laparoscopic surgeon. aim: intestinal malrotation (im) without midgut volvulus in adults is a rare clinical entity, which is the result of an incomplete rotation of the small bowel during embryogenesis, due to the nonlysis of the ladd bands. these ligaments spread between the duodenum and caecum and do not allow the gastrointestinal tract to take its normal position into the peritoneal cavity. im appears in to - newborns and is usually asymptomatic. diagnosis is usually made in the first month, and presents with findings of an acute abdomen, small bowel ileus and volvulus. im in adults is a rare entity. most of the times it is asymptomatic, but it can cause chronic abdominal discomfort and constipation. we present the laparoscopic management of an adult patient with intestinal malrotation. methods: our patient, a year old female, presented to the emergency room with a -month history of abdominal pain and nausea. all blood tests were normal. an abdominal mri showed intestinal malrotation without volvulus. due to persisting symptoms, she underwent a diagnostic laparoscopy with complete lysis of the ladd bands. the only unusual finding was a slight oedema of the duodenum. results: her symptoms settled postoperatively and she was discharged on the nd postoperative day. since her discharge, she has not developed any similar abdominal pains or complaints. conclusions: symptomatic intestinal malrotation in adults is an unusual clinical entity, but it is definitely one of the differential diagnoses we need to consider in case of chronic abdominal symptoms. the management consists of the division of the ladd bands, and this procedure can be performed safely with laparoscopy. many small intestinal obstructions are due to adhesions after laparotomy, but small bowel obstructions without history of open surgery is relatively few. in diagnostic imaging such as preoperative ct examination, the cause is diagnosed to some extent, but details are sometimes unknown unless operative observation is actually made. in many institutions, laparoscopic surgery is also actively introduced into the operation to relieve bowel obstruction, and its effectiveness is beginning to be recognized. we examined the usefulness of laparoscopic surgery for patients with small bowel obstruction without history of laparotomy from experience in our hospital. aim: from december to october , we searched cases of laparoscopic surgery for a small bowel obstruction without previous laparotomy at our hospital, and clinical findings, surgical results, and postoperative course were examined. results: there were ten cases. eight men and two women. the median age was years ( - yrs.) . reasons for intestinal obstruction were adhesions cases, internal hernia cases, persimmon stones case, small intestine tumor case. four cases of adhesions were emergency surgery. there were cases of emergency surgery and waiting surgery. five laparoscopic operations were completed and five cases during laparotomy transition. the median surgical operation time was min ( - min), and the median bleeding amount was g ( - g). there was no fatal case after operation, only one complications of ileus. the median length of hospital stay was days ( - days) . conclusion: laparoscopic surgery for intestinal obstruction with no history of laparotomy was thought to be a safe and effective procedure. although the transition to laparotomy would be higher in case of emergency, but there was no case of large incisional laparotomy. conclusions: laparoscopic surgery for sbo reduces postoperative complications and contributes to shortening the postoperative hospital stay and to decreasing the rate of recurrences, although it is a retrospective study, which is a safe and a useful approach. furthermore, first episode of sbo without previous operation seems to be an appropriate indication for laparoscopic surgery. background: postoperative adhesion after abdominal surgery may cause intestinal obstruction, chronic pain, or female infertility, which constitutes the major problems after surgery. adhesion formation are reported to be reduced by laparoscopic surgery and the use of anti-adhesion barriers. seprafilm composed of sodium hyaluronate carboxymethylcellulose bioresorbable membrane has been widely used to date, especially in open surgery. the characteristics of seprafilm, which is easily stick when wet, conversely brittle when dry cause it difficult to deliver into the abdominal cavity via the small incision in laparoscopic surgery. therefore, seprafilm is not much used in laparoscopic surgery. although various methods of insertion of seprafilm have been reported, some need special devices, or some acquire skill. methods: we adopted the pre-moistening technique for the replacement of seprafilm in consecutive cases of laparoscopic gastrointestinal surgery. a sheet of seprafilm was cut into equal pieces. to soften the sheets, one of the pieces was placed on a folded wet gauze until it became naturally curled then it was reversed, and the same procedure was repeated. softened sheet is easily to deliver into the abdominal cavity via a small incision by pushing with digital finger. moistened sheet expands naturally in the abdominal cavity. one or two pieces were needed to cover the incision. this process took only a few minutes. results: in all cases, the sheets were successfully introduced into the abdomen and spread widely enough to cover the incision. there have been no adverse effects, no postoperative complications, or gastrointestinal obstruction due to adhesion in the observation period of median two years. conclusions: short term outcomes were good after applying this technique. however, to record the incidents of intestinal obstruction and chronic pain, over years observation is indispensable. long term follow-up studies are required to clarify the usefulness of the anti-adhesive barrier in gastrointestinal surgery. b. east, rd department of surgery, motol faculty hospital, prague, czech republic aim: since when the ipom acronym was used for the first time our views at intraperitoneal mesh positioning has changed several times. despite growing evidence on its possible long term consequences it is still preferred method at some centres for large number of patients. the aim of this study is to point out the pitfalls of this method but also show that ipom is a good technique but only for highly selected cohort of patients. methods: this is a review of the literature focusing on the indications and complications of ipom pointing out controversies among the published articles over last two decades. some mesh material characteristics are being discussed as they are basic for understanding this complex and highly sensitive issue. results: a wide range on indications of ipom from little umbilical to large incisional hernias is advocated by many. however, some opinion leaders promoting this technique as universal and ideal for everyone just few years ago are advising to avoid it if possible lately. a necessary overlap has also been questioned recently. despite improving anti-adhesion barriers and methods of fixation in may a surgical mesh has become classified as risk class iii by the eu parliament and council on medical devices hoping to prevent physiomesh like incidents in the future. the need for post market registries and long term follow up is obvious. conclusion: us as surgeons implant a mesh in our patients and therefore we should be aware of its possible long term effects. no mesh on the market has a long term safety evidence especially in the intraperitoneal space. ipom is a good technique but possess a significant risk of long life complications and therefore should be spared only for those unfit for other methods of repair, patients with too high mesh infection risk, obese or older patients. introduction: acute appendicitis in elderly patients is relatively uncommon and could represent an underlying neoplasm. hence patients over the age of are often referred for a follow-up colonoscopy after management of acute appendicitis. the current routine use of computed tomography (ct) scans in the evaluation of suspected acute appendicitis in elderly patients prior to surgery coupled with intra-operative findings at laparoscopy question the role of follow-up colonoscopy for these patients. aims: to determine the role and optimal timing of colonoscopy in early detection of colorectal neoplasia after treatment of acute appendicitis in elderly patients. methods: all patients aged years and above with confirmed appendicitis admitted to our hospital during the period / / to / / were included. follow-up colonoscopy, diagnosis of colorectal neoplasia and its location in this patient cohort was evaluated. results: number of people aged and above in olol who had appendectomies from the dates / / to / / = . out of them / ( %) had full colonoscopy within years of the appendectomy.of them of the colonoscopies done were maleand were females. / ( %) of these colonoscopies were completely normal. colonoscopy identified colorectal carcinoma in ascending colon ( . %). other pathologies identified included: benign polyp ( %), polyp with low grade dysplasia ( %) and others ( . %) (lymphocytic colitis, ulcerative colitis, medication related ulceration, diverticulosis, melanosis coli, haemorrhoids). conclusions: in elderly patients above years of age: there may be an increased risk of colorectal cancer after acute appendicitis. only % of this patient cohort underwent colonoscopy after appendectomy. the current recommendations suggest the need for follow-up colonoscopy in elderly patients post acute appendicitis. further studies are needed to decide whether routine colonoscopy is indicated after acute appendicitis patients over years. introduction: it is generally accepted that the main aetiology of appendicitis is obstruction due to appendicoliths in adults and lymphoid hyperplasia in children. in contrast, incidental appendicoliths have been reported to occur in up to % of the asymptomatic population. controversy still exists regarding the association of appendicolith and appendicitis. is the appendicolith a causative factor or merely an incidental finding? aims: to determine the association between the presence of appendicolith and acute appendicitis (perforated or non-perforated) vs healthy appendix. methods: we collected the data retrospectively from the electronic records of all appendicectomies performed between january and december in our institution. data collected included: age, sex, appendix histology and the presence of appendicolith. interval or incidental appendicectomies were excluded from this study. we analysed the data using spss software version . results: during the study period appendectomies were performed (males: , females: , age range: - years). cases were histologically confirmed cases of acute appendicitis and of these, were perforated. a normal appendix was identified in cases. the remaining cases were due to chronic appendicitis, sub-acute appendicitis, lymphoid hyperplasia, parasitic infestation, and neoplasm. appendicolith was found in cases, of which were found in a normal appendix and were found in an inflamed appendix. out of the cases of appendicolith with normal appendix: cases were aged between and years old, cases were aged between and years old and case was aged between and years old. out of cases of appendicolith with acute appendicitis, cases were aged between and years old, cases were aged between and years old and case was aged over . conclusions: appendicolith may merely be an incidental finding and is not the primary cause of appendicitis. no significant correlation between gangrenous/perforated appendicitis and the presence of appendicolith. contrary to popular belief appendicoliths are more common in paediatric appendicitis than in adult cases. further research is recommended. over the last years, patient satisfaction surveys have gained increased popularity. nowadays, respect for patients' needs is central to our health care system. hospitals use patient satisfaction surveys to assess quality of care. many hospitals routinely survey patient satisfaction but relatively little data has been published. our acute surgical assessment unit operates from am to pm monday to friday and in its first year saw surgical patients, of whom were discharged and were admitted to the hospital for further management. aims: to assess the levels of satisfaction of patients attending asau at our lady of lourdes hospital. methods: a random sample of patients seen in the asau was surveyed to determine their level of satisfaction and the experience they had whilst attending asau. a novel self-reported patient satisfaction questionnaire was developed and used to assess patients' opinion regarding the treatment they received, the doctor's explanation of their condition, the waiting time and the service in asau. also the questionnaire encouraged patients to suggest improvements to the service. aim: sintestinal obstruction is a very common cause of presentation to an emergency department. the most common cause in patients with prior abdominal surgery are adhesions, but the list of differential diagnosis is large. internal hernia is a very rare cause of obstruction, with a reported incidence of between . and . %. the herniation related with broad ligament defects is even more uncommon. methods: we report the case of a -years-old woman with antecedents of liver transplant, tubal ligation and appendectomy. the patient was admitted refering abdominal pain in the epigastrium of h duration, accompanied by nausea and vomiting. on physical examination, abdomen was depressible, tender in the right low quadrant, without evidence of peritoneal irritation. laboratory studies were normal except for an elevated leukocyte count with a left shift. computed tomography (ct) revealed dilated small bowel loops with a transition point in right lower quadrant. radiological diagnosis was intestinal obstruction, with fibrous adhesion as the most probably aetiology. management was conservative at the beginning, with intravenous hydration, nasogastric tube and administration of gastrografin (diatrizoate) without a good response. results: at h, an exploratory laparoscopy was perform, finding dilatation of small bowel loops and a cm defect in the right broad ligament in which a segment of ileum was herniated. ileal segment was liberated without evidence of ischemia. the hernial defect was closed by laparoscopy with simple silk stitches. the postoperative course was excellent, tolerating oral feeding next morning. the patient was discharged h after surgery. conclusions: internal hernias of the broad ligament are an extremely rare cause of intestinal obstruction, but must be added to the differential diagnosis for female patients due to the risk of intestinal strangulation and perforation. even if clinical and radiological diagnose is difficult, ct is the best tool to delineate the cause and location of the obstruction. laparoscopy allows reduction of the hernia and closure of the defect with minimal invasiveness. because of that, the laparoscopic approach of bowel obstruction should be considered as the first choice if there is the suspicion of an internal hernia, without signs of necrosis or perforation. the laparoscopic approach is a safe and effective tool in the management of postoperative complications. it is well tolerated in critically ill patients and avoids respiratory and wound related morbidity associated with laparotomy. it also reduces diagnostic delay and a considerable number of unnecessary laparotomies, with a high resolution rate and minimal morbidity. it thus represents a valid and necessary alternative in surgeon's armamentarium. in the management algorithm of our institution we always choose the laparoscopic technique as the fisrt tool in case a reoperation is necessary. , small bowel obstruction ( . % vs . %), and colorectal cancer obstruction ( . % vs . %) was found higher for acs unit group, and also progressively higher during the last years. conclusion: according to our study, laparoscopic approach in abdominal emergencies shows an upward trend, and surgeons from acs units seem to have higher rates of laparoscopy than general surgeons in emergency procedures. background: incarcerated and strangulated hernias present a major problem in emergency medicine. there is scarce data about the role of laparoscopy in the management of these patients. laparoscopic repair offers the benefits of the ability to survey the incarcerated organ and to evaluate its viability, apart from the obvious advantages of laparoscopic surgery. the use of mesh repair in these emergent operations is also a major concern, due to the un-sterile conditions in which they are performed. objective: to evaluate the safety and short-term efficacy of laparoscopic emergent repair of incarcerated hernias. methods: retrospective review of prospectively collected data of all the patients who underwent emergent laparoscopy due to an incarcerated hernia between november and october . results: during the study period, patients underwent emergent laparoscopy due to incarcerated hernias ( females, males). had incarcerated inguinal hernias, and had incarcerated umbilical hernias. mean age was . . all inguinal hernias were repaired in the tapp approach, and using an absorbable mesh. all umbilical hernias were repaired using the ipom approach. patients had bowel obstruction, had incarcerated omentum, and one patient had incarcerated urinary bladder. patients underwent resection of an ischemic organ ( bowel, urinary bladder, omentum). mean hospital los was . days. during the follow up period there were no mortalities, and no recurrences. one patient had a wound infection that resolved with antibiotics. conclusion: laparoscopic emergent repair of incarcerated hernias is a safe and feasible approach. further studies with longer follow up time need to be conducted, in order to evaluate the added benefit of the laparoscopic approach. gibraltar is a small overseas british territory with a residential population of approximately , inhabitants, that increases up to , daily due to incoming tourists and cross-frontier workers. as a geographically isolated center we have to provide a varied service including emergency surgery, and elective operating such as colectomies, gastrectomy's etc. one of the challenges faced is the limited stock of red blood cell (rbc) units within gibraltar and reliance on platelets (plt) from across the border from spain. given the immanent brexit we need to prepare for the challenges we will face in these times of political and distribution uncertainty. a prospective audit of all blood use within gibraltar was carried out over months. the number and type of units requested, the number of units given, the speciality, location and indication for requests was recorded. introduction: the use of laparoscopic surgery in abdominal emergencies, such as in trauma, has had a slow acceptance. the advantages with this approach include less postoperative pain, faster recovery, quicker return to everyday activities, and fewer complications. we have collected the cases and indications of laparoscopy in abdominal trauma in the main hospitals in the andalusian capitals and compared with the national registry material and methods: a total of patients who underwent laparoscopic surgery in the main hospitals of seville, cordoba, malaga, cadiz, huelva, jaen, granada and almeria were analyzed. they have been compared with the traumas archived nationally by the spanish association of surgeons taking into account age, sex, score of the american society of anesthesiologists, hemodynamic stability and mechanism of injury. the intra and postoperative variables were compared between groups. results: at the national level, the main cause of abdominal trauma were traffic accidents, therefore, it was the patients who had a greater number of laparoscopies ( . %), followed by stab wounds ( , %) and run over ( . %). in our series, the average age of the patients is years and % are male. only eco-fast was performed in % of the patients, being positive in . % of the cases. as they were stable patients, in % of the cases a tac was possible. in our data, % of the laparoscopies were performed for therapeutic purposes as well as being diagnostic, thus avoiding a posterior laparotomy. conclusion: slaparoscopic surgery for abdominal trauma, either blunt or penetrating, is safe and technically feasible in hemodynamically stable patients. we found that laparoscopic surgery was associated with shorter operative time, lower estimated blood loss and faster return to normal diet. based on our findings we establish the indications of laparoscopy in these patients aims: submucosal aneurysm of small intestine is extremely rare, but its rapture can be lifethreatening. due to the unstable hemodynamics and unknown site of bleeding, emergency laparotomy has been widely performed for the rupture. we will present case reports and show the strategy for minimally invasive treatment for ruptured aneurysm. methods: we experienced two cases of ruptured submucosal aneurysm resected by laparoscopic surgery. case is a -year-old male who was taken to our er with massive hematochezia. ct showed arterial bleeding in the small intestine and angiography revealed bleeding from the ilial artery. selective embolization using gelatin sponge and micro coil was performed and hemostasis was obtained. video capsule endoscopy found the hemispheric elevated lesion with protrusion at the top in the ileum. using balloon assisted enteroscopy, the site of aneurysm was marked with injecting india ink, which allows surgeons to accurately and easily identify the part of small intestine with aneurysm. subsequently, a single incisional laparoscopic assisted partial ileectomy was performed for the purpose of definitive diagnosis and preventing re-bleeding. the ileum with aneurysm was easily identified in laparoscopic exploration owing to the marking, and it was taken out from the incision to perform resection. case is a -year-old female who was transferred to our emergency department with sudden onset of massive melena. ct and angiography were perfomed, and bleeding from the rd jejunal artery were confirmed. subsequently, therapeutic embolization was performed in the same way as case . enteroscopy revealed submucosal elevation similar to case in the jejunum. we carried out endoscopic tattooing, followed by single incisional laparoscopic assisted partial jejunectomy. results: the operative time in case and case were min and min, respectively, and the amount of blood loss was both ml. the postoperative course was uneventful in both cases. case was discharged on the postoperative day , and case was on postoperative day . conclusions: our experience indicates that ruptured submucosal aneurysm of the small intestine can be effectively managed by a laparoscopic surgery with combination of therapeutic embolization and enteroscopic evaluation, which is safe and minimally invasive. background: laparoscopic bilateral inguinal hernia repair may be completed with one large selffixating mesh crossing the midline in front of the bladder. no studies have investigated in detail whether preperitoneal mesh placement induces temporary or more lasting urinary symptoms. methods: urinary and hernia related symptoms were evaluated preoperatively and postoperatively at , and months in patients using the iciq-mluts questionnaire and eurahs-qol score. results: voiding symptoms and bother scores were unchanged at or months, but there was significant improvement at months compared with preoperative findings (symptoms p \ . ; bother score p \ . ). incontinence symptoms improved at month (p \ . ) but not at or months, with a bother score significantly improved at month (p \ . ) and months (p \ . ). diurnal and nocturnal frequency did not change significantly postoperatively, but months nocturnal bother score was decreased (p \ . ). eurahs-qol scores showed significant improvement in all domains for all measurements compared to previous measurements. postoperative symptoms were improved at months, compared with preoperative pain scores (- . ), restriction of activity (- . ) and cosmetic scores (- . ) these findings were statistically significantly (p \ . ). at months, there were no patients with severe discomfort (score = ) for any of domains. no recurrences were diagnosed with % clinical follow-up at months. conclusion: placing a large preperitoneal self-fixating mesh for bilateral groin hernia repair did not cause new urinary symptoms and demonstrated significant improvement in voiding symptoms at months. incontinence and nocturnal bother score were significantly improved. introduction: tep/tapp hernia repair is an increasingly widely used surgical methods for minimally invasive treatment of inguinal hernia. tep advantages to tapp are noincision of the parietal peritoneal sheet, therefore no need for its recovery-sewing or sticking at the end of the procedure, and no need for attachment of the prosthetic mesh to the structures of the anterior abdominal wall, which results in a reduction in the financial cost of operation.various types of meshes with different characteristics are used, depending on the surgeon's preferences.the aim of this study is to highlight mesh-related postoperative complications, which can be serious and life-threatening. material and methods: a retrospective cohort study of cases of unilateral or bilateral tep and tapp hernia repair performed at the university hospital for the period - with a study of early and late postoperative complications potentially causally related to the implanted prosthetic mesh and methods of their treatment. results: for a -year period tapp ( bilateral) and tep ( bilateral) have been performed. three complications (clavien-dindo iva, ivb and v) were found, of which were early postoperative (up to pod)-one in tapp- pod small bowel adhesive ileus due to suture dehyscense of the peritoneal sheet and adhesion of a bowel loop to the surface of polypropylene mesh.one in tep- pod-a large preperitoneal hematoma with haemorrhagic shock at years old female in anticoagulant therapy-an open revision of the preperitoneal space and definitive haemostasis; followed in pod established bladder lesion from erosion from the edge of self-locking polypropylene mesh. suture and drainage performed, but the patient died of decompensation of concomitant diseases. a late complication- months after bilateral tep-erosion of soft polypropylene mesh of sigma (probable undetectable lesion of the peritoneum) with faecal peritonitis-hartmann procedure with laparostoma followed by restitution but persistent chroniosepsis with established abscess in retzii. months after-revision with abscess incision and extraction of infected meshes. discussion: use of biologic meshes is quite expensive, however synthetic non-resorbable meshes implanted in preperitoneal layout is a prerequisite for specific severe postoperative complications. inguinal hernia repair is one of the most performed procedure all over the world, with more than million procedures performed each year, it represents one of the top three most performed procedures. the lichtenstein procedure is one of the first procedures that a young trainee in general surgery learn, not only for its reproducibility and for the great numbers of procedures that could be done in each department, but also because during inguinal hernia repair the trainee learn a lot of skills which are the basis of major surgical interventions. the surgeon's performance for any procedure could be evaluated by way of established learning curves that can predict the minimum number of procedures required to reach the same intra and post-operative outcomes as an experienced surgeon performing the same technique. the aim of our multicentre study was to analyse how many cases are required to stabilize operating time (ot) and intra and post-operative complication rates over the course of the learning curve period for a lichtenstein procedure. from january to december all lichtenstein procedures from four different institutions were recorded in a prospective maintained computer database. the results of the first consecutive procedures performed by three different trainees (group a; group b; group c) were compared with the same numbers of procedures by two senior surgeons of the same institutions (group e, group f). cusum analysis was performed to evaluate the achieving of learning curve. no differences in terms of biometric and hernia type were recorded between the five groups. cusum analysis showed that the trainees achieve the learning curve between the - procedures. no intra or post-opertive complications were recorded during the training period.in conclusion after our analysis we found that at least procedures are needed for the trainees to achieve the learning curve for lichtenstein procedures. background: since its first description in the s, the total extraperitoneal (tep) technique has established itself as a popular endoscopic method for the repair of inguinal hernias. the tep repair is generally viewed as a technically-demanding procedure requiring adequate experience to minimize and handle complications. in this case report, we describe an uncommon complication of urethral injury, which was successfully repaired laparoscopically. case report: mr r is a year old gentleman with no significant past medical history who presents to the department of general surgery, tan tock seng hospital, with a two-month history of a reducible right inguinal hernia, associated with some tenderness. an ultrasonography confirmed the diagnosis of a fat-containing indirect right inguinal hernia. in view of persistent pain, mr r was counseled for a laparoscopic repair of his right inguinal hernia. as mr r was able to empty his bladder just prior to surgery, no urinary indwelling catheter (idc) was inserted. an infra-umbilical incision was made to access the posterior rectus sheath and a balloon was used to bluntly dissect the pre-peritoneal plane. on inspection of the operating field, persistent pooling of blood was noted in the retropubic space. careful inspection revealed a defect in a tubular structure just inferior to the bladder neck. an idc was inserted, which confirmed a . cm defect in the pre-prostatic urethra. decision was made for primary repair using absorbable sutures in two layers. the bladder was subsequently filled via the idc, which did not reveal any leak. we then completed the right inguinal hernia repair using a mesh. mr r made an uneventful recovery and was discharged on post-operative day with instructions to keep the idc in-situ for two weeks. the idc was removed after two weeks and a micturating cystourethrogram was performed, which showed no filling defects along the urethra and no contrast leaks. discussion: though uncommon, urethral injuries can be a complication of laparoscopic tep repair. the key to managing these complications is in the early identification of such injuries intra-operatively. with early recognition and careful assessment, such complications can be managed laparoscopically with minimal post-operative morbidity. aim: the purpose of this study is to report surgical technique and outcome of hybrid tapp procedure (a combination of tapp and ipom) for inguinal hernia patients complicated with preperitoneal space adhesion. methods: hybrid tapp procedure is applied if peritoneal dissection or closure of the peritoneum is difficult due to severe adhesion. the peritoneum should be dissected as much as possible. for the site where adequate dissection was achieved, the collagen mesh is placed outside the peritoneum. in the part where dissection was difficult it is placed inside the peritoneal cavity. in order to prevent mesh migration, the mesh should be directly fixed to the cooper's ligament with a tacker. for this purpose, the peritoneum around the cooper's ligament must be well-dissected, even if it is strongly adhered, so that the ligament can be exposed. the crucial points in the hybrid tapp procedure are fixation of the mesh and prevention of the bowel herniation into the preperitoneal space. at the site where peritoneal dissection is possible, the mesh is directly fixed on the fascia using a tacker. if it is difficult, the mesh is placed in the peritoneal cavity and fixed over the peritoneum. if there is a risk of migration along with peritoneum, transcutaneous full-thickness fixation can be performed using non-absorbable sutures. the preperitoneal space should be closed tightly as soon as possible in order to prevent the bowel herniation into the preperitoneal space. at closure of the preperitoneal space, the peritoneum is fixed on the collagen mesh using non-absorbable sutures. objective: show a tapp approach using a self-fixating mesh( x cm. progrip tm laparoscopic self-fixating mesh, medtronic) with bipolar peritoneal defect sealing, avoiding the use of tackers and performing an easy and sutureless peritoneal closure. material and methods: years old male, asa ii, medical history of beta-latacm allergy, high blood pressure, dyslipidemia and bilateral knee surgery. diagnosed of bilateral inguinal hernia at consultation due to inguinal disconfort. surgical site infection prophylaxis with iv vancomycin. balanced general anesthesia. supine decubitus position with shoulder supporting to allow a forced trendelemburg. degree optical device with trocars disposition: one mm umbilical trocar and mm trocar in both flanks, same distance and height to umbilical trocar. peritoneal opening and flap creation with monopolar energy, blunt maneuvers and pneumoperitoneum dissection. anatomical landmarks identification(cooper's ligament, epigastric and iliac vessels, hernia defect and spermatic cord elements). reduction of hernia sac content(pseudosac in this case, direct hernia) and complete peritoneal dissection to achive a correct mesh placing. mesh is folded in parts(one inferior part, two superior parts) in vertical axis outside the abdomen to facilitate the posterior intraabdominal maneuvers. introduction: into abdominal cavity with grasping forceps and correct unfolding mesh assesment: medially(pubic bone), caudal(cooper's ligament) cranial(more than cm of hernia defect/ deep inguinal ring) and lateral(anterior superior iliac spine). finally, we use a bipolar forceps to close de peritoneal defect. in order to facilitate this step, its necessary to decrease pneumoperitoneum pressure and to use the grasping forceps to bring together both peritoneal flap edges prior to bipolar energy sealing. results: min. surgical procedure. h hospital discharge, no complications. routine outpatient follow up(week, month, months and month later) with an epididymitis episode months after surgery(treated with oral ciprofloxacin). conclusions:-this procedure is an easy implementation technique once the intraabdominal mesh unfolding procedure control is reached.-the use of a self-fixating mesh avoid the use of tackers and its potential disadvantages(e.g. increasing postoperative pain).-bipolar peritoneal sealing offers a quick, easy, cheap and safe peritoneal closure, avoiding the contact of the mesh with the viscera in the same manner. results: we performed procedures within patients. the average age was years. twenty six percent of hernias were bilateral, , % were inguinoscrotal and % in the right side. the median asa score was . the conversion rate was , %. the average duration of the procedure was , min min. overall morbidity was %. there were seromas ( , %) . on -year follow-up, one recurrence ( , %) was found and chronic postoperative pain in one case . we had no mortality. in the univariate analysis, male sex, inguinoscrotal hernias, hernias classified as nyhus a were significantly associated with overall postoperative morbidity. a chronic obstructive pulmonary disease was the only variable significantly associated with the occurrence of medical complications. conclusion: given these results, the tapp technique is a good alternative in the treatment of groin hernias. however, enhancing this approach is essential to reduce the operating time and the postoperative outcomes. introduction: studies have emphasized the impact of a strong safety culture on patient outcomes. consequently, many interventions focus on improving the safety culture, of which teamwork and safety climate are important ingredients. it is known that differences in culture and safety attitudes may also impact teamwork. implementations of safety interventions, such as a ' black box', are dependent upon these differences. the aim of this study was to assess the safety culture at the operating theatre complex, along with the theatre staff's attitude towards a specific quality improvement intervention, a black box in the operating room as a tool for structured team debriefing. methods: the validated dutch version of the hospital survey on patient safety culture was administered to all healthcare professionals working in the operating room complex at one academic medical centre. this survey was supplemented with questions regarding the use of a 'black box', a medical data recorder in the operating room, to measure the staff's attitude towards this quality improvement tool and its potential contribution to patient safety. aims: the aim of the study was to compare two methods of treatment of dunbar syndrome: thelaparoscopic release of median arcuate ligament alone and the hybrid method consisting ofsurgery and percutaneous stent implantation to celiac trunk. methods: we performed laparoscopic release of ct in the department of general, mini-mallyinvasive and elderly surgery in olsztyn in - . all of patients suffered from severepain of abdominal cavity before the surgery. three patients underwent doppler percutaneousangioplasty of the ct with stent implantation one month after the laparoscopy. results: all patients reported relief of symptoms in the first days after the operation. in two cases fromboth groups, there were a complete remission of the symptoms. in one case respectively,there was an improvement. there were no postoperative complications. the results of both methods do not show the differences therefore the surgery alone seems tobe a safe and feasible procedure. it increases the comfort of the patient and brings theopportunity for normal functioning. the method of wedge resection of lungs in patients with limited forms of chemo-resistant pulmonary tuberculosis is developed. in order to evaluate the efficacy, patients underwent surgery (the main group). for comparison, the data on similar operations in patients, made according to the traditional method (with the help of a cardboard weaving machine yo- ) were selected. compared the duration of the stage of resection itself, the frequency of need for additional hemostasis of the parenchyma sutures, the degree of deformation of the pulmonary tissue in the seam area, the frequency of postoperative complications and reoperations, the duration of postoperative inpatient treatment. the developed method, in comparison with the traditional one, has the following advantages: simultaneously leak proofness and hemostasis with minimal electrothermal damage to tissues are provided and there is no need for additional hemostasis, there are no negative effects of manual stitching of parenchyma of lung with abandonment of foreign material, a significant reduction in the duration of wedge resection of the lung from . to . min, a decrease in the number of postoperative pulmonary-pleural complications is achieved by . % and caused by them reoperations-by . %, shortening the duration of postoperative inpatient period of treatment from . to . days. introduction/aims: laparoscopy is a diagnostic and therapeutic resource that is largely used in elective gastrointestinal surgery due to its well-known advantages over the classic open approach. nevertheless, there is still some discussion about its application in emergency surgery. our aim is to analize the use of the laparoscopic approach by the members of the surgical emergency unit from our medical center. methods: a descriptive research based on the data of patients who required emergency surgery, that was performed by the members of the surgical emergency unit of a spanish hospital between november and may , was conducted. these data were analyzed according the pathology that motivated the surgical procedure and the chosen form of surgical approach (open versus laparoscopic). results: out of the patients in whom emergency surgery was performed, suffered from a pathology that actually allowed the laparoscopic treatment. laparoscopy was used in . % of these patients. according to pathology, the most common were acute appendicitis and cholecystitis, in which the laparoscopic approach was used, respectively, in % and % of the cases. regarding other less frequent pathologies, such as gastroduodenal perforation, bowel obstruction, diverticulitis and pancreatitis, laparoscopy had a less significant role. according to the year, a general tendency to increase the use of the laparoscopic approach was found, most notably in the cases of acute appendicitis and cholecystitis (with rates above % in ). conclusions: despite our positive results in the terms of the implementation of the laparoscopic approach in emergency surgery, there is still room for improvement, especially in regards of the less common pathologies. furtheremore, additional studies are needed in order to identify the factors that have had an effect, in favour or detriment, in the development of emergency laparoscopy in our center. aims: laparoscopic surgery, which produces small scars, has become widespread. when performing surgery through small laparoscopic incisions, a surgeon manipulates tools inserted into the abdomen through ports. for minimally invasive accurate procedure, the port as the pivot point should be stabilized on the abdominal wall. however, these laparoscopic incisions are loaded while manipulation because it is difficult for the port to be fixed on. thus, it is necessary for the patient friendly manipulation to be fixed the port mechanically. we developed a new pivot restraint device (prd) attached to a trocar for guiding the tool. the purpose of this study is to evaluate both of reducing the operating time and the load of the port with the prd experimentally. methods: the prd uses gimbal mechanism for two rotating axes and a linear guide mechanism for the insertion axis though into the forceps. in the experiment, the left hand forceps with or without the prd and the right hand forceps without the prd were set on the training box. the box had a measuring system created with a pressure sensitive sensor for the continuous force (resolution . n, fps) applied to abdominal wall fulcrum. the experiment task was performed as following three steps. ( ) the surgeon lifted the g weight for s at the initial position using the right hand forceps. ( ) the weight was transferred from the right hand forceps to the left hand forceps, and held for s. ( ) the weight was moved to the predetermined position, held for s, and returned to the initial position. the surgeons were five endoscopic specialists and five non-specialists. the operating time and the time ratio exceeded n for the left hand forceps were measured. two grouped datasets with or without the prd were compared using two-sided t-test. results: the prd was associated with both of reducing the operating time ( . s vs. . s; p \ . ), and the load of the port ( . % vs. . %; p \ . ) at the statistical analysis. conclusion: the prd could be used for reducing the operating time and the load of the port in minimally invasive accurate procedure. background: pathophysiological changes during laparoscopic surgery and positive pressure pneumoperitoneum (pp) may include (beside cardiovascular changes) elevated intra-thoracic as well as intracranial pressures. however, the possibility of physiological and functional cerebral impairment under pp is still debated. aim: to study the effects of pp on brain activity during different modes of anesthesia and ventilation during laparoscopic cholecystectomy (lc). patients and methods: thirty patients undergoing elective lc were divided to those who were ventilated by intermittent positive pressure ventilation (ippv, pt.) and by high frequency jet ventilation (hfjv, pt.). in those under hfjv we used total intravenous anesthesia (tiva). in those under ippv we either used inhalational anesthesia or tiva. intra-ocular pressures were detected in both eyes, trans-cranial doppler was used to measure the changes in flow of the middle cerebral artery, and cerebral oxygenation (o saturation) was measured too. each parameter was detected during anesthesia before surgery, several times during surgery under pp and after co evacuation. a novel computerized signal analysis by a continuous recording through a single electrode was done to explore cerebral cognitive activity during surgery. results: all surgeries went uneventful and without complications, pp was set to mmhg, and each patient was positioned in a degree anti-trendelenburg posture. cerebral perfusion and oxygenation were not changed significantly during pp. intra-ocular pressures decreased during anesthesia and increased during pp, but to a lesser extent under tiva. however, pressures during pp did not exceed pre-surgical values. we did not observe changes in cognitive brain activity during pp, although enhanced cerebral activity was seen under hfjv. conclusions: increased intra-abdominal pressure during laparoscopic surgery was not accompanied by decreased cerebral functions, maybe due to cerebral circulatory auto-regulation. changes in cerebral cognitive functions under hfjv might be explained either by the different cerebral effects of tiva in comparison to inhalational anesthesia, or due to dissimilar hemodynamic changes during hfjv. aims: gallstone ileus (gi) is a rare complication of cholelithiasis and accounts for . - % of small bowel obstructions. intermittent and non-specific presentation often results in late diagnosis. the triad of rigler is pathognomonic (pneumobilia, small bowel obstruction and ectopic gallstones), so an image test is usually mandatory in order to assure the diagnose. our aim is to expose our experience regarding this topic to show that a minimally invasive approach is feasible in selected cases. methods: since january we treated cases of gi, of whom ( %) underwent laparoscopic surgery. in all cases a ct was made to reach diagnosis. enterolithotomy alone is our preferred procedure for the resolution of this pathology. here we present a descriptive analysis of our data in those cases where a laparoscopic treatment was attempted. epidemiological variables, surgical technique, postoperative complications, days until hospital discharge, recurrence, etc. has been collected. results: % of patients were female( ) and % male ( ). mean age was . size of gallstones varied from to mm and ct located them all in the ileum. two conversions to open surgery were made ( %), in one case because the gallstone could not be found and in the other case due to the need of an intestinal resection. in two cases ( %) la aparoscopic-assisted surgery was performed using a pfannestiel incision for the gallstone extraction and enterorrhaphy. only one case was total laparoscopic approach ( %). two cases needed an intestinal resection and anastomosis, one of them was complicated with a leak that needed reintervention. there were two cases of recurrence during the follow-up time. hospital stay varied from to days, mean of days. conclusion: the widespread use of ct facilitates early diagnosis with high sensitivity detecting rigler's triad. a totally laparoscopic procedure might be ideal for patients specially with solitary stones even though a laparoscopic-assisted approach is an easier technique for surgeons with less experience in laparoscopic surgery. although experience in minimally invasive surgical treatment of gi is still developing, it may be recommended in selected cases and experienced hands. introduction: most of surgical interventions in hospitals in the world, where laparoscope is used, it is common that the vision inside the human body is constantly interrupted by fogging in laparoscope tip. the laparoscope fogging is caused by the difference of temperatures between the optic tip and the abdominal cavity. material and method: we replaces the traditional laparoscope for the ehs (endoscope heater system) with resistance between the internal and external tube that maintains the temperature of laparoscope at ( - °celsius) without modifying the external architecture of traditional laparoscope. results: ehs does not generates any waste like other anti-fog systems, like liquids, plastics covers or electric heater. reduces intervention time, can keep same instruments or accessories for the intervention. all of the above means a saving of resources with have a positive environmental impact. conclusions: the discomfort transmitted by surgeons about the fogging in laparoscopy tip make success of the product and it will replace the current laparoscope which is fogged. aim: synchronous locally-advanced low rectal cancer and prostate adenocarcinoma represent a rare condition and a challenging situation for colorectal surgeons and urologists. the simultaneous resection of both adenocarcinomas after long-course chemoradiation therapy combines two major surgical procedures associated with a potentially increased postoperative morbidity. in the other hand, simultaneous resections minimize the risk of difficult dissections, which are expected if the two procedures are scheduled sequentially. in the past decade, robotic-assisted minimally-invasive surgical techniques have been increasingly used to treat both rectal and prostatic malignancies. especially in case of prostatic malignancy, the robotic approach is considered the treatment of choice because it is associated with significantly lower blood loss and transfusion rate, and much greater functional outcomes compared to laparoscopy. methods: we present the case of a -year-old male patient (bmi: . ) diagnosed with a histologically proven locally-advanced rectal adenocarcinoma (ct an ) located at cm from the anal verge and concurrent histologically proven prostatic adenocarcinoma [gleason score of ( ? )] located in the postero-basal right lobe. the preoperative total-body computed tomography (ct) scan showed no evidence of metastatic disease. after discussion in a multidisciplinary meeting, the patient received a long-course neoadjuvant chemoradiation therapy (ncrt). at the restaging positron emission tomography / magnetic resonance imaging (pet-mri), the rectal lesion was classified as ymrt n . preoperatively, the surgical difficulty was assessed as high, based on the calculation of the eumarcs score (equal to / ). moreover, due to the high-risk status of the prostate cancer (gleason ), it was decided not to preserve the neuro-vescular bundles during the radical prostatectomy. results: the patient was operated on after weeks from completion of ncrt by using the da vinci robot system si with a single docking approach, as previously described, in order to address both cancers. conclusions: this video shows the main surgical steps of the simultaneous robotic resection of the low rectal adenocarcinoma first, of the prostatic carcinoma then, and the mechanical colo-anal anastomosis followed by drain positioning and ileostomy. this video demonstrates the perioperative safety and feasibility of the minimally invasive robotic approach in case of extended and challenging oncologic resections. general surgery, rambam medical center, haifa, israel year old, male patient presented with melena, without abdominal pain, nausea or vomiting. patient underwent colonoscopy and tumor was found in ascending colon (near the hepatic flexure). biopsy from the tumor has showed moderately differentiated adenocarcinoma. his blood laboratory examinations were within normal limits except of hgb level- . . cea and cea - were normal. abdominal computed tomography was normal . patient underwent da vinci robot-assisted right hemicolectomy with extracorporeal anastomosis. total operating time was min. three days after operation patient started regular diet and was discharged home on day four. final pathology result confirmed diagnosis of moderately differentiated adenocarcinoma. introduction: one of the goals of colorectal surgery is to decrease the number of leaks once an anastomosis has been performed. this life-threating entity after elective surgery has been related to the clinical history of the patients, the location of the tumor and to technical reasons, specially due to tension in the anastomosis or to lack of vascularization. tension could be identified during surgery, while vascular supply is evaluated by the surgeons based on a subjective analysis of the color of the colon/ileum. fluorescence tries to make these subjective parameter more objective in order to avoid an anastomosis with lack of vascularization, decreasing the numbers of leaks related to this factor. patients and method: the study presents a quasi-experimental analysis made from january to october in two hundred and eighty-five patients who underwent elective colerectal surgery, performing either a colo-rectal, ileo-rectal or intracorporeal ileo-colic anastomosis. vascular supply was eveluated using indocianyne green (icg) in one hundred and forty-five patients, while one hundred and forty subjects were operated in a previous period without using this technology, being considered the control group. the number of time that the attitude changed and the number of leaks were collected. results: out of the cases performed, were right colectomies (rc), left colectomies (lc) and rectal excision (re). in % the transection line was changed ( , % in rc, , % in lc and , % in rr) . in comparison with the control group, the icg group had a significantly less indicence of anastomotic leak compared to the control group ( , % vs. , %, p = , ), lower rate of terminal stoma after reoperation ( , % vs. , %, p = , ), a shorter length of hospital stay ( days vs. days, p = , respectively), and a low morbidity and mortality. conclusions: the rate of leaks after colorectal surgery decrease using icg to detect the proper transsection line before to perform the anastomosis in comparison with control group. these findings might influence in the final results although it is necessary in the future to find a system that provides greater objectivity by quantifying icg. aims: anastomotic leaks continue being one of the most important complications when a colorectal surgery is performed. this complication is usually related to the level and type of resection, the patient clinical history and surgical technique, where tension and vascular supply are the most important. indocyanine green (icg) fluorescence angiography seems to be helpful in order to evaluate the vascularization at the resection margins. methods: we have collected data on colorectal procedures that were performed by the same surgeon using icg fluorescence angiography to evaluate vascular supply to the anastomosis. in order to asses in which of the different type of colorectal procedure has more value to be used, we analyzed the type of surgical procedure, the percentage change in the resection margin and the number of anastomotic leaks (al). results: all of the cases were performed by laparoscopic approach: left colonic resection (lc), right colonic resection (rc), splenic flexure partial resection (sf), low anterior resection with partial mesorectal escision (lar), ultra low anterior resection with total mesorectal escision (ular) and total colectomy (tc). there was a change of transection line (ctl) in lc ( , %), rc ( %), sf ( , %) and ( , %) in rectal anastomosis (lar, ular and tc). as far as al we found: lc ( , %), rc ( %) and , % in rectal procedures. lc, sf and rectal procedure showed more ctl and less al, while rc showed less ctl and more al. conclusion: icg fluorescence angiography as an additional tool to try to reduce the anastomtic leak rate seems to have more value in the procedures that involve the left colon and the rectum, since that is where we have observed the greatest number of ctl, this could be explained by the riolan's arcade and the variability of the vascular anatomy. however, it seems that this is a line of research should continue developing with longer and larger studies, so in that way we can have more significant results. retrorectal tumors ara rare and often found incidentally. the majority of retrorectal tumours are benign, but they have potential for malignant transformation and therefore should be resected when found. a case of a -year-old female patient with a retrorectal tumor is showed. the tumor was found incidentally on ct scan of the abdomen for evaluation of non specific right side abdominal pain. a mri was also performed and imaging was informed as a probably congenital retrorectal tumor (tailgut cyst) there was no evidence of involvement or invasion of other structures the tumor was palpable at rectal examination. a transanal minimally invasive surgery (tamis) approach was proposed. preoperative preparation was done with a full mechanical and oral antibiotic bowel preparation. preoperative parenteral antibiotics werw administred. under general anesthesia, lithotomy position. the contour of the tumor is not visible due to the small size. palpation of tumor and placement of clips to lolocate was done. placement of gel point path and rectal insufflation. a longitudinal incision was made to the posterior left side of rectal wall. the insufflation of the perirectal extraperitoneal space allowe for excellent exposure of the tumor. the tumor was disected with ligasure. then the tumor was extracted transanally.the proctotomy was closed in a single layer with reabsorbible monofilament continuous suture (pds). no complications after the procedure. the patient was discharged at days. discusion: traditionally, the retrorectal tumors have been resected using a posterior parasacrococcygeal approach, an abdominal approach or a combined abdominal and posterior approach. with the advent of minimally invasive surgery, laparoscopyc approach has been described too. however, tamis approach is feasible, with low pain, morbidity, fester recovery and excellent cosmetic (no scare) results. it can be accomplished using standard laparoscopic equipment, with transanal access. we think that perhaps it could be the gold standar approach for this tumors. aimes: robotic-assisted laparoscopic surgery (rals) is a promising advanced technology that can overcome the inherent limitations of conventional laparoscopic surgery (cls). its advantage includes free-moving multijoint forceps, a motion scaling function, high-quality three-dimensional imaging, and stable camera work by an operator. this study aimed to clarify the short-term outcomes of rals for rectal tumors. methods: this study group comprised patients who underwent rals for rectal tumors (cancer in patients and gastrointestinal stromal tumor in patient), excluding ones with distant metastasis from november through december . the clinicopathological findings and short-term outcomes in rectal tumors were analyzed. results: the median operative time was min ( - ). the median console time was min with a median blood loss was ml ( - ). conversion rate was . % ( / ). the median postoperative hospital stay was days ( - ). patients ( . %) had postoperative complications. patients ( . %) had lymph nodes metastases. the mean harvested lymph node was . . the r resection rate was % ( / ). conclusions: these results suggest that rals for rectal tumors is safe and feasible, and the perioperative outcomes are acceptable. introduction: anastomotic healing defects are a feared complication which might have a fatal impact on the patient. fundamental conditions for proper anastomotic healing include sufficient blood supply. fluorescent angiography using indocyanine green in the spectrum of near infrared light facilitates the monitoring of tissue perfusion during a surgery. aim: a presentation of the results of our non-randomized study in which we assessed prospectively obtained data from a perioperative assessment of anastomosis perfusion by fluorescent angiography using indocyanine green during robotic rectal cancer surgery. method: thirty patients with rectal cancer, who underwent a robotic resection with primary anastomosis, were consecutively included in the study between april , and june , . the study included patients facing a least invasive surgery with a guaranteed payment by a health insurance company. during the surgery, we monitored and assessed the quality of the perfusion of the resection line of the sigmoid colon and subsequent anastomosis by means of fluorescent angiography using indocyanine green in the spectrum of near infrared light. the data were obtained prospectively and subsequently analyzed. results: between april , and june , , we consecutively included rectal cancer patients in the project: men and women. monitoring of the perfusion of the resection line and anastomosis was successful in all cases and perfusion quality was satisfactory across the sample. perfusion insufficiency requiring a change in the resection line level or anastomosis adjustments was not detected with any patient. in two cases ( . %) of tme, we gave up the planned protective ileostomy owing to quality perfusion of the anastomosis. one patient ( . %) suffered from defective anastomosis healing without clinical symptomatology (type a). we found no technical complications related to fluorescent angiography or undesirable effects due to the application of indocyanine green. conclusion: even though we did not register insufficient perfusion in our sample and hence we did not have to change the resection line level or adjust the anastomosis, we may state that fluorescent angiography performed by an experienced colorectal surgeon may potentially reduce the frequency of complications linked to defective anastomosis healing.supported by mo aims: the aim of our study is to demonstrate whether robotic surgery has any influence on the reduction of complications in the aged population undergoing rectal cancer. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer. we divided our population in groups: under year old, between and year old and above year old. we recorded complications in each group intra and post procedure. qualitative variables were expressed in terms of absolute frequencies and percentages and mean values and standard deviation were used to express quantitative variables. the analysis of data was applying fisher's exact test or chi-squared test for qualitative variables and variance analysis or student'-t test for quantitative variables. statistically significant values of p \ . underwent multivariate logistic regression analysis. results: the present study included patients ( males).seventy seven patients were under year old, patients were between and year old and patients were above year old. the analysis showed conversion rates of . %, . %, . %, and complication rate of . %, . %, and . % in each group. univariate analysis showed no differences between the three groups. nevertheless, there were statistical differences from bmi, asa and neoadjuvant therapy. in multivariant analysis only neoadjuvant therapy was significant. conclusions: robotic approach do not decrease complications in elderly population. introduction: it has been described the advantages of total transanal mesorectal excision (tatme), with better visualization and access to the lower rectum. we use this access whith the gel point path device, to repair a rectovaginal fistula with stenosis of low rectal anastomosis in two patients, that would be difficult by conventional abdominal approach method: we show our surgical technique for repair a rectovaginal fistula with stenosis of low rectal anastomosis in two female patients operated due to rectal neoplasia. one of the patients underwent prior chemo-radiotheratpy. rectoscopy and image test was performed at the patients prior the intervention. no recurrence signs are recorded at mri.we describe the operation technique: a new anterior rectal resection was performed with a combined transanal (gel point path) and abdominal minimally invasive approach. redo anastomosis whith eea stappler was performed, vaginal repair and epiploplasty. the intervention was especially laborious due to the fibrous tissue. pathology: fistulous path without tumor infiltration in the two patients. at two months, a opaque enema show permeability and absence of leaks in the two patients. the ileostomy was closed at three months. discusion: we believe that transanal access through the gel point path can be a good option for rectovaginal fistula and stenosis of low rectal anastomosis, allowing a better visualization and acces, and making more easy a very difficult intervention. introduction: tamis or transanal minimally invasive surgery for polyp resection has increased fame for several situations in which adenomas with or without dysplasia cannot be removed with conventional colonoscopy. in this video we show the step by step technique performed with the da vinci xi system. material and methods: in this video we show the setting and the location of the patient-side cart and the arms to perform the resection of polyps in different patients and how to develope the procedure. results: after placing the patient-side cart the arms are connected to ports and the camera, double fenestrated grasper and scissors are connected to the arms through a transanal gel-port device. a line is described around the polyp with monopolar energy to determine the place of the dissection. the scissor is exchanged by a robotic harmonic wrist instrument and the complete dissection is performed. the wound is closed using a robotic needle holder and a suture. results: transanal robotic surgery could be safely performed after a standardized technique is stablished. aims: robotic rectal cancer surgery has demonstrated to obtain at least the same results than laparoscopic surgery. however, robotic surgery is associated with high rates of costs, specially when conversion to opened surgery occurs. the goal of this study is to create a predictor nomogram of conversions for robotic rectal cancer surgery. methods: we performed a retrospective analysis of a prospective database of patients who underwent robotic surgery for rectal cancer from october to november . we performed a bivariant analysis and detected the variables which were related with the conversion: body mass index (bmi) and the t. we divided the patients of the population in two groups depends on obesity (bmi of kg/m ) and on t (t - /t - ). we registered conversions in each group calculating the pretest risk. we performed likelihood index (lr ?/-) for under and above kg/m of bmi, adding in a second step the lr of t; obtaining the prediction index for four groups by using a standardize nomogram. results: the present study included patients ( males). were under bmi of kg/m and above. regarding t, were with a tumor of t - and with t - . the analysis showed a conversion rate of the statistical sample of %. univariant analysis showed significative differences in the bmi (p = . ) and t (p = . ). a nomogram was performed; as regards the bmi, the positive likelihood index in the group of bmi [ a prediction index of conversion of % (lr ? , ) and in bmi \ the prediction index of conversion is % (lr- , ). adding the t group data, for bmi [ and t - the conversion prediction rate is . % (lr- , ); for bmi [ and t - the conversion prediction is % (lr ? , ). bmi \ and t - the conversion prediction is % (lr- , ); imc \ and t - , the conversion prediction is %. conclusion: a standardize nomogram with the variable bmi and t facilitates the selection of patients for robotic surgery in rectal cancer avoiding conversion to open surgery. background: d-laparoscopy is proven to improve performance in dry laboratory settings, especially for novice surgeons due to better depth perception. however, the benefits for experienced laparoscopic surgeons are still discussed. aim: the aim of this study is to compare the results of right hemicolectomy (rc) using a conventional ( d hd) laparoscopic system with rc performed using a d laparoscopic system in terms of duration, complications and results. material and methods: from all laparoscopic right hemicolectomies performed in our clinic we selected all procedures performed by the same team of consultant surgeons using the same technique and divided them in groups. the study group comprised of all patients operated using our d einstein vision . system; all other patients which were operated using our standard wolf hd laparoscopy system comprised the control group. all patients were retrospectively analyzed in terms of patients characteristic, or time, duration of operation, intra-and postoperative complications, length of hospitalization, pain score, necessity of analgesics and number of lymph nodes retrived. risk factors for complications (bmi, smoker, diabetes, copd, bph) were also registered. results: there were patients included in the study group, while the control group comprised of patients. mean operation time in the study group was . min in the study group, while mean or time was . min. mean operation time in the control group was . min, while mean or time . min. one reintervention was noted in the control group and two in the the study group; no conversion to open surgery was noted. there were no significant differences regarding patient characteristics, pain score, wound complications, hernia rate, length of hospitalization or number of lymph nodes removed. conclusions: there were no significant differences regarding the outcome of rc using d laparoscopy; total or time was significantly higher in the study group due to the time needed to set up the d-laparoscopy unit. this is biased by the fact that the d system needs to be set up manually while the conventional hd system is integrated in the or. also, there was no significant difference in complication rate. background/purpose: robotic approach can be a treatment option for patients with pelvic recurrence after primary resection for rectal cancer. however, data regarding patient selection, complication rates, and oncologic outcomes are rarely reported. we aimed to present initial experience and to evaluate feasibility, safety, and oncologic outcomes of robotic salvage surgery for recurrent rectal cancer. methods: ten patients who underwent robotic salvage surgery for local recurrence at the anastomotic site, lateral pelvic side-wall, or lateral pelvic lymph nodes (lpns) were retrospectively evaluated from a prospectively maintained database. results: two patients underwent pelvic mass excision with en bloc resection of anastomosis and redo-anastomosis, and eight patients underwent lateral pelvic lymph node dissection (lpnd) for lpn metastasis; one of these eight patient underwent additional en bloc resection of anastomosis. all patients achieved r resection. the median operation time was min and the median estimated blood loss was ml. there were no conversions. as for intraoperative complications, one patient experienced ureter injury during lpnd because the metastatic lpn was closely abutting to the ureter. the median hospital stay was days. in six patients who underwent lpnd, the median number of harvested lymph nodes was (range - ) and the median number of metastatic lymph nodes was (range - ). with median follow-up months, one patient developed lung and pelvic recurrence at months after salvage operation and seven patients remained in disease-free state at the last follow-up. conclusion: initial experience of robotic salvage surgery for pelvic recurrence in rectal cancer indicated that it is safe and feasible. therefore, the robotic approach can be considered as a treatment option for the treatment of local recurrence in selected patients. introduction: there is uncertainty regarding the effects of simulated patient death. several reports showed increased cognitive load and poorer learning outcomes, and others increased performance without causing stress to learners. we have not found any report studying the impact of animal death in the simulation lab. methods: this was an observational cohort study to assess the emotional and cognitive load of surgeons who experienced animal death in the simulation lab. seventy-four faculty and residents from different surgical specialties training minimally invasive surgery participated in the study. one cohort consisted of surgeons whose animal died during surgery, and the other by those whose animal survived. emotions were assessed using the scale for mood assessment and cognitive load with nasa task load index. results: twenty percent of participants experienced mortality while training anti-reflux surgery ( cases) and other procedures ( cases). causes of death included intraoperative pneumothorax (n = ), hemorrhage (n = ), and cardiac dysrhythmias (n = ). participants exposed to animal death had higher levels of sadness and anxiety, and lower levels of happiness (p [ . ). cognitive load was slightly higher in the exposed cohort (p [ . ). conclusions: these findings suggest that mortality in the animal lab do not have a significant effect on cognitive workload and emotions of surgeons training complex laparoscopic procedures. introduction: the visuospatial profiles of expert laparoscopic surgeons remain unaccounted in the current literature for as the influence of visuospatial ability on laparoscopic learning has mainly been investigated in medical students or novice surgeons and using simulators as means of performance measurement. such knowledge is critical, as without understanding how clinical experience may impact visuospatial processes in surgeons, we hinder our efforts to utilize the available knowledge to support surgical education for the future. this study is aiming to explore the development and influence of visuospatial processes on intraoperative laparoscopic learning. method: the study reports the interim baseline results from the ongoing longitudinal study throughout a -year period of training on laparoscopic surgery. data from surgeons including residents undergoing training were captured and compared to specialists who are working in departments of general and visceral surgery at two large hospitals. the mean experience of the surgical residents was years. the mean laparoscopic experience among the senior surgeons is years, with each surgeon performing an average of laparoscopic procedures per week. visuospatial ability was tested using mental rotation test (mrt), guay visualization of views tests (gvvt), spatial perspective taking and spatial orientation test (ptost) and pictorial surface orientation (picsor). spearman correlation coefficient was used in this study with a p-value of significance at \ . . results: senior surgeons have an overall good visuospatial profile, in the sense that they performed close to optimum on all measurement scales. the spearman rho revealed a significant correlation between scores on gvvt and picsor (r = . , p = . ) and between ptost and picsor (r = -. , p = . ). a significant correlation between years of laparoscopic experience and ptost score was also observed (r = . , p = . ). when comparing residents and senior surgeons, no significant difference on the mrt was observed (m = . , sd = . ), nor between baseline scores of senior surgeons and resident surgeons on all tests. conclusion: the results of this study carry important clinical and theoretical implications, as the results hint towards the idea that intraoperative laparoscopic experience lends little to no influence over the development of visuospatial ability. learning models and laparoscopic technical skills, how to adapt each case to improve objectives: according to da. kolb learning is the result of how people perceive and then process what they have perceived. the aim of this study is to identify the personal characteristics of learning in of the participants in a course of laparoscopic technical skills according to the styles described by kolb. methods: between june and november , participants performed a h course distributed over five consecutive days performing laparoscopic manual intestinal anastomosis in endotrainer. they all filled in kolb's learning style test adapted to spanish. the anastomoses were performed in 'ex-vivo' swine intestines. in each anastomosis we evaluated the quality at the end and execution time. the test and quality variables were analyzed through statistical studies. results: in our study, % of the participants were women and % wew men. %were staff surgeons and % were resident. the median age among residents was years and among the staff years. the most frequent learning model in the sample studied was converging ( %). the predominant model among women was assimilating ( %), which, however, represented only % in men. in men, converging model was predominant ( %). among the staff, the most frequent model was diverging ( %). adaptation style prevailed among residents ( %), being rare among the staff ( %). the mean time of the anastomosis was min for both the adapter model and the assimilator, min, for the convergent and divergent models. the quality of the anastomosis performed by each participant was % for the adapter model, % for the assimilator model, % for the convergent model and % for the divergent model. the predominant style in our study was convergent. among women, the most frequent model was assimilator wheras in men it was the least frequent. in the residents, the most frequent model was adapter however, it was very rare in adjuncts. among residents we do not find divergent styles. the highest quality of the anastomosis was achieved by those who worked with an assimilating style. knowing previously the training style we can individualize the teaching methodology in order to improve competences. aims: assess whether laparoscopic appendicectomies (la) are a superior option to open appendicectomies (oa). specifically, comparing the time taken, complication rates and whether it is more appropriate to perform an la overnight, as opposed to oa. finally, to find out how a range of outcomes differs between different grades of surgeon. methods: an information request was sent to the clinical coding department to derive patient identification numbers for all appendicectomies over a ten-month period ( total surgeries). these numbers were then inputted into the hospital information system where the electronic operation note is present, and specific outcomes were derived and analysed. results: % of operations were oa and % were la. mean la times for consultants, sas and spr were . , and min respectively and oa , and min respectively. their respective conversion rates were %, % and %. oa had a complication rate of . %, la was . %. conclusion: oa are performed more than la. spr doctors had the slowest completion times for la but the lowest conversion rates. sas doctors had the fastest completion times for la and oa but higher conversion. la takes longer than oa but has lower complication rates; key factors when performing at night. key statement: laparoscopic appendicectomies require more surgeon-hours and have the potential to be converted to open, however the rates of complications and serious complications are significantly lower. background: paper based resources have been the standard sources for information for centuries. however, more and more people (patients and staff alike) are looking online for information. while the internet often provides excellent resources, there is often conflicting and confusing material of doubtful veracity. trainee staff and patients/carers should be able to access reliable resources whenever and wherever they are. the aim of this project was to create a high-quality resource fulfilling these needs. aim: we present a video demonstrating our integrated colorectal education website ( http://www.colorectaleducation.com/). our approach: high quality health care provision requires highly trained staff as well as wellinformed patients. information resources for these two groups are usually accessible from different repositories. our integrated website provides a common platform for all those involved in colorectal surgery, to use, learn and reflect on. users are directed to separate sections for patients and colorectal professionals. multiple disclaimers prevent patients accidentally stumbling across clinical/ operative information, whilst providing access to those who wish to do so. trainees struggle with balancing their educational needs with their service commitments. this website gives them the opportunity to view detailed operative training videos on the go. many of videos are chapter based allowing them to stop and re-start with ease. modules are also available for nurses providing them access to relevant educational material. the modular design of the website allows us to build upon it with more topics planned to be added over the next eighteen months. the resource also has detailed chapterised videos for patients due to undergo various colorectal procedures. all have been approved by a multi-professional panel including patients and are designed to provide information, offer support and to allay any anxiety. videos with the care pathway and previous patients' experiences are accessible on demand. conclusion: on demand information has now become the norm with the use of smart phones/ tablets. this website provides patients, surgical trainees and other healthcare professionals access to information and education in a clear and reliable format anywhere in the world. colorectal education, on demand and just a click away! objective: in the last decade the growing interest in robotic surgery is evident as shown by several published articles. the aim of the present study is to evaluate the main outcome of a single center experience and to describe the organizational system we have progressively established in our center in order to improve the development of robotic program in all surgical area. materials and methods: we report a case series of patients who underwent robot-assisted surgery at sanchinarro university hospital since the beginning of the program (october ) until november main patient demographic characteristics, type of surgery, peri and postoperative data and follow-up were evaluated. results: a total of robotic procedures were performed for a total of patients. the prevalence of malignant disease was %. a total of pancreatic surgery were performes; liver resections (mean operating time: min); gastrectomy (mean operating time min); esophagectomy (mean operating time: min); colorectal resections ( rectal resections, sigmoidectomy hemicolectomies right, left colectomy) (mean operating time: min); nissen procedures (mean operating time: min), esofagheous myomectomy for achalasia (operating time: min); adrenalectomy (mean operating time: min); three biliary surgery for benign desease, splenectomy. eight partial resection of the duodenum, one yeyunal resection, one mesenteric cyst resection and retroperitoneal tumor have been performed. conversion rate was %, total morbidity have been %. there has been no peri and postoperative mortality up to days after surgery. the average hospital stay and intensive care were respectively days (range - days) and . days (range - days). conclusions: the organizational model defined in our center is facilitating the constant and progressive development of the robotic program. a broad and flexible availability of the robotic system, a progressive increase of young surgeons joining this technology as well as the institutional and departmental economical effort are the points with which the robotic system may increase its development in a surgical department. aims: endoscopic surgery has been widespread in the field of general surgery. however, in japan, there is no standard program for endoscopic surgery training, and its competency has not been considered for the acquisition of board certified surgeon. the purpose of this survey was to investigate the current situation of endoscopic surgery training and autonomy of young surgeons for endoscopic surgery in japan. methods: the survey was planned to target general surgery members of the japan society for endoscopic surgery (jses) who was post graduate year or less. after approval by the ethics committee of jses, the request for the participating in survey was mailed to object members. questionnaire responses were available in print or online media. the contents of the questionnaire consisted of items, about the conditions of endoscopic surgical training, experienced case number, and the self-assessment of autonomy from to point by zwisch scale in specific procedures of endoscopic surgery. results: the total response rate was . % ( / ). sixty five answers were excluded due to inadequate response and answers were analyzed. of the questionnaire respondents, % were male and % were female. the ratio of board certified surgeon was %. although % of the teaching hospitals had simulators for basic training of endoscopic surgery and % of the respondents practiced basic skill of endoscopic surgery, only % teaching hospitals had specific training programs for endoscopic surgery. the surgeons who operated cases of laparoscopic appendectomy and inguinal hernia repair and cases of laparoscopic cholecystectomy, right hemicolectomy and sigmoidectomy, felt confident to perform each procedure independently. regarding with laparoscopic rectal resection and gastrectomy, even though the surgeons who had cases of experience, they didn't had confidence to perform those procedures independently. conclusions: this study is the first national survey to investigate the status of endoscopic surgery training in japan and the autonomy of young surgeons for endoscopic surgery. in order to develop a training system for not only basic skills but also advanced procedures of endoscopic surgery, cooperation of each teaching hospital, academic surgical society, medical specialty board is necessary. currently there is a debate about what is the most optimal work schedule for residents of general surgery, it is important to respect the free time of residents to avoid burnout, however it is also important have enough exposition to clinical cases that allow a satisfactory development in the clinical practice. this becomes even more important when we talk about the learning of surgical skills. this is where the laparoscopic simulation industry opens a large area of opportunity, for a reasonable price it is possible to practice basic laparoscopic skills without compromising patient safety. this is a pilot study that was carried out during the period from january to june , in a public hospital in monterrey, nl, mexico, the composition between the execution of the standardized exercises of the fls (fundamental laparoscopic surgery) in an endoscopic simulator was performed to residents of general surgery (from first to fifth year) hrs before being on call vs these same residents post call. a series of questions was asked to each resident in each measurement, so in this way they answered the same questions twice, then a comparison of the results of both questionnaires was made. the results of the exercises were assessed and rated by the same person using the criteria established in the fls for the scores of each exercise and for the final grade. an average age of years was obtained, measurements were taken of residents of which are male and female. on average, the residents before be on call performed the exercises with h of having slept while the post call performed the exercises with . h of having slept, the residents before be on call had on average . h without sleep while the post call had h without sleep. the average number of hours worked per week is h, measured by the time in and out of the hospital. in this study, conclusive results were obtained regarding the null relationship of sleep deprivation with the performance of laparoscopic skills in surgical residents. aim: 'precision cutting' is one of skills tasks of the fundamentals of laparoscopic surgery (fls) program, which is cutting a circle on a piece of gauze under laparoscope and assessed by completing time (maximum time limit: s). there is no definition of quality of the final product. the aim of this study is to develop an assessment tool of laparoscopic precision cutting and test its reliability. method: an assessment tool of laparoscopic precision cutting was developed with four items based on completion, degree of deformation, degree of being pulled, and overall appearance of the final product of laparoscopic precision cutting by experts' meetings. the scale of each item was points likert scale. a descriptive sheet with a legend and a text description for each scale (fig) was attached for assessors' reference. for our high school entry medical students, they gained hands-on experiences of laparoscopic skills first time by attending a -hour course at minimally invasive surgery training center, national taiwan university hospital (ntuh). we invited students to participate this study after this training. we collected participants' final products of ' precision cutting' station and assessed them by using this assessment tool. this study was proved by institutional review board, ntuh (irb no: rinb). results: students were enrolled between february to june . two non-medical assessors and a senior surgeon were invited to assess the products. the mean score and cronbach' s alpha value of each item were as followed: completion . ± . , . ; degree of deformation . ± . , . ; degree of being pulled . ± . , . ; and overall appearance . ± . , . . conclusions: in summary, we successfully developed an assessment tool for laparoscopic 'precision cutting' and showed its reliability. the tool could provide qualitative descriptions for objective feedbacks. validating this tool in a large scale is undergoing. purpose: to evaluate whether the participants who experienced this scenario could recall an interventional scenario for testing trainees' situational awareness and intra-operative decision making when they participated this training again. methods: we designed an iodm training course for junior surgical trainees and nurses by using live pigs since sep . in the first simulation, we created an interventional scenario and then provided an educational session. a researcher disconnected the ekg monitor on purpose for creating a scenario that the pig would lose vital signs when the team nearly finished a diagnostic laparoscopy. if the team did not aware the situation after . min, a researcher would remind the team (fig). we used a new developed assessment tool of iodm and an assessment tool for nontechnical skills for surgeons (notss) for self-evaluations and objective assessments. we also discussed with them about their reactions while encountering this interventional scenario. results: between sep to june , teams participated this training and experienced this interventional scenario. fourteen nd year surgical trainees have experienced it before. only one participant ( %) recalled it and made a quick decision while encountering this interventional scenario again. the results of iodm assessment and notss did not show statistical difference comparing their self-assessments in the first and second year. based on the analysis of the discussions, most of them remembered this this interventional scenario and reminded themselves to react it properly before the simulation. however, when they were the primary surgeon of diagnostic laparoscopy, they focused on performing this procedure and tutoring their junior trainee. they had no capacity in their brain to notice the change of vital signs. in addition, although they increased their situation awareness in clinical settings after the st time iodm training, they did not show this ability in the simulation. conclusions: recalling of an interventional scenario for testing situational awareness of surgical trainees was very poor ( / , %) among the nd year surgical trainees. qualitative analysis of discussions showed their brain capacities were occupied by performing new procedures and tutoring others. how to enhance trainees' situational awareness should be addressed. aims: a well-designed learning curve is essential to measure the progress of surgical abilities. learning curves are very important to test the skills of trainees. however, there are still no welldefined criteria for developing good learning curves. as a result, many authors use subjective evaluation criteria. the purpose of this review is to analyse this field of surgical education and to identify the key criteria for good learning curves. methods: learning curves were investigated in the field of laparoscopic and robotic minimally invasive surgery. surgery of appendectomy, cholecystectomy, cholectomy, inguinal hernia repair and gastrectomy were considered. the type of surgery, the year of publication, the design of the study, the surgeon's experience (resident, young or senior), the surgical technique, the number of patients involved in the study and the suggested learning curve by the different studies were taken into account. in the selection of articles, more importance was given to those based on the activity of young surgeons or residents. results: the literature analysis showed conflicting results. the different learning curves for the same surgery may be due to the different evaluation criteria considered. only a few studies investigate the learning curves of young surgeons and residents. conclusions: the data available in the literature on learning curves are contradictory. several factors need to be evaluated in order to create more accurate learning curves. we suggest the introduction of checklists with a score for each parameter to be examined, in order to develop more objective and standardized learning curves. aim: the uk training programme for transanal total mesorectal excision (tatme) has completed its first round of training. the study aim was to design a reporting platform that provided trainees with video-assisted feedback in a clear, concise and useful manner to support their training. methods: an established method of video analysis called observational clinical human reliability analysis (ochra) was used to assess the surgical performance of the trainees during their clinical tatme cases. a reporting form for the ochra results was designed identifying areas of difficulties in each procedure and providing error reduction mechanisms. this was piloted during the national training programme for tatme in the uk. results: the ochra reporting form underwent three modifications before the content and format was agreed upon. the final version is divided into three sections: a. case details, b. ochra findings, and c. suggested error-reducing mechanisms. for part b the tatme procedure was divided into four phases of the operation: . pursestring, . rectotomy, . tme dissection, and . connected phase when the abdominal and transanal teams work together synchronously. for each phase, ochra findings described the most frequently occurring technical inaccuracies/errors, number of consequential errors/adverse events and the most frequent and serious consequences encountered. suggested error-reducing mechanisms in part c were developed and established by an expert workshop and individual interviews with international surgeons experienced in tatme.trainee and mentor feedback stated that the reporting form had a clear format, easy to follow and understand. the error-reducing mechanisms were particularly useful and allowed the trainee to focus on improving specific technical aspects in their subsequent cases. conclusion: video analysis using ochra can provide a wealth of information on surgical performance, especially for trainees at the start of their learning curve. as an exploratory study, validation of the reporting platform is required; however, its potential to offer detailed, individualised feedback to enhance training is promising. laparoscopic pelvic surgery training program-using a new concept d-printed versatile pelvi-trainer r.c. elisei , f. graur , c. popa , e. mois , l. furcea , n. al hajjar general surgery, bistrita emergency county hospital, bistrita, romania; general surgery, regional institute of gastroenterology and hepathology ,,prof. o. fodor,,, cluj-napoca, romania pelvic laparoscopic surgery (rectal, urological, or gynecological laparoscopic surgery) is an advanced surgery which require advanced skills, not easy to acquire. there are a lot of training programs for advanced laparoscopic skills but many of them are not affordable for most of surgery residents in eastern europe, where the training programs are far behind from those in western europe. because of that those training programs need to be improved and optimized. in the european union we want equal and high skilled surgeons. this is why we designed a new concept of pelvi-trainer, a versatile one in order to offer the residents the possibility to achieve advanced laparoscopic skills like perfect coordination, precise movements, ability to cut and suture after a well defined route, all of them in the pelvis tight space. we d-printed this pelvitrainer which has multiple characteristics: cheap and easy to produce, easy to be used, versatile because offer the possibility to achieve the skills named above, and many others, but also to train on real ex vivo animal rectum (suine model). we also believe that with a proper training a medical student and a young surgery resident are able to achieve the same skills like experienced surgery residents or specialists. in order to demonstrate that we need a study to compare the time to perform or more exercises in this new concept pelvi-trainer by the medical students, young and experienced residents and surgery specialists. what we want to achieve with this training program project is to have more and more skilled surgeons in advanced laparoscopy and an equal laparoscopic surgery training all over the country, close to the level of training in the western europe. also we want this training program to make a standardization of the pelvic laparoscopic surgery training first in our country and then in other countries if possible. aims: the objective of this systematic review is to provide an evidence-based overview of the different components of laparoscopic training curricula, emphasizing the value of objective forcebased assessment and how this in implemented in modern laparoscopic training. methods: bibliographic databases of pubmed and embase were searched till april to identify studies reporting on evidence-based laparoscopic skills training. abstracts of retrieved studies were reviewed by two authors independently and those meeting the inclusion criteria were selected for full-text review. results: the search yielded a total of individual records. a total of articles were included. the articles were divided into nine different categories, which include 'metrics', 'benchmark criteria', 'measurement systems', 'timetable', 'training modalities', 'camera settings', 'training tasks', 'serious gaming', and 'competition'. a descriptive analysis of the data is provided. motion analysis parameters, such and path length and time are frequently validated and used for assessment. the results of validation studies on tissue manipulation parameters, such as maximum force and mean force show proved their discriminating power between different levels of proficiency. however, implementation of these metrics remain restrained. conclusions: numerous studies on laparoscopic skills training have been conducted over the years. nevertheless, no consensus is reached towards the use of objective assessment tools. although the value of validated metrics is described well, implementation of objective metrics is limited. we recommend to consider objective force-and motion metrics for feedback and assessment during laparoscopic skills training. surgery, regional institute of gastroenterology and hepatology, cluj-napoca, romania; anesthesiology, university of agricultural sciences and veterinary medicine, cluj-napoca, romania; radiology, regional institute of gastroenterology and hepatology, cluj-napoca, romania aims: the aim of the study was to create a new easy learning method of swine liver anatomy for residents in training. based on human liver surgical anatomy we put 'face to face' the similar structures and also the differences using ex vivo porcine models and ct reconstructions from live pigs. methods: having in mind the human liver anatomy, in the first stage we used data obtained from dissection of twelve porcine liver models to create an anatomical pattern, which summarized the most important surgical information. in the second stage, anatomical data obtained from ct scans of twelve living anesthetized pigs were analyzed. the ct reconstructions and volumetry data were added to the gross anatomy pattern to create a more complex learning module. results: the residents established the most frequent description of swine liver anatomy by putting together the information from ex vivo model dissection. the liver parenchyma is divided into four main anatomic lobes: left lateral, left medial, right medial and right lateral. all those lobes are connected only in the posterior part, which allows a very good separation between them by deep fissures. just as in humans, we found eight distinct segments with independent vascularization and biliary drainage. portal vein has a specific 's' shape; in most cases hepatic artery was found like a trifurcation and extrahepatic biliary tree has a very thin wall. in the right hemi-liver, the inferior vena cava passes through the liver parenchyma. most frequent, we found five hepatic veins which are running completely intraparenchymal. the imagistic data offered a very useful d reconstruction with anatomical positions of the vascular-biliary tree and liver segmentation and gave us the possibility to create practical scenarios for resections. perhaps the most important information was to discover and see the section plan and to calculate the volume of the remaining liver after resection. conclusions: the anatomical-imagistic pattern based on \ i[ex vivo \/i [ model disections combined with imagistic data offers a unique mindset before intervention. the concept 'human \ i[vs \/i [ swine' to create an easy method of learning for residents in training can be applied to swine liver anatomy. the learning of surgery is traditionally based on the behaviourist model . goals are set, standards of care fixed, with regular assessments of the level achieved. the teacher exercises control over the student, imposing rules and models, supported by 'reinforcing' actions (reward or punishment). the theory of skinner's program of education, from , is reflected in surgical learning. it foresees a gradual progression by level of difficulty, following a transmission-imitation model . these theories seem currently outdated to face the new challenges of medicine and surgery and to keep up with technological developments. bruner, one of the theorists of the constructivist model, proposed in a method of collaborative learning between those who teach and those who learn. the goal of the method was to improve strategic problem solving. the comparison between various perspectives (between teacher and student), allowed the learner to better absorb knowledge and improve critical thinking. in kapur published on the theory of 'productive failure'. this model makes the error of a single person useful for all his colleagues, privileges the practice of theoretical knowledge, contextualised learning as opposed to abstract learning, and 'guided' practice compared to a 'guided' theory. bruner and kapur's systems favour creativity, critical analysis of a problems origin, and the practical use of knowledge. they represent a hypothesis of learning, based on constructive discussion and a continuos 'give-take' feedback system. in order to put these new models into practice in the clinical context, one may hypothesise and propose the adoption of a formal discussion of clinical cases that are complicated or difficult. thereby making the theoretical lessons more collaborative, intuitive and inclusive. in the surgical field, one could adapt such a concept to surgery simulation, virtual reality and anatomical models. aim: large hiatal hernias have a surgical indication when the patients suffering disabling symptoms such as anaemia, dyspnea, chest pain, gastric reflux. several studies showed that in the case of large hernias the placement of a prosthesis was safe and could protect against recurrence. mini-invasive surgery is the preferred approach for hiatal hernia repair and anti-reflux procedure and the toupet fundoplication has been shown to be the best surgical technique for the hiatal hernias repair.the laparoscopic approach is currently the surgical gold standard but is burdened by technical difficulties especially in the case of large hiatal hernias. the robotic system is designed to overcome some technical difficulties of laparoscopy and the studies available in literature report the safety and effectiveness of the robotic approach in complex hiatal hernias repair. methods: we present the case of a grade iv hiatal hernia treated with a robotic approach in a years old woman (bmi: kg/m ). the medical history consisted of a road accident with a probable mechanism of deceleration, three years before. the patient had been suffering from dyspnea for three years. due to the recent discovery of an anaemia, the patient was subjected to an endoscopic examination with the identification of a voluminous grade iv hiatal hernia. a subsequent computed tomography (ct) scan showed also the partial herniation of the transverse colon. results: the patient underwent to surgery by using the da vinci robot system siÒ (intuitive surgical, sunnyvale, usa) with a single docking approach. the surgery consisted in the liberation of the hernial sac, the placement of a goretex prosthesis and the packaging of a toupet fundoplicatio. the surgery was performed without complication. conclusions: the robotic approach in the hiatal hernia surgery seems to be a valid alternative to laparoscopy, especially in complex cases. the surgical ability in robotic surgery is of paramount importance. general thoracic surgery, kawasaki municipal hospital, tokyo, japan aim: video-assisted thoracoscopic surgery (vats) with carbon dioxide (co ) for mediastinal surgery is known to improve the visualization of medaistinal space. we report our experiences with two cases that underwent vats thymectomy using co insufflation under the one-lung ventilation general anesthesia by double lumen tube. methods: the instruments that were used for vats thymectomy were only the -mm -degree rigid thoracoscope, maryland jaw energy device, cotton made-dissectors, and straight endoscopic grasping forceps. they were used through sealed ports designed for laparoscopic surgery. lowpressure co insufflation set at mmhg were used for compression of surround tissue of mediastinal tumor during the releasing procedure. results: the patients were an -year-old male and a -year-old female. thoracoscope with the mmhg co insufflation provides excellent visualization of the medaistinal space and operation could be done smoothly without any hemodynamic compromise. their pathological diagnoses were thymic cancer and thymoma, type b . the operative times were min and min. the postoperative courses were uneventful and the patients were discharged on day th and rd . conclusion: we have just begun to routinely use co insufflation for mediastinal tomorectomy and present our early experiences of successful vats thymectomy by utilizing co \ su \/su insufflation. aims: this retrospective study aims to evaluate the feasibility of single-incision thoracoscopic surgery (sits) for primary spontaneous pneumothorax (psp), using a novel multichannel port (x gateÒ). methods: between october and november , ten patients who underwent sits using x gateÒ. nine patients were male and was female, with mean age of . ± . years old. a . cm incision is placed in the middle axillary line on the th or th intercostal space, depending on the lesions. postoperative outcomes of these patients were compared with those of patients with psp who underwent conventional three-port video-assisted thoracic surgery (vats). results: there were no conversions from sits to vats. mean operative time of sits group was significantly shorter than that of three-port vats group ( . ± . min vs . ± . min, p = . ). mean number of staplers used in surgery was . ( ) ( ) ( ) ( ) in sits group and ( ) ( ) ( ) ( ) ( ) in vats group (p = . ). mean duration of postoperative drainage was also shorter in sits group ( . ± days vs . ± . days, p = . ). no recurrence and wound infection were observed in sits group. conclusion: sits using x gateÒ is feasible when performed for selected patients with psp. x gateÒ provides good visualization of intrapleural space and esthetic outcomes, as well as a superb maneuverability by decreasing mutual interference of surgical instruments. although conventional three-port vats for psp is well established, sits using x gateÒ can be a permissible alternative. further examinations are required to evaluate efficacy of sits using x gateÒ. aims: haemorrhage remains a leading cause of potentially preventable death in trauma. in particular non-compressible torso haemorrhage is approximated to cause - % of mortality in civilian trauma patients with otherwise survivable injuries and % in war setting. we performed a literature review to assess the potential for using endovascular stenting in traumatic venous injuries and explore the evidence of their efficacy and safety with different venous injury patterns. methods: systematic online search of pubmed performed using key words'endovascular stent', 'venous injury', trauma, penetrating, blunt, abdominal and pelvic. inclusion criteria included all studies that explored the use of endovascular stents following traumatic abdominopelvic venous injuries. english language studies were used. results were presented according to prisma guidelines. results: of the studies generated by the search,there were only four case reports in the literature documenting the use of endovascular stents in traumatic venous injuries dating back to and most recently . the four cases included three retrohepatic ivc injuries, two secondary to blunt trauma and one penetrating; whilst the final case a blunt injury at the ilio-caval bifurcation. all four cases reported successful deployment of stents via the femoral or internal jugular veins, with subsequent resolution of haemorrhage. length of time taken for stent insertion ranged from to min. three of four patients made full recoveries and discharged from hospital, with one patient subsequently dying of a brain injury independent of the successful venous stent insertion. no complications were reported at up to months follow up in remaining cases including stent leak, stenosis or migration. conclusion: endovascular venous stents have been used successfully in managing complex abdominopelvic traumatic venous injuries. in particular retrohepatic venous injuries refractory to hepatic packing and vessel embolization, which are not amenable to direct surgical repair due to anatomical location. however before endovascular stenting can be added to the arsenal of interventional radiologists for abdomino-pelvic trauma, further development of stents custom made for venous injuries as well as prospective studies examining their long term safety and outcomes is needed. tracheal papilloma is a rare neoplasm growing from the tracheal or bronchial epithelium and has no specific clinical presentations. this is a -year-old female who complained of progressive dyspnea for about months. physical examination was unremarkable and the there was no abnormal finding by the chest plain film. chest computed tomography was arranged and revealed a mass lesion located at the tracheal lumen with more than % luminal obstruction. we used fiberoptic bronchoscopy to evaluate the airway and found a mass lesion with pedicle originated from the posterior tracheal wall. cryotherapy was considered for the tumor mass removing to establish a patent airway. the pathologic report revealed tracheal papillomatosis without any malignant component. dyspnea was immediately improved and the patient chose closely observation after the bronchoscopic cryotherapy. aims: recent advances in laparoscopic surgery, both in techniques and instrumentation material, have led to the emergence of innovative technological fields, among which robotic surgery stands out.one of the handicaps of this surgery is its high cost as well as the long learning curve. in this stage a new tool arises, the flexdex semi robotic arm, which combines the precision and the range of movements of robotic surgery with the greater availability, simplicity of use and learning of conventional laparoscopic surgery.the objective of this study is to evaluate the efficacy and safety of the flexdex device in different laparoscopic procedures. methods: flexdex's is a three-axis gimbal technological device integrated in a conventional laparoscopic instrument that translates the surgeon's hand, wrist, and arm movements from outside the patient into corresponding movements of an end-effector inside the patient's body.the greater accessibility provided by the flexdex allows the surgeon to perform sutures in areas of difficult access where mobility with conventional laparoscopic instruments is not optimal. the comfort of the surgeon remains fundamental in any type of surgery, even more when we are in anatomical locations with complex access, especially for the realization of sutures. here is where surgical innovation instruments such as flexdex provides ergonomic comfort for the surgeon and improves the patient's safety, especially in high-risk situations, such as when performing anastomosis. results: we present a prospective series of laparoscopic procedures carried out by the same surgical team being the initial experience in our environment in the use of the flexdex semi robotic arm for the realization of complex anatomical sutures.this is a case series of patients to whom different surgical techniques requiring manual suture have been performed. these being tapp procedures, nissen-type fundoplicature and reinforcements of colorectal anastomosis. it is important to note that in none of the cases complications were recorded conclusions: flexdex can provide an excellent alternative to the robotic systems in complex surgical procedures, offering surgeons the precision and control they desire while maintaining the balance of cost, outcome and patient benefit. background: a new single-port device (fsis-flexible-single-incision-surgery) is presented. this new platform has three working channels, two for rigid instruments and one for the flexible endoscope. the channel for flexible instruments offers a pneumatic sealing to avoid the air's leak of the cavity (abdomen, rectum, vagina) . in this study the preclinical data are shown testing the feasibility and safety for laparo-endoscopic instruments. methods: experimental evaluation of feasibility and safety in two stages. in the first stage a working channel with pneumatic sealing was tested in simulators to use a flexible endoscope. in the second stage (animal model) the single incision device that makes possible to use laparoscopic instruments and flexible endoscopes was tested. the measured variables were: time of the procedure, co employed, adverse intraoperative events, grip's losing, losing of pneumatic sealing, feasibility and safety of the procedure for the surgeon. results: the hysterectomy and double adnexectomy was done with a median time of . min. the median of the co consumption was . litres. only in one case ( . %) the surgeon had problems with the abdominal navigation of the endoscope that was easily solved. the grip's lose wasn't a major problem. the median size of the skin incision was . cm. the median surgeon' score for the feasibility was and for the safety was . . conclusions: the surgeons considered that the use of the device was very feasible and safe. the fsis-device is a universal platform for single-incision-surgery for surgeons and gastroenterologists and for abdominal, rectal and vaginal access. aim: despite the near-infrared fluorescence (nirf) via the intravenous administration of indocyanine green (icg) improves the visualisation of the cystic duct (cd) and the extrahepatic biliary tract (ebt), the back fluorescence of the liver reduces the signal-to-noise ratio.we have modified the technique of nirf cholecystocholangiography with intragallbladder icg injection by using the arrow-karlan tm balloon cholangiography catheter instead of the purse string at the gallbladder's fundus. this procedure allows a high rate of visualisation of the ebt, with few cases of icg leakage.aim: of this study is to confirm the feasibility of this different technique and to analyse the icg spillage from the gallbladder and to identify the ebt. methods: we enrolled nine patients undergoing laparoscopic cholecystectomy for cholelithiasis. the gallbladder was perforated with the cholangiogram catheter, the balloon inflated with . ml of saline and tightened. the bile was drained and the icg bolus injected. a titanium clip was the placed on the catheter strict closely to the gallbladder in order to prevent the catheter dislocation. results: the cd and the ebt were visible before dissection in / and / patients respectively. after dissection the cd was visible in all the patients and the ebt again in / patients. there was only one icg spillage due to a tardive positioning of the clip. in a case of inflamed gallbladder this technique helped in the identification of the dissection plane. conclusions: our preliminary results of this ongoing study confirm the feasibility of this different approach as a possible alternative to the purse string and a good visualisation of ebt. introduction: robotic-assisted surgery is a promising technique for overcoming the limitations of laparoscopic surgery, especially with regards to complex and advanced surgical procedures. here, we describe the establishment and implementation of our robotic upper gastrointestinal (gi) and hepato-pancreato-biliary (hpb) surgery program within our center of excellence for minimally invasive surgery as well as the first-year results. method: robotic-assisted surgery was performed using the davinci xi surgical system tm and performed by two surgeons specialized in minimally invasive surgery (db and tk). our robotic surgery program of upper gi and hpb surgery was established in three steps: ( ) first, surgical procedures with easier degree of difficulty were performed robotically, including cholecystectomy, minor gastric resections and fundoplications. ( ) then, pancreatic distal resections, enucleations, adrenalectomies and atypical liver resections were robotically performed, as procedures with moderate degree of difficulty. ( ) finally, advanced and highly complex procedures were performed, including right hemihepatectomy, complex pancreatic head resections (including portal vein resections), total gastrectomy and esophagectomy. data collected from july till july were retrospectively analyzed with regard to conversion rate, morbidity (clavien dindo grade £ ) and mortality. results: within the first year, a total of robotic assisted upper gi and hpb resections were performed. the first step of establishing our robotic surgical program included eight procedures. here, conversion rate, morbidity and mortality were %. within the second step of establishment procedures were performed. conversion rate, morbidity and mortality were %, % and %. the last step included of advanced and highly complex procedures. these procedures resulted in a conversion rate of %, % morbidity and % mortality. conclusion: our stepwise approach enables a safe implementation of a robotic surgical program for upper gi and hpb surgery with low morbidity and no mortality even for highly complex procedures. however, highly complex procedures required a high conversion rate, which might be caused by the early stage of experience. the standard surgical procedure of choledochal cyst is a complete excision of the cyst with rouxen-y hepaticojejunostomy and laparoscopic surgery had been increasingly used. this is still a challenging way to perform anastomosis due to the small diameter of bile duct and the possibility of bile leak or stricture. robotic system can overcome the shortcomings of laparoscopy with providing three-dimensional view, magnification, and articulated instruments. from jan to dec , patients underwent robotic cyst excision and hepaticojejunostomy by single surgeon. we reviewed the clinical data and compared with laparoscopic outcomes of early (from to ) and late (from to ) group, retrospectively. patients of robotic series were all female with mean age . years and bmi . . the mean size of cyst was . . cm, and todani type ia , ic and iva , respectively. total trocars were used with robotic working arm and assist and camera. the mean operative time . ± . min, and it was similar with late laparoscopic group ( ± . min) and significantly shorter than early group ( ± . min).there were no open conversion in robotic and late laparoscopic group, however, the early laparoscopic group involved % of conversion rates. the hospital length was ± . days in robotic group, and it was similar with late group ( ± . ) and more shorter than early group ( . ± . ). in robotic series, postoperative complications occurred patients. one case included cholangitis which was resolved after conservative treatment. bile leakage was developed in patient, and treated with drain that inserted intraoperatively. last cases showed incisional hernia at postoperative months, and was corrected by laparoscopic herniorrahphy. complications (n = ) in late laparoscopic group included hepaticojejunostomy stricture and stone, bleeding of jejunal branch, portal vein thromobosis, acute pancreatitis, and adhesive ileus. there were no mortaility case in any groups.robotic surgery of choledochal cyst is a safe and feasible option with short-term results that are comparable to laparoscopic approach. general surgery, sanchinarro university hospital, madrid, spain background: the incidental detection of benign to low-grade malignant small pancreatic neoplasms increased in the last decades. the surgical management of these patients is still under debate. the aim of this paper is to evaluate the safety and feasibility of robotic enucleations. methods: we retrospectively reviewed our prospectively databases from november . demographics, pathological characteristics, perioperative outcome, and medium-term follow-up of patients who underwent robotic pancreatic enucleations were collected. results: patients were included. the mean age of the patients was years ( - ). the median body mass index was ( - ). ten lesions were located in the pancreatic head, in the pancreatic body, in the pancreatic tail. operative time was min (range - ), no intraoperative transfusion were needed and in one patient conversion to open approach was needed. in three patients grade b pancreatic fistula occurred. the mean postoperative stay was , days. conclusions: robotic enucleation is a feasible and safe approach, with low incidence of morbidity. the results of surgical treatment of patients with pulmonary tuberculosis were evaluated depending on the prevalence of the tuberculosis process and the type of surgical intervention used. according to the results of the questionnaire, people operated on pulmonary tuberculosis in the period from to years ago, the frequency of cases of tuberculosis reactivation, the complicated course of the remote postoperative period, as well as the mortality and causes of lethal outcomes were assessed. it was found that after sublobular resection and lobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistula- . %, cardiovascular insufficiency-in . % operated. the mortality rate was . % with a total clinical efficacy of . %. after combined resection and bylobectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. after pneumonectomy, treatment failure was noted at . %, relapse of tuberculosis- . %, pleural empyema- . %, bronchial fistulae- . %, cardiovascular failure- . % operated. the mortality rate was . % with a total clinical efficacy of . %. robotic reduced-port splenectomy using single-site platform j.h. lee background: in the era of minimal invasive surgery, single incision laparoscopic splenectomy can offer some advantages compared to conventional laparoscopic splenectomy. but it requires expertise in minimally invasive techniques due to technical difficulties. the da vinci robotic reduced-port splenectomy using single-site platform permits greater freedom of movement and higher levels of accuracy than previous laparoscopic surgery through two small incisions. methods: we performed a retrospective review of all patients who underwent robotic reduced-port splenectomy using single-site platform at our institution between january, and november, . one cm periumbilical incision was made for glove port insertion and the other incision was made at left side of abdomen for additional mm port insertion.the surgical technique is much same as open procedure. short gastric artery was ligated, firstly. splenic artery and vein were ligated individually. during the surgery, any stapling device was not used. vessel sealer was used for hemostasis and mobilization of spleen. a specimen was removed through umbilical port site within lap-bag. result: eight patients ( female and male) with median age of . years underwent robotic reducedport splenectomy using single-site platform (one case with combined robotic cholecystectomy for gall bladder stones without additional trocar). the indications were; hematological disease (n = ), splenic mass (benign n = , malignant n = ). preoperatively measured spleen size was ranged . cm to cm (mean cm). there were no intraoperative complications and open conversion. mean operative time was min. (range - min) including docking (mean min) and console time (mean min) mean blood loss was under ml. mean hospital stay was . days after surgery. one patient underwent oral anticoagulation therapy only for portal vein thromobisis without any symptoms, and thromobisis was resolved at month follow-up ct scan. there were no clavien-dindo class iii or above postoperative complication. conclusions: robotic reduced-port splenectomy using single-site platform seems to be feasible and effective. it seems to overcome certain limits of previous robotic or conventional single-site laparoscopic splenectomy and single-site only robotic splenectomy. we think mm additional port allows to use endo-wrist da vinci instruments such as vessel sealer which enhances dissection efficiency andsafety of procedures. aims: inguinal lymph node dissection carries an important risk of post-operative complications, mainly related with wound complications and long term lymphedema. the minimally invasive approach aims to reduce the morbidity of this procedure, avoiding the traditional groin incision but still allowing a full access to the lymph node basin. the authors aimed to describe their videoassisted inguinal lymph node dissection (vilnd) cases, comparing the surgical outcomes with a sample of open inguinal lymph node dissection (oilnd) cases. methods: we performed a retrospective descriptive study that compared the data from patients submitted to vilnd since (the year in which this technique was first performed in our institution) with the patients submitted to oilnd in and . gynaecologic and urologic malignancies were excluded. the statistical analysis was performed using spssv Ó, with a p value \ . indicating statistical significance. results: a total of cases of inguinal lymph node dissection were analysed, . % of which vilnd (none of them requiring conversion to the open approach). melanoma was the primary tumour in % of patients. the vilnd and oilnd groups had no statistically significant difference between them regarding age, body mass index, smoking status or the reason for lymph node dissection-clinically detected lymph node vs. positive sentinel node biopsy. the mean of isolated lymph nodes in the vilnd ( . ) and oilnd ( . ) groups was also not statistically different (p = . ). there was no difference in the rate of post-operative seroma, wound dehiscence or lymphedema. the rate of surgical site infections was higher in the oilnd group- % vs. . % during post-operative hospital admission (p = . ); . % vs. . % after discharge (p = . ). conclusions: in our population of patients we conclude that the main advantage of the videoassisted approach regarding surgical morbidity lies in the reduction of the infection rate, as the published literature also confirms. the equivalent number of lymph nodes retrieved in both groups points toward the oncological safety of the minimally invasive procedure, that we hope to study further in the future after a longer follow up period. objectives: to evaluate the clinical feasibility of tumor localization technique with radio-frequency identification (rfid) clip marker methods: we developed the proto-type rfid integrated endoscopic clip (rfid-clip) and probe to detect it on serosa surface during the laparoscopic surgery. a pig weighing kg was used as the specimen for the in-vivo test. endoscopist performed the application of the rfid-clip on porcine gastric mucosa. after then, the surgeon tried to find the location of rfid-clip using the detection probe and marked with the electrocautery. after the gastrectomy with cm margin (each to proximal and distal), we confirmed the prediction of rfid-clip location and accuracy of resection. results: rfid-clip location was detected and recorded on the exact site of clip application. detection range was very short and we confirmed there are almost no differences between actual clip location and our prediction. this result might arise from using the low-frequency rfid tag to increase the accuracy through reduction of the range. however, some rfid-clip were not detected because of the issue of clipping trouble, not rfid tag. conclusions: this is a basic study to evaluate the clinical usefulness and feasibility of the new localizing technique. we confirmed the possibilities of this system and it could be the helpful option to provide the information of exact location for the minimally invasive surgery or early gastrointestinal tumors. background: the advantages of laparoscopic posterior retroperitoneal adrenalectomy (lpra) have been described in the literature. the aim of this study was to compare the clinical outcomes of lpra and robotic posterior retroperitoneal adrenalectomy (rpra) and determine the differences that could affect the outcomes. methods: we retrospectively analyzed adrenalectomy cases at asan medical center from to . there were lpra and rpra cases, and their clinicopathological features and surgical outcomes were compared. results: in lpra, there was a positive relationship between operation time and male gender, early period of experience, adrenal tumor size, and pheochromocytoma. in rpra, adrenal tumor size and pheochromocytoma were the only factors affecting the operation time. when the adrenal tumor size was = . cm, the operation time of lpra was shorter than that of rpra (p = . ). when the tumor size was [ . cm, there was no significant difference in the operation time of lpra and rpra (p = . ). conclusions: rpra is a feasible and technically safe approach for benign adrenal diseases. the use of rpra could benefit patients with large tumors and provide comfort by overcoming the factors contributing to a longer operation time in the laparoscopic technique. methods: twenty years experience at the american university of beirut medical center for laparoscopic adrenalectomy. a total of cases were done laparoscopically with no conversion and minimal complication. the average operative time is mins.the video will show the various steps used for lap redo (lt) adrenalectomy for a cm pheochromocytoma using the lateral position and through trocars. attempt to remove the pheochromocytoma in iraque was complicated by cardiac arrest treated successfully and patient referred to the american university of beirut medical center. results: patient had smooth postoperative course following laparoscopic adrenalectomy and patient discharged days later with no complications. conclusions: even large adrenal masses can be completed laparoscopically in advanced experienced centers in laparoscopy. surg endosc ( ) aims: the adrenocortical of uncertain malignancy neoplasm is a spectrum of classification for adrenal tumors whose histopathological diagnosis is uncertain. clinical case: we present a year old patient with constitutional syndrome and severe hypercortisolism and hypokalemia reason why she was admitted to icu for episodes of ventricular fibrillation. no other medical history of interest except refractory hypertension to treatment. the tc showed a left adrenal mass of . . cm with microcalcifications, areas of necrosis and hemorrhage, no infiltrating, without disease to distance. the surgery was a laparoscopic left adrenalectomy with no evidence of infiltration and no lymph nodes. the histopathology lesion presented a dense proliferation cellular of cortical type, with incomplete fibrous, without vascular or capsular invasion, with a % ki ; positivity vimentin and cd . all epithelial markers, were negative. all this leads to the diagnosis of a neoplasm of uncertain malignancy potential adrenocortical. during the postoperative period, the patient presents a crisis of adrenal insufficiency that was treated with intravenous replenishment corticoidea and later orally with good clinical response. discussion: the adrenal carcinoma has a low incidence ( . %), incidence peak around the years, the most frequent is the mixed secretory. they are - % of the adrenal incidentalomas. it is usually presented to the diagnosis as a locally advanced tumor with metastases (to liver, lung, retroperitoneal ganglia and bone). may present clinically due to hormonal hyperproduction; or be non-functioning tumors. the adrenal carcinoma poses a great difficulty at the time of the diagnosis pathological, and includes as differential diagnosis to other abdominal tumors. the distinction between corticoadrenal adenoma and adrenal carcinoma is sometimes difficult, so it has been defined a spectrum of intermediate category called adrenocortical neoplasm of intermediate or uncertain malignancy. it is obtained with the weiss criteria, being necessary at least of them for confirm the diagnosis of adrenal carcinoma. this category has a low risk of local recurrence or metastasis, but it needs a narrow follow-up. conclusion: adrenal carcinoma of uncertain malignancy implies a new category in those tumors of difficult classification. aims: multiple endocrine neoplasia type (men ) is an autosomal dominant disorder with an estimated prevalence of per , in the general population. among patients suspected to have a pheochromocytoma, the diagnosis is rarely confirmed and only % is presented bilaterally. we present bilateral laparoscopic adrenalectomy in patients with men . method: a -year-old woman with a family history of medullary thyroid cancer and breast cancer. personal history: hypertension, medullary thyroid cancer, breast cancer, laparoscopic cholecystectomy. appendectomy. after a study by endocrinology and suspicion of bilateral pheochromocytoma, discussing the case in a multidisciplinary committee, bilateral adrenalectomy was decided by laparoscopic approach. selective alpha- -adrenergic blocking agent (doxazosin) were utilized before surgery. under general anesthesia left adrenalectomy was performed first in right lateral decubitus position. mmhg pneumoperitoneum was started with the verres needle and trocars ( mm umbilical, mm subxifoid and mm left subcostal).once dissection was completed the gland was placed in a plastic bag and extracted through one of the trocars incisions, then the position of the patient was changed to left lateral decubitus for the right adrenal approach. another right subcostal mm trocar was used. adhesiolysis of previous cholecystectomy was performed to right adrenal approach. adrenal veins were divided between metallic clips.no drainage was employed. results: the procedures were successfully performed without conversion. surgical time was min and hospital stay was days. had a clinical reversion with control of blood pressure monitored by endocrinology conclusions: currently, the laparoscopic approach is the technique of choice for the management of adrenal pathology.lateral decubitus transperitoneal approach is the procedure of choice in most cases. bilateral laparoscopic synchronous adrenalectomy is feasible and safe with good results as in our patient. traditionally the treatment of hyperparathyroidism for patients with familial hyperparathyroidism was subtotal parathyroidectomy or total parathyroidectomy and auto transplantation. in the era of minimally invasive parathyroidectomy, the removal of only abnormal glands guided by preoperative localizing studies has been suggested. aims: this systematic review aimed to investigate the role of focused minimally invasive parathyroidectomy in the treatment of patients with familial hyperparathyroidism. methods: electronic databases were searched with the search terms 'men i', 'familial hyperparathyroidism', 'men a','hyperparathyroidism-jaw tumor syndrome', 'parathyroidectomy', 'minimally invasive ', for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria. results: thirty five possibly relevant studies were identified. abstracts were reviewed and fifteen articles were excluded. twenty studies, that met inclusion criteria were retrieved in full text and included in the systematic review, including three retrospective cohort studies i.e. two presenting data on meni associated hyperparathyroidism and the third study on familial hyperparathyroidism and seventeen small case series or case reports. the two retrospective studies on meni hyperparathyroidism included patients treated either with focused minimally invasive parathyroidectomy or with the conventional approach. these studies presented conflicting data with one supporting and the other negating the focused minimally invasive parathyroidectomy due to the failure of localization studies to identify enlarged parathyroid glands in a great number of patients. conclusion: undoubtedly, the idea of minimally invasive parathyroidectomy in patients with hereditary and familial hyperparathyroidism is interesting. this idea is especially challenging in the case of meni. existing data suggest that focused mimimally invasive parathyroidectomy is feasible under the condition of exact preoperative localization studies. the main advantage of this approach is the minimization of the risk of postoperative hypoparathyroidism. however, data are limited and further research is needed before valid conclusions can be drawn on the suitability of this approach. objective: resection of pheochromocytomas is a challenging procedure due to hemodynamic lability, tumor vascularity and malignant potential.given the technical challenges for resection of large pheochromocytomas, there were hesitations about using the laparoscopic approach for these tumors during the first decade of laparoscopic surgery. however, improvement in imaging modalities,better pharmacological preparation,advances in anaesthesia and laparoscopic surgery rendered laparoscopic surgery for pheochromocytomas safe and efficient. our aim was to evaluate surgical outcomes in patients with pheochromocytoma and to validate the role of laparoscopic surgery in the treatment of these tumors. design: a total of procedures for pheochromocytoma were performed between january -september . the preoperative diagnosis, operative details, complications, length of hospital stay, morbidity and follow up were retrieved from the hospital records of patients who underwent adrenalectomies for benign and malignant adrenal tumors in the same period. preoperative localization was established in all patients with computerized tomography (ct) or magnetic resonance imaging (mri), while iodine - -metaiodobenzyguanidine(mibg) scan was reserved for ambiguous cases where paraganglioma or metastatic disease was suspected. endocrinological evaluation and complete adrenal dynamic testing were performed to determine whether the tumor was functional or not. results: eighty-seven tumors were removed from patients. one patient with meniia underwent bilateral resection of pheochromocytomas in two stages. tumor size in laparoscopic procedures ranged from . cm to . cm (mean . cm). forty-three patients had benign disease, potentially malignant (based on pass), malignant with metastasis. eight were in the context of a familial syndrome. sixty -eight patients underwent laparoscopic adrenalectomy, patients had open approach from the start for recurrent pheochromocytoma or large benign tumor, patient had open approach due to inoperable malignant pheochromocytoma and patients had conversions from laparoscopic to open procedure. nine patients received sodium nitroprusside intraoperatively to treat hypertension. one patient developed pulmonary embolism, and succumbed month later. there were no recurrences for the benign tumors during the follow-up period. conclusions: laparoscopic resection of pheochromocytomas despite its increased level of difficulty compared to that of other adrenal tumors, is a safe and effective procedure. aim: the concept 'large' in transperitoneal lateral laparosopic adrenalectomy (tlla) has been evolving along time, ranging from to - cm depending on different authors. on the other hand, some authors discourage laparoscopic surgery in larger tumors due to the increased risk of malignancy in those larger than - cm, referring to malignancy in out of or cases. paragangliomas are rare tumors originated in extra-adrenal chromaffin cells, with an incidence of - cases per million inhabitants. they can appear in any location between neck and pelvis. sympathetic paragangliomas are usually functional and catecholamines producers. we present a movie of surgical intervention of a -year-old patient who, in study for refractory hypertension, presented paraganglioma producing norepinephrine, whose approach was performed laparoscopically. -year-old woman studied by nephrology for refractory hypertension. on physical examination, only obesity standed out. in blood exams, levels of normetanephrine were observed in plasma of pg/ml and aldosterone pg/ml. abdominal scintigraphy was performed in which there was no evidence of increased activity at adrenal level. abdominal ct shows retroperitoneal extra-adrenal tumor of inter-aortocava location immediately below renal vessels with dimensions of . . cm. after preparation, she was operated. laparoscopic access was performed under exhaustive monitoring. an heterogeneous, polylobulated tumor of cm, located interaortocava, intimately adhered to left renal vascular pedicle, was observed. a cattell-braash and kocher maneuver was performed, with exposure of inferior cava and aorta to iliac bifurcation. complete tumor excision was performed after clipping arterial and venous tributary branches. after the operation, the pacient presented favorable evolution being discharge on the second postoperative day with good control of blood pressure levels. laparoscopic approach of retroperitoneal paragangliomas is a safe technique, which allows minimally invasive access, with consequent improvement in postoperative results. the exact location of lesions and their relationships with surrounding structures, as well as their functional behavior, are very important when considering the best therapeutic strategy for these patients. we present the case of a -year-old obese male patient referred for adrenalectomy after being diagnosed with left adrenal incidentaloma. abdominal mri showed a . / . / . left adrenal mass with normal hormonal levels. after preoperative workup, the patient underwent standard laparoscopic adrenalectomy. the lateral to medial dissection and mobilization of the spleen and pancreatic tail was difficult due to the abundance of peritoneal and pararenal fat. the anatomy was peculiar: the bulky pancreatic tail was located well inferior to the splenic hilum and was visible throughout the intervention and the spleen was quite elongated-long axis = cm. the exposure of the adrenal gland was therefore cumbersome. the operating time was min and blood loss ml. the abdominal drainage was maintained for h. before discharge the patient underwent a control abdominal us examination that only showed a thin line of left pleural fluid. the patient was readmitted days after discharge for chest pain, fever ( . °c) and malaise with no abdominal signs. the emergency ct scan diagnosed left basal pneumonia with minimal pleural effusion and a / cm fluid collection between the spleen and diaphragm while the blood test showed leukocytosis. the patient was treated for pneumonia with an apparent clinical benefit for three days and lowered white cell count but his condition worsened during the forth day. repeat abdominal us demonstrated that the abdominal collection increased in size therefore the patient underwent emergency surgery. during laparoscopic exploration, the collection was unveiled as being pancreatic juice (more than times the normal serum levels of lipase and amylase). after thorough lavage, two drainage tubes were positioned in the left subphrenic space. the postoperative course was uneventful under antibiotic treatment for pneumonia and pancreatic antisecretory medication. the patient was discharged after days with minimal pancreatic drainage and the drainage tube was extracted after more days. the aim of the study was to develop the algorithm and the choice of the method of endoscopic treatment of a combined pathology of uterine leiomyoma and adenomyosis depending on the reproductive plans. methods: the study involved patients with a combined pathology of uterine leiomyoma and adenomyosis. indications for conservative myomectomy were: the size of the uterus is more than weeks. pregnancy; multiple leuomatous nodes and adenomyotic foci up to cm in size; hemorrhagic and pain syndromes, anemia, compression of the adjacent organs; suspected node malfunction; submucous leiomyoma deforming the uterine cavity with foci of adenomyosis; subserous, cervical isthus nodes and foci of adenomyosis; the presence of endometrial hyperplasia, tumors of uterine appendages; growth rate of uterine leiomyoma more than weeks pregnancy for the year; the growth of uterine leiomyoma on the background of drug treatment; infertility associated with leiomyoma and uterine adenomyosis.the laparoscopic myomectomy of the subserous node on the 'leg' with a size of more than cm and nodes of more than cm of intramural location is shown with an interest in preserving the organ.the hysterectomy is indicated for women after years of age who insist on hysterectomy, with a combination of uterine leiomyoma with atypical endometrial hyperplasia. results: the conservative myomectomy and removal of adenomyotic foci were performed in ( . %) patients: from hysteroscopic access- , vaginal access- , laparoscopic access- , abdominal access- in the presence of reproductive plans.the hysteroscopic myomectomy was performed in ( . %) patients, hysterectomy in ( . %) patients: from laparoscopic access- , from vaginal access- , from abdominal access- in the absence of reproductive plans. conclusions: the choice of surgical treatment of uterine leiomyoma and adenomyosis depends on the reproductive plans of the woman and the severity of the lesion.the laparoscopic method of treating a combined pathology of uterine leiomyoma and adenomyosis in the presence and absence of reproductive plans is a priority for women. surgery, policlinico ,,paolo giaccone,,, palermo, italy background: breast cancer in females represents the most frequent neoplasm in all age groups. the risk of getting breast cancer (mc) increases with age. the brca and the brca genes (tumor-suppressor genes, autosomal dominant transmission at high penetrance) alone justify from % to % of cases of hereditary breast cancer. methods: from january to june we have analyzed patients with brca mutation. all patients had in common a genetic mutation of brca or brca tumor suppressor genes. results: the frequency of germline mutation on brca ( patients: %) was identical to brca gene ( patients: %). of the analyzed patients were women ( . % of patients) brca and brca , and men ( . %) all with brca mutation. conclusions: prophylactic surgery must be seen as a way to put the patient in the condition to implement the most appropriate treatment. further studies will be necessary to support the validity of prophylactic surgery in patients with mutations in brca and brca genes. introduction: laparoscopic hysterectomy is a safe surgical technique for removing the uterus with or without including the ovaries and fallopian tubes. laparoscopic surgery of endometrial cancer is a safe method, with the mean time of recovery being two days only. material-method: the case of a yr old woman with metrorrhagia and anaemia (ht , %) due to adenocarcinoma of the endometrius is presented. the patient underwent a laparoscopic hysterectomy and oophorectomy. trocar ports were used during the procedure (a mm transumbilical port, similar to the port used in single incision laparoscopic operations, two mm ports at the level of the anterior superior iliac spines, and a mm port in the middle of the imaginary line between the pubic symphisis and the umbilicus). the uterine vessels and the uterine ligaments were ligated and dissected by using a thermal energy source. the patient's postoperavite course was uneventful. the patient continues to be in good condition, months post-surgery. conclusion: laparoscopic hysterectomy seems to be a safe method for addressing endometrial cancer, as it offers the surgeon a better surgical field, is tissue friendly and causes fewer postoperative complications. it is considered to be a less traumatic operative method, as due to zooming in the picture there is greater accuracy in handling the tissue, and blood loss is minimal. m. shahin background: hysterectomy is one of the most frequently performed surgical procedure. though there are three approaches in hysterectomy (open, vaginal and laparoscopic), still there are controversies regarding the optimal route for performing it. methods: this prospective comparative study included obese patients subjected for panhysterectomy as a treatment. the forty-two patients were allocated into two groups: group (a) subjected to laparoscopic pan-hysterectomy, group (b) subjected to open pan-hysterectomy. results: there was significant difference between the two groups regarding mean operative time, blood loss, analgesic requirements and hospital stay, while no significant difference regarding intra-operative complications. conclusions: laparoscopic hysterectomy in obese patients has emerged as a viable, safe and better alternative to open hysterectomy amongst appropriately trained surgeons. general: endometriosis in the inguinal region is rare. the usual presentation is that of a woman in the reproductive age group. it accounts for . - . % of patients affected by endometriosis. the groin swelling is usually slow growing, painful with exacerbations during menses. the incidence of inguinal endometriosis on the right side is - % as compared to the left. aim: to present our laparoscopic approach for the treatment of the diagnostic dilemma. case presentation: a -year-old woman presented with a palpable mass in the right groin. the swelling was associated with a dull aching pain. the patient was suffering from increasing pain over the swelling during menstruation. she had undergone cesarean section some years ago and the scar had healed by primary intention. mri scan revealed a nodular hypoechoic lesion at the level of the internal inguinal ring with the absence of vascular flow around the lesion. results: since inguinal endometriosis was in the differential diagnosis and it may be associated with pelvic or intraperitoneal endometriosis, a laparoscopic approach was decided. the procedure was successfully completed laparoscopically following the transabdominal preperitoneal approach. the endometriosis was found, after dissecting the internal inguinal ring, firmly adhered to the round ligament. it was excised en bloc with the round ligament. a preperitoneal polypropylene mesh was inserted to protect for future inguinal hernias due to extensive dissection at the level of the internal inguinal ring. no intraperitoneal endometriosis was appreciated. histopathology revealed endometriosis of the round ligament. the patient was uneventfully discharged the next day. on follow up the patient was asymptomatic. conclusions: round ligament endometriosis is a rare entity. it is a disease of specific interest to the physician. it can be confused with an inguinal hernia and thereby pose a diagnostic dilemma. we recommend considering endometriosis in the differential diagnosis of groin swellings in women. the transabdominal preperitoneal approach is feasible and safe in the hands of an advanced laparoscopic surgeon. introduction: sentinel node biopsy is the newest accepted method for surgical staging of early stage endometrial and cervical cancer. aim: to evaluate the role of the technique of indocyanine green (icg) identification of the sentinel lymph nodes in cases of early endometrial cancer. material and method: five patients with early endometrial and cervical cancer were introduced in a prospective study. icg was locally injected during the laparoscopic exploration. novadac pinpoint near to red technology was used. guided biopsies were performed into the marked sentinel nodes and histological results were evaluated. results: sentinel lymph nodes were easily identified by using icg and near-infrared technology. technical details are described. no associated complication was encountered. conclusion: sln mapping using icg in uterine cancers is demonstrated as an effective and safe procedure. laparascopic extraction of an intraperitoneal gossypiboma following c/s and a retroperitoneal gossypiboma following pyeloplasty n. ozlem general surgery department, ahievran university, kirsehir, turkey gossypibomas are forgatten foreign bodies,iatrogenic.their symptoms are different where they are. they extracted with laparotomy in the past but now we can some article mentioned their extraction was made with laparoscopy. case : y o female has abdominal pain after c/s for . years. a gossypiboma was extracted with laparoscopy above umblicus.a superficial surgical site infection existed,drained,subsided. case : yo m had a pyleoplasty operation years ago.a gossypiboma was extracted with retroperitonescopy,no postoperative event. basibuyuk et al reported retroperionescopic extraction of a gossypiboma from single port in first time.althoug every effort taken the incidence of foreign body detected in the body is about . - . %.they are most frequently localized in the intraabdominal cavity followed by tracheobronchial area,pleural cavity,pararenal area,vagina,spinal chord, neck, femur,breast,bladder,pancreas,and they may cause local irritation,and infection.tactile sense is absent in laparoscopy. all radiologic examinations(usg ct pet mri etc) be used to detect.we used usg ct.in the end laparoscopy make the diagnosis and remove gossypibomas in our cases with less postoperative pain and cosmosis. justo et al the computerized tomography (ct) scan is the most useful method for diagnosis; however, sometimes the preoperative diagnosis remains uncertain even after the imaging exam. in that case, laparoscopy arises as a valuable diagnostic tool, as well as a prompt treatment option. concerning gossypiboma, prevention is preferred rather than treatment. notwithstanding, there is no highly reliable prevention system. counting sponges is a method based on staff communication during the surgery with only % sensibility. routine surgical postoperative x-ray (spox) constitutes an early detection system, but the need to incorporate a radiopaque marker and to expose the whole surgical field to maximize its efficacy limits its use. more recently, electronic dispositives based on barcode detection and other technological adjuncts for counting sponges are being developed. none of these prevention systems are reliable when used alone. our education and research clinic was a state hospital before. no surgeon followed above instruction.but now we use all. multiple procedures and surgical teams, long operations and non-elective operations are the evidenced risk factors.c/s operation was learned full opened of ostium of cervix of the patient. urology, japan, nagoya, japan aims: some scoring systems have been suggested to standardize the renal tumor characteristics. among them, renal score is widely used in partial nephrectomy. whereas diameter-axis-polar (dap) score was developed to be more significantly related with postoperative renal function. our study compared dap score with renal score in robotic partial nephrectomy (rpn) outcomes. methods: records of patients who underwent rpn at nagoya daini red cross hospital between april to october were analyzed retrospectively. those include three oncocytomas. accordingly, we calculated the estimated glomerular filtration rate (egfr) just before rpn and month postoperatively in patients. we compared two nephrometry scores with warm ischemic time and change in egfr. results: in our institution, four surgeons performed rpn. according to dap score, patients were high, were middle and were low. according to renal score, were high, were middle and were low. the median warm ischemic time was min ( - ). the median egfr decreased from . ( . - . ) to . ( . - . ) ml/min/ . m . there were no significant differences in warm ischemic time and percentage change in egfr between renal score groups (p = . and . ) but significant differences between dap score groups (p \ . and p \ . ). univariate and multivariate analyses were used to identify factors influencing postoperative renal function. that confirmed that dap score was independent poor predictors of change in egfr after rpn. conclusions: dap score is simpler estimate system than renal score. our study suggested that dap score is a useful scoring system for preoperative evaluation of renal tumor for rpn. further investigation is needed to better understand preoperative dap score. aims: retroperitoneal primary tumors comprise a great variety of neoplasm with different histological typologies, with insidious clinical symptoms and little specificity in most cases. its diagnosis is established through imaging tests and anatomopathological study is needed so complete surgical resection is the treatment of choice. the aim of the video is to demonstrate the safety and efficacy of the minimally invasive approach in patients with retroperitoneal lesions. methods: a -year-old female patient who, in the course of an abdominal pain at the right iliac fossa suspected of possible acute appendicitis, is diagnosed with a right retroperitoneal tumor, compatible with primary neurogenic tumor on a ct. radiographic imaging is a key component of the evaluation of a patient with a retroperitoneal mass, a ct scan is necessary to evaluate the primary site as well as to rule out metastatic disease. after complete biochemical study, nonfunctioning tumor is determined. the study is completed with mri where the lesion is located below the right kidney, in front of the right psoas muscle and lateral to the inferior vena cava, and without contact with these structures. ??it is in intimate contact with the ovarian vein. the complementary tests and iconography of interest of the case are exposed. surgical intervention is proposed with a laparoscopic approach. results: full minimally invasive approach in left lateral decubitus position: trocars-lateral laparoscopic transabdominal approach. laparoscopic liberation of the right colon, kocher maneuver until the inferior vena cava is visualized, identification of a tumor of approximately cm in the right infrarenal region, lateral to the right ureter, which includes the gonadal vessels. resection of the tumor in block with margins previous dissection and clipping of the proximal and distal gonadal vessels with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. definitive result of the specimen: leiomyosarcoma, grade of the fnclcc with negative margin. the laparoscopic approach is a safe and effective technique in the approximation of retroperitoneal tumors, a radical oncological criterion is always needed with correct margins of resection especially in those of uncertain etiology. we started endoscopic thyroidectomy using the lifting method in and have developed single incision endoscopic thyroidectomy (siet) via chest (c-) or axillary incision (a-) by our original retractor since . we created a new approach in . recently, we have applied this method to parathyroid surgery. in this study, we present our method and results in parathyroid surgery with regard to surgical outcome and patients' complaints. method: endoscopic parathyroidectomy of c-siet was performed in patients with hyperparathyroidism (primary , secondary ) in new approach (mean age , male female ). single parathyroid adenoma was diagnosed using ultrasonic device, preoperatively. the patient is placed in a supine position with the neck extended. mm vertical incision is made in anterior chest. flexible endoscope (olympus co. japan) is used through mm trocar detached the retractor. in new approach, the parathyroid and thyroid are exposed through the avascular space between sternal head and clavicular head of sternocleidomastoid muscle. both of the skin and sternal head are lifted up by our original retractor (takasago medical co. japan). parathyroid adenoma behind the thyroid is resected using an ultrasonic scalpel. i would like to present our c-siet procedure. results: no scars in the neck were left in all cases. benign and hemi lateral parathyroid adenoma sized from mm to mm (mean: . mm) were operated. mean operation time is min. in new approach. there was no complication. parathyroid hormone levels decreased in all patients immediately after operation. conclusion: it is a little possible to make recurrent nerve palsy in this approach. new approach is useful to operate and make the working space wider without stress to find out of parathyroid adenoma. our original retractor can be introduced easily in most hospital, because it is not so expensive. most of women satisfied cosmetic results because of hidden scars. objectives: radiofrequency ablation (rfa) is a novel and developing technique for the treatment of parathyroid hyperplasia/adenoma in the context of secondary hyperparathyroidism (hpt) to chronic kidney disease (ckd) and there is little literature on the subject. the purpose of this study is to determine its usefulness by contributing a case carried out in our hospital. methods: we selected a case of secondary htp in a patient of years old with ckd who presented a parathyroid adenoma detected clearly by ultrasound scanning. the patient was dismissed for surgery due to high surgical risk due to his comorbidities. rfa of a right inferior parathiroid adenoma was performed. intact parathyroid hormone (ipth) was measured before arf and min after de procedure, calcium and phosphorus were measured the day after. the treatment was considered effective if ipth levels decreased at least % min after rfa and calcium levels decreased the day after. results: ipth level before rfa was pg/ml. ipth level after min of rfa was pg/ ml, this meant a % reduction (normal values - pg/ml). calcium levels were from . at the baseline to . the day after (normal values . - . mg/dl) and phosphorus from . to . mg/dl (normal values . - . mg/dl). the patient presented dysphonia as a complication that improved with corticosteroid therapy. we are currently waiting for the next analytical controls at , and months after the proceidure. conclusions: rfa of parathiroid adenomas for treating secondary hpt in patients with ckd is feasible in selected patients. this treatment may reduce the morbidity that surgery supposes, it is developed in an outpatient regime avoiding hospital admission and this contributes to a reduction of health costs. however, a longer follow-up is necessary to verify the good results in our case. splenectomy is one of the treatment strategy for advanced portal hypertension due to liver cirrhosis. after splenectomy, thrombocytopenia is dramatically ameliorated, and liver function parameters have also been improved in several clinical settings. however, the mechanism underlying such a phenomenon remains unclear. the aims of the present study was to analyze histological changes of the liver after splenectomy in human, and to speculate the underlying mechanism. subjects and methods: cirrhotic patients with hepatocellular carcinoma (hcc) who had undergone laparoscopic splenectomy prior ( weeks- months) to hepatic resection were analyzed (n = ). non-tumorous liver specimens obtained at hepatectomy were histologically investigated. liver tissues from cirrhotic hcc patients who underwent only hepatectomy were used as controls (n = ). results: after splenectomy, significant leukocytosis, especially increase in monocytes, was observed in addition to thrombocytosis. in the non-cancerous liver tissues, many round-shaped cd -positive macrophages accumulated after splenectomy, while this phenomenon was merely observed in patients without splenectomy. the macrophages were cd ? (m marker) and cd -cd ? , suggesting their anti-fibrotic population. the accumulated macrophages existed around fibrous scar as well as ck ? epcam ? cells spreading out from the ductular reactions (dr). as a result, the number of ki -positive hepatocytes significantly increased after splenectomy. the amount of platelets detected in the liver did not change even after splenectomy. finally, remarked attenuation of the established liver fibrosis was detected after relatively long duration. the accumulated macrophages expressed metalloproteinase (mmp)- and fibroblast growth factor (fgf)- , suggesting these molecules may possibly participate in resolution of established fibrosis and hepatocyte proliferation. conclusion: splenectomy in cirrhotic patients with portal hypertension ameliorate liver fibrosis, and stimulate liver regeneration. the mechanism possibly include hepatic accumulation of anti-fibrotic cd -positive macrophages and stimulation of dr-derived ck ? epcam ? progenitor-like cells. in patients with advanced splenic fibrosis, splenectomy could be a feasible therapeutic modality. the paper tries to establish the role and the opportunity of using laparoscopy in regard with abdominal contusions, as well as its indications or contraindications, combined in a therapeutic algorithm. we analyzed two groups of patients with abdominal contusions divided over two -year periods, - ( patients) and - ( patients) respectively. we have separated the two periods because starting from we have established a strategy for dealing with cases of abdominal contusions where we included diagnostic and / or therapeutic laparoscopy and nonoperative management. the investigation was done by fast echography, ct scan, simple abdominal radiography, peritoneal lavage puncture, and sometimes arteriography. in the second period we determined the diagnostic and therapeutic laparoscopy indications: suspicion of hollow or parenchymal organ injury, or mesentery injury, the presence of hemoperitoneum or fluid in the peritoneal cavity in a stable patient without major hemorrhage, apparent with unique injuries, without immediate vital risk and without other associated severe trauma. we have associated in this last period the nonoperative management for patients with grade and lesions of parenchymal organs that do not have fluid in the peritoneum, or only a very discreet quantity. in the first period, all patients were treated by classic surgery, resulting in unnecessary laparotomies where no visceral lesions were revealed. in the second period, we applied non-operative management to patients out of , patients with grade and splenic injuries, and patients with grade and hepatic lesions. diagnostic laparoscopy was performed in cases, in of them without evidence of lesions, and in other cases of grade lesions no therapeutic action was required. therapeutic laparoscopy was required for one case of splenectomy and one of hepatorrhaphy. diagnostic laparoscopy is useful in abdominal contusions, if certain indications are followed and in selected patients. in our study, with the introduction of modern therapeutic strategies, unnecessary laparotomies were completely avoided, some lesions being even treated by laparoscopy. the new algorithm introduced allowed % of patients to avoid laparotomy. aims: about cases of splenic hamartoma have been described in the literature since it was first described by rokitansky in , it is a rare benign tumor. it is usually a casual finding in laparotomies or autopsies. they are usually asymptomatic, but there are few symptomatic splenic hamartomas and they can be associated with haematological alterations, being in some cases associated with spontaneous splenic rupture and acute abdomen, two thirds of them have multiple tumors. there are no specific data that allow the preoperative diagnosis of this entity, which is performed after the anatomopathological study of the surgical specimen, which must be extracted entirely, this together with the size of the spleen makes the laparoscopic approach difficult. the aim of this video is to demonstrate the surgical technique of a complete laparoscopic approach for this type of lesions, without the need for assistance laparotomies (handport). methods: clinical case: a -year-old man admitted to internal medicine due to fever and left lumbar pain. additional explorations of interest are discussed, including: thrombopenia of probable peripheral origin secondary hypersplenism (fna of bone marrow), ct: splenomegaly with splenic masses, which deform the splenic contour, compatible with atypical hemangiomas, without being able to discard other vascular splenic tumors. results: complete semi-laparoscopic approach, trocars, multilobulated splenomegaly ( x cm.), mechanical vascular section, complete bag extraction after minilaparotomy on the left flank. the patient presented a successful postoperative recovery, being discharged on the th po day. abdominal ultrasound at st week with portal vein thrombosis, which resolves after treatment with heparin. definitive result of the specimen: multiple splenic hamartoma. asymptomatic one year after surgery. the laparoscopic approach is a valid and effective alternative to splenic benign tumor lesions. the size does not contraindicate this type of approach, although the complete extraction of the spleen is recommended for its pathologic study. we recommend eco-doppler control per week, given the risk of portal thrombosis with an existing laparoscopic post-splenectomy. objectives: splenic cysts are a rare entity, currently described between - cases in literature. a female patient's case is hereby presented, giant splenic cyst treated by conservative laparoscopic surgery obtaining good results. method: years old female, without any relevant medical history, examined after abdominal pain on the left hypochondriac region, nausea, postprandial swelling and mass sensation. after exploration the presence of such mass was ratified, the rest of exploration found no relevant findings, no record of previous traumatism nor any other relevant incidence. diagnosis was made through ultrasound and computerized tomography, the existence of a big splenic cyst is confirmed, cm by cm, on the superior section of the spleen, negative results after parasitism test, normal haemogram, coagulation and biochemistry levels. patient was intervened using laparoscopic surgery, performing the deroofing technique on the cyst (two liters of orangey amber serous liquid that was sent for analysis) as well as extirpation of superior wall of the cyst, which was sent to pathological anatomy, a saline solution was used to cleanse the cavity, omentum and drainage were then set in place. results: patient evolved satisfactorily, hospital discharge and drainage withdrawal after h. regular check-ups, after and months, patient presents no symptoms nor recurrence. pathological anatomy confirmed primary splenic cyst and the extracted liquid as cystic. conclusion: splenic cysts are primary ( %) or secondary ( %). diagnosis is performed through imagery tests, cat scan the being standard test used. regarding her treatment there is no clear consensus, due to the fact that up to a few years ago, complete splenectomy was the recommended treatment, techniques with preservation of the spleen are currently being widely recommended through laparoscopy in literature. among the conservative techniques percutaneous aspiration, with or without the injection of a sclerosing agent, partial splenectomy, marsupialisation, cystectomy, decapsulation, unroofing or fenestration can be found. the main issue is recurrence rates. few cases of primary giant splenic cysts treated by laparoscopic decapsulation can be found in literature, this treatment being simple and quick to perform, resenting a recurrence rate lower than other techniques such as aspiration and marsupialization. introduction: technology's progress and its application in the minimally invasive surgery of the thyroid gland offers us new surgical approache's like the transaxillary approach. this new technic still unusual in our environment and has recently begun to be incorporated into our surgical practice. the objective of this case is to explain step by step how to carry out a right transaxillary thyroidectomy and emphasize in the most relevant tips to take into account. also we going to review the main limitations we observed so far. statement of the case: we present the case of a -year-old woman referred for evaluation of a left thyroid nodule without associated symptomatology. the blood test shows normal thyroid profile. cervical ultrasound is performed identifying a . cm single right nodule with welldefined edges and presence of peripheral vascularization . no other nodules are identified. fna of the nodule describes a bethesda iii. after evaluation we decide to perform a left transaxillary thyroidectomy. discussion: surgical treatment of the thyroid gland by transaxillary approach may be indicated in previously selected patients, offering the advantages from minimally invasive techniques (shorter recovery time, shorter incision length, etc.). surely, more evidence and experiencie is required to make a better assessment of minimally invasive approaches in thyroid surgery. surgery, taipi city hospital, yan-ming branch., taipei, taiwan; surgery, taipei city hospital, taipei, taiwan the first endoscopic thyroidectomy was performed in using a cervical approach. since then, various remote-access method, have been developed for thyroid surgery to avoid scarring of the neck. trans axillary approach(taa),bilateral axillo-breast approach(baba),and retroauricular approach(raa) are common in use. the main benefit of these procedure is that there are no visible scar that is one of the drawbacks of conventional kocher's incision. however,these methods require more dissection and longer operation time than conventional thyroidectomy transoral thyroidectomy(tovet) is a new approach and has become popular in recent years, however,most surgeons peformed a single procedure because of the limited patients and the learning periods sine ,more than cases were performed,patients received endoscopic thyroidectomy(et) procedure at our hospital. we compare the surgical procedure of bilateral axillo-breast approach(baba) with transoral vestiblar approach(tovet) in our hospital both performed by one single surgeon .the surgen has expended eaqual amounts of time with these two procedures. the patient seletion process,operation time, operation procedure and approach,learnig experience, consmetic effect,onaologic consideration and surgical outcome were discussed yhroughly. presenting a case of a thyroid metastasis from an ovarian carcinoma, we conducted a review of the literature without finding similar reported cases. case: a -year-old woman consults for progressive asthenia, weight loss and ascites. abdominal ct finds a conglomerate in the pelvis involving the ovaries and peritoneal implants, the largest up to cm. an omental epigastric lesion biopsy and paracentesis is performed resulting in adenocarcinoma and omental metastasis from ovarian neoplasm, associated with ca of . patient starts neoadjuvant therapy with carboplatin-paclitaxel. in image controls there is a favorable response. three months later, intervention was carried out; laparotomy hysterectomy ? double anexectomy ? omentectomy ? appendectomy ? pelvic and paraaortic lymphadenectomy.the anatomopathological study shows a low-differentiated endometrioid carcinoma, omentum infiltration and absence of metastatic lymphatic involvement. while getting the maintenance treatment with bevacizumab the patient presented symptoms of arthritis and hypercalcemia was detected ( . ) with pth . a gammagraphy was performed and an increased uptake area was detected in the lower pole of rtl, suggestive of a parathyroid adenoma. we initially proposed the possibility of performing radiofrequency ablation but in a previous thyroid ultrasound we visualize nodular lesions in rtl compatible with adenoma and a mass in the superior mediastinum that seems to correspond the area of greatest uptake in the gammagraphy so finally the procedure is dismissed and surgery is proposed. during the intervention we found a hard consistency nodule in the inferior pole rtl and lymphadenopathies of hard consistency in right vi area that are sent for intraoperative anatomopathological study with the result of adenocarcinoma metastasis without identifying origin. a total thyroidectomy, parathyroidectomy and central ganglion drainage is performed with the result of a parathyroid adenoma, lymphatic invasion of ovarian-grade latent carcinoma and extensive vascular permeation by carcinoma of the thyroid. the patient maintains oncological treatment with carboplatin-caelix. in the last follow-up, the pth and calcemia remains normal. conclussion: although some cases of neoplasic thyroid involvement associated with struma ovarii have been published, no cases similar to the one described are found, neither in our experience, which is why it is an exceptional case. the aim of the study was to evaluate the effectiveness of the use of embolization of the splenic artery in order to prevent portal bleeding. methods: the study included patients, who had esophageal varices bleeding, which developed as a result of decompensated cirrhosis of the liver of various etiologies of classes b and c according to child-pugh. patients were divided into groups. the main group included ( . %) patients who underwent endoscopic ligating of bleeding varix and in order to prevent recurrence of bleedingembolization of the splenic artery with gianturco coils. the comparison group consisted of ( . %) patients who received only drug therapy. to assess the effectiveness of the treatment, the patient's condition was monitored for months. results: the average age of patients in the comparison group was . ± . years. using only drug therapy, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. the average age of patients in main group was . ± . years. performing endoscopic ligation of bleeding varices, we stopped bleeding in ( . %) patients. in all cases, at the end of treatment, we received an improvement in clinical and laboratory parameters. ( . %) patients died. the duration of treatment was . ± . days. a statistical analysis of mortality and duration of treatment revealed a significant difference (p \ . ) between the groups in both indicators. after splenic artery embolization in all cases managed to achieve a reduction in blood flow of - %. after months among patients in the comparison group, bleeding relapse occurred in ( . %) cases. in the main group, this indicator was . % ( patients). the indicator in the main group was significantly (p \ . ) different from the same indicator in the comparison group. conclusion: performing embolization of the splenic artery in patients after endoscopic hemostasis of variceal bleeding allows to reduce the pressure in the portal system, which in turn leads to a decrease in the frequency of bleeding recurrences. thoracoscopic esophagectomy for aortoesophageal fistula y. ebihara, t. shichinohe, y. kurashima, s. murakami, surgery ii, hokkaido university, sapporo, japan background: aortoesophageal fistula (aef) is an uncommon but one of highly fatal conditions. there are surgical, endoscopic and interventional radiological treatment options, however, definitive treatment is the surgical intervention. video-assisted thoracoscopic surgery (vats) has been gradually accepted as a substitution for thoracotomy to reduce the invasiveness of the surgery as radical surgery for esophageal cancer. we aimed to evaluate a feasibility of vatsesophagectomy (vats-e) for aef in this study. introduction: achalasia is the most common motility disorder of the esophagus. heller's cardiomyotomy associated with a antireflux technique is the treatment of choice in patients with this disease; however, a small group of patients could present a recurrence of the symptoms being necesary a new surgery, what is an important challenge for most of the surgeons. we report the case of recurrence after a laparoscopic miotomy and dor fundoplication as a paradigm for the appropiate management in this kind of patients. methods: a years old female, who underwent a previous miotomy and a dor fundoplication in due to an achalasia.six years after surgery, the patient showed epigastric pain and dysphagia. the study of the patient was performed with: barium swalow, phmetry, manometry, ct-scan and mri showing a recurrence of her disease.the patient was transfered to our center where she underwent a new surgery.the key points of the new surgery includes the next steps: dissection of the previous adhesions, dissection of the dor's partial fundoplication, avoid dissection of the anterior esophageal wall at the leve lof the hiatus (the area of previous myotomy) in order to avoid perforation of the esophagus, lateral and posterior dissection of the distal esophagus, lateral myotomy at the rigth wall of the esophagus and a toupet's funduplicatury. all of thisis procedures are done under intraoperative endoscopy in order to confirm a good passage to the estmach and to identify a perforationic supervision. results: following theseis steps several patients have been operated in our center with excellent results. in all of these cases, including the patiente presented previously, the symptoms have dissapeared. conclusions: achalasia is a rare motility disorder of the esophagus, being recurrences an important challenge for surgeons. a great proper therapeutic strategy using the different diagnostic exams and the supervison by a group of experts in this kind of entity are the basis in order to obtain good results in these situations. aims: re-do fundoplication is usually performed for recurrent reflux symptoms due to wrap failure or recurrent hiatus hernia. conversely, persistent dysphagia may occur early due to tight wrap/crural repair which should be avoided by good surgical technique. a small group of patients however may suffer progressive dysphagia due to weakening motility (especially in older patients), fibrosis of the wrap or a combination of the two. this video demonstrates the successful treatment of this problem with a laparoscopic conversion from nissen to posterior toupet fundoplication. a year old man underwent an uncomplicated laparoscopic nissen fundoplication in with complete resolution of reflux symptoms. he re-presented years later, still free of reflux but suffering progressive dysphagia and troublesome regurgitation. investigations demonstrated intact wrap and no mechanical obstruction, but confirmed low-amplitude peristalsis. a trial endoscopic dilatation improved symptoms for days before recurrence, suggesting likely wrap fibrosis (which would reduce elasticity and impede passage of food bolus), justifying consideration for a conversion from nissen to toupet. results: this video demonstrates the expected adhesions between fundoplication and inferior surface of left lobe liver, mobilisation and division of the nissen fundoplication, and reconstitution of a posterior toupet fundoplication. the patient made a good recovery and was discharged the following day. three-and six-month follow-up confirmed complete resolution of symptoms with no recurrence of reflux. conclusion: laparoscopic re-do surgery for late-onset progressive dysphagia is a safe and viable option. patients must be thoroughly investigated and carefully selected for an appropriately tailored procedure. they should also be advised of the increased risks associated with re-do surgery. the anatomy can be unpredictably distorted by variable adhesions and this operation should therefore only be performed by laparoscopic surgeons experienced in both primary and re-do fundoplication. methods: i report unusual iatrogenic injury of cervical esophagus that resulted with complete resection post total thyroidectomy for papillary ca of thyroid patient presented days post surgery to our center. the video will show the steps used to treat this unusual complication by neck exploration, laparoscopic trans hiatal esophagectomy with creation of gastric tube with preservation of the right gastroepiploic artery and the neck anastomosis between the cervical esophagus and stomach. were open and minimally invasive esophagectomies. of the patients, were for squamous cell carcinoma, were adenocarcinoma and were of other histological diagnosis such as gastrointestinal stromal tumor and schwannoma. the median length of stay for patients who underwent minimally invasive esophagectomies was days ( to days) while the median length of stay for patients who underwent open esophagectomies was days ( to days). the minimally invasive group had a shorter icu stay of day. for day morbidity, the minimally invasive esophagectomy group had patients who encountered anastomotic leaks, with post operative pneumonia while the open esophagectomy group had patient with anastomotic leak, patient with post operative stricture and patient with delayed gastric emptying. there were mortalities in the minimally invasive group while there were no mortalities in the open group. conclusion: our data show that patients who underwent minimally invasive esophagectomies had a shorter duration of hospitalization with similar perioperative morbidity rates. minimally invasive esophagectomy is a viable surgical option for a select group of patients. aims: there has been an increasing tendency towards minimally invasive surgery for esophageal cancer. our aim was to evaluate the results of the thoracoscopic approach (ta) and compare them with the ones of open approach (oa) at our institution. methods: retrospective review of all patients who underwent esophagectomy due to esophageal cancer (adenocarcinoma or squamous cell) between and were included. patients with siewert iii tumors and those who didn't need a thoracic approach were excluded. results: during the study period were performed esophagectomies, through ta. in . % of these, the abdominal stage was done by laparoscopy. when comparing ta versus oa, there were no statistically significant differences in the baseline characteristics of the two groups (mean age, median body mass index, ecog performance status, asa score, smoking status, diabetes mellitus, pulmonary disease, histologic type, clinical staging and neoadjuvant chemo and radiotherapy). regarding outcomes, there were no significant differences in need of intraoperative transfusion, median intraoperative blood loss, operative time and length of stay. although not significant, in ta group there was a tendency for higher overall morbidity ( . % versus . %, p = . ); major morbidity-ctcae - ( . % versus . %, p = . ); anastomotic leak ( . % versus . %, p = . ) and re-intervention rate ( . % versus %, p = . ). on the other hand, in ta group there was a tendency (although not significant) towards lower rate of respiratory complications ( . % versus . %, p = . ), lower rate of r margins ( . % versus . %, p = . ) and higher median of lymph nodes removed ( versus , p = . ). conclusions: in our series, outcomes of ta were similar to oa, with a tendency towards lower respiratory complications, lower rate of r margins and higher number of lymph nodes removed in ta group. the impact of these findings in survival remains to be seen. the tendency towards higher morbidity may be related to the learning curve, since this were the first cases performed at our center. background: esophagectomy is a surgical procedureburdened by a high morbidity rate. the effect of minimally invasive (mi) approach on elderly patients is still not clear. aim: of this study was to analyze the impact of mi approach on post-operative course according to the patient age. methods: a consecutive series of patients underwent to elective oncological esophagectomy between and . all data were entered into a prospective database. patients submitted to -flield or trans-hiatal esophagectomywere excluded andonly ivor-lewisopen, hybrid or totally minimally invasive esophagectomywere. patients were stratified according to age in groups:group a(= years) patients, group b ([ and \ years) and group c (were = years) .clinical and pathological factors influencing surgical outcome were evaluated. complications were classified according to clavien-dindo (cd). results: as expected outcomes worsened with patients age(cd = b: . % group a, % group b and % group c. p = . ), mortality ( % group a, % group b and . % group c. p = . ) and length of stay ( days group a, days group b and days group c. p = . ).a statistically significant higher incidence of anastomosticleaks was observed among patients submitted to totally mi esophagectomy in group c vs a and b that were respectively , %, % and %. major respiratory complications were not statistically different among these three sub-group. conclusions: old age has a significant impact on outcomes afteresophagectomy. in this subset of patients a mi approachcould also increasepostoperative morbidity. elderly patients should be carefully selected before to be submitted to mi esophagectomy. introduction: esophagectomy is a major surgical procedure with morbidity and mortality related to the patient's condition, stage of the disease, complementary treatments, and surgical experience. minimally invasive esophagectomy (mie) may lead to a reduction in perioperative morbidity and mortality with very good quality of life. material and method: we present the experience of the center of excellence in esophageal surgery regarding totally mie through thoracolaparoscopic modified mckeown three-stage approach followed by esophageal reconstruction by gastric intrathoracic pull-up and cervical esophagogastric anastomosis used for the treatment of thoracic esophageal cancer. results: in the last years, mie was performed initial, in our clinic with extracorporeal preparation of the gastric conduit with reduced lung complications and hospital stay. we introduced the totally minimally invasive esophagectomy with laparoscopic-assisted feeding jejunostomy using a d high definition camera. operative times were: thoracic- min, abdominal- min and cervical- min with a total of min. the augmented d high definition image provided an excellent visual field, that allowed an accurate identification of dissection plans and extensive periesophageal and perigastric lymphadenectomy. the short-term outcomes of the totally minimally invasive esophagectomy procedure were very encouraging with early feeding on jejunostomy and the control of cervical anastomosis was usually performed in the th day postoperative and the patients were discharged in the th day postoperative without any symptomatology. at the first and third-month follow-up was not reported any major complications. the long-term oncological results are being evaluated. conclusions: the totally minimally invasive approach using advanced technology of endoscopic surgery allowed for these patients a simple postoperative evolution, no major complications, and a good recovery after an extensive surgery. the solid experience in open esophageal surgery of the upper gastrointestinal surgeons provides a fast learning curve of complex minimally invasive surgical procedures with reduced perioperative morbidity. long-term follow-up should confirm the results from the literature regarding the survival, which is expected to be for these patients at least equivalent with outcomes after open esophagectomy. introduction: esophageal fistulas, benign or malignant, represent a real challenge for the surgeons and gastroenterologists, regarding the treatment and the outcome. in these cases, endoscopic treatment is the first line approach, being less invasive and sometimes avoiding the need for surgery. this includes clips, stents, glue and even suture. material and method: we have analyzed esophageal fistulas in patients with benign or malignant pathology, diagnosed and treated in the first months of . the management of this complication included a self-expandable esophageal metallic stent. we have evaluated the diagnosis, the surgical intervention, the timing until the development of the leak, the localization and management of the fistula. results: were postoperative leaks and spontaneous esophageal fistulas. the localization was cervical in one case, thoracic in cases and abdominal in cases. for the postoperative fistulas, in patients the treatment included at least one surgical reintervention with lavage and drainage, beside the insertion of an esophageal metallic stent. in the other cases, endoscopic treatment and antibiotic therapy was enough. in cases, the stent migrated needing repositioning. days mortality was %, both patients from postoperative group. conclusions: esophageal fistulas represent a severe complication, usually in patients already immunocompromised. endoscopic management, including self expandable esophageal metallic stent, can be the main approach, by stopping the contamination and by permitting the early per oral feeding. disadvantages include the possibility of migration and the need of removal after - weeks. methods: five hundreds and one patients with esophageal cancer who underwent mie from to at our department were eligible. we considered the risk factors of complications of pneumonia, anastomotic leakage, and hoarseness after surgery, and the risk factors of difficulty of surgery. results: the risk factors of postoperative complications in univariate analysis were more than years old (odds ratio: . , p = . ), more than ii in asa-ps (odds: . , p \ . ), more than g of bleeding (odds: . , p = . ), more than min. of operation time (odds: . , p \ . ), and colon reconstruction (odds: . , p = . ). the one in multivatiate analysis was more than ii in asa-ps (odds: . , p = . ). the risk factors of much bleeding were colon reconstruction (odds: . , p \ . ), and more than of lymph node dissection (odds: . , p = . ). the risk factors of long operation time without cervical lymph node dissection were neo-adjuvant therapy (odds: . , p \ . ), more than of lymph node dissection (odds: . , p = . ), and colon reconstruction (odds: . , p \ . ). the ones with cervical lymph node dissection were more than pstage iii (odds: . , p \ . ) and more than of lymph node dissection (odds: . , p = . ). conclusions: considering those risk factors, we should perform perioperative management more carefully. method: sa -year-old man with a tobacco and alcoholic habit was suspended for years, under treatment for arterial hypertension, who consults for a logical dysphagia of months of evolution. he is diagnosed of stenosing esophageal distal third epidermoid carcinoma txn m . it is decided to place a prosthesis that is effective and subsequent neoadjuvant qt-rt, after weeks of its completion the surgery is performed. results: the surgery is performed in times, initially by laparoscopy. the esophageal hiatus and the greater curvature are dissected preserving the right gastroepiploic, and lymphadenectomy of the celiac trunk with pedicle section of the left gastric. gastric plasty is performed with a section of lesser curvature towards fundus. it is continued by thoracoscopy. a section of the azygos vein is performed, dissection of the esophageal middle and lower third and lymphadenectomy. gastric plasty is promoted, proximal esophagus section and latero-lateral intrathoracic gastro-oesophageal anastomosis. the anatomopathological study reports ypt and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: ivor-lewis endoscopic surgery is safe and meets oncological criteria in selected patients with distal esophageal neoplasia and performed by an experienced esophagogastric unit. background: the rates of thoracoscopic esophagectomy performed in the prone and left lateral decubitus positions are similar in japan. we retrospectively reviewed short term outcomes of thoracoscopic esophagectomy for esophageal cancer performed in the left lateral decubitus position under artificial pneumothorax by co insufflation in a single institution. this study aimed to evaluate the feasibility of applying this procedure. methods: between july and december , patients with esophageal cancer underwent thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation. the thoracic procedure is performed as follows:the lymph nodes around the right recurrent laryngeal nerve are dissected. on the cranial side, the lymph node dissection is advanced to the level of the inferior thyroid artery. then, the assistant rotates the trachea toward the ventral side, and the lymph nodes around the left recurrent laryngeal nerve are dissected. the middle and inferior mediastinal lymph nodes are dissected including supradiaphragmatic lymph nodes and the dorsal lymph nodes around the thoracic descending aorta. then, the esophagus is transected using an automatic suture device. finally, the tracheal bifurcation area lymph nodes are dissected. we retrospectively analyzed these patients. results: the completion rate of thoracoscopic esophagectomy was . %, and the procedure was converted to thoracotomy in five patients, due to hemorrhage,severe adhesion. the mean intrathoracic operative time, intrathoracic blood loss, and number of dissected mediastinal lymph nodes were . min, . ml, and . , respectively. postoperative complications included pneumonia ( . %), anastomotic leakage ( . %), and recurrent nerve paralysis ( . %). postoperative ( d) mortality was / ( . %) due to ards and nomi, respectively. conclusions: standardization of the procedure for thoracoscopic esophagectomy in the left lateral decubitus position under artificial pneumothorax by co insufflation, with a standardized clinical pathway for perioperative care led to favorable surgical outcomes. introduction: recently thoracoscopic surgery has become widespread even in chest procedure in thoracic esophageal cancer surgery. as an advantage of minimally invasive esophagectomy, it is possible to perform sophisticated procedures due to its magnified visual effects. on the other hand, short-term perioperative safety and oncological safety are still unclear. in cases where abnormal anatomy or comorbidity in the thoracic cavity is observed, it is thought that it is necessary to carry out thoracic surgery which ensures safety while keeping in mind the transition to transthoracic surgery. here, we report on esophageal resection of the thoracic esophageal cancer accompanied by a mm saccular aneurysm inside the aortic arch. patient: a -year-old man visited a nearby doctor with a chief complaint of discomfort during swallowing. upper gastrointestinal endoscopy examined middle cervical esophageal cancer and received referral to our hospital. ct revealed a mm saccular aneurysm inside the descending aorta in contact with the thoracic esophagus. preoperative diagnosis was middle thoracic esophageal cancer; -iic ct bn m stageia (uicc th). we performed thoracoscopic esophagectomy and lymph node dissection as curative surgery. the anterior surface of the aorta was exposed from the lower mediastinum and descended ascendingly, reaching the lower end of the saccular anus at the head level of the lower pulmonary vein. peeling off the esophagus dorsal side along the margin of the saccular sac and performing esophageal resection. conclusion: we reported thoracoscopic esophageal resection for thoracic esophageal cancer with chest descending aortic saccular aneurysm. thoracoscopic surgery, which can fully exploit close magnification effect, seemed to be useful for anatomically disqualified cases. introduction: anastomotic leakage from oesophagojejunal (oj) anastomosis after total gastrectomy is associated with a high morbidity and mortality rate. leakage rates reported vary between ?% and ?% but lack of consensus in management. in the past, it often required surgical intervention or radiologically abscess drainage that will keep patients fasted with external drain for a long duration. recently, variable endoscopic options-oesophageal stents, clips, fibrin glue and endoluminal vacuum therapy had been introduced with variable outcomes. here, we presented a case of oj anastomotic leak management with combination innovative endoluminal and radiologically technique to insert double pig-tailed catheter. aim: to introduce the feasibility of double pig-tailed catheter for drainage and management of oj anastomosis leak. a year old man presented with two months history of dysphagia. upper endoscopy (ogd) showed suspicious cardio-oesophageal lesion. histology biopsy confirmed with adenocarcinoma. ct-scan of thorax, abdomen and pelvic showed irregular thickening at cardiooesophageal junction with regional lymphadenopathy. no distant metastases. he underwent uneventful d total gastrectomy. on th post-operative day, patient had spike fever and newly developed atrial fibrillation. urgent ct-thorax, abdomen and pelvis with oral omnipaque. it showed lower mediastinal gas-containing fluid adjacent to oj anastomosis within the left retrocrural space suspicious for leak. ogd evaluation showed pin-hole oj leak. guidewire inserted via endoscopy into left retrocural space under radiologically guidance. double pig-tail fr cm subsequently inserted via seldinger approach over guide wire. the proximal end of pig-tail pushed into left retrocural space and distal end positioned into efferent jejunal limb with crocodile jaw through endoscope. diluted contrast injected and passed down to efferent limb with minimal leak. outcome: after double pig-tail insertion, patient started on clear feed on st day post-insertion. one week later, he was started on full feed. repeat upper endoscopy and stent removal done two weeks later. contrast injection showed small blind ended sinus tract from anastomosis toward left pleural space without obvious leak. conclusion: radio-endoscopic is a novel minimally invasive technique that allows insertion of double pig-tailed internal drainage to control oj anastomosis leak. it allows early enteral nutritional feeding and avoid external drainage. background: the number of gastric cancer (gc) survivors, especially long-term survivors, is increasing. how best to evaluate the diseasespecific survival (dss) of gc survivors over time is unclear. we aimed to assess changes in the conditional survival of patients with gc after curative intend gastrectomy and the evolution of the impact of well-known risk factors. methods: clinicopathological data from , patients who underwent curative intend resection for gc at four specialized centres (three in china and one in italy) and from the surveillance, epidemiology, and end results (seer) database were retrospectively analysed. changes in the patients' -year conditional disease-specific survival (cs ) were analysed. we used time-dependent cox regression to analyse which variables had long-term effects on dss and devised an accurate, dynamic dss predictive model based on the length of survival. results: the median follow-up time was months, and disease-specific death occurred in , cases ( . %). the dss of the patients after surgery was dynamic, and most of the disease-specific deaths occurred within the first years after surgery. based on -, -, -, -and -year survivorships, the cs of the population increased gradually from % to . %, . %, . %, . %, and . %, respectively. subgroup analysis showed that the cs of patients who had poor prognostic factors initially demonstrated the greatest increase in postoperative survival time (e.g., n b: . %- . %, ? . % vs. n : . %- . %, ? . %). time-dependent cox regression analysis showed the following predictor variables constantly affecting dss: age, the number of examined lymph nodes, t stage, n stage and site (p all \ . , years after gastrectomy). the influence of prognostic factors on dss and cs changed dramatically over time. based on data from several large global centres, we developed an effective model for predicting the dss of gc patients based on the length of survival time. this model can provide personalized long-term follow-up strategies for patients. methods: we retrospectively analyzed clinicopathological data for rgc patients who underwent radical gastrectomy from centers. the prognosis prediction performances of the ajcc th and ajcc th tnm staging systems and the trm staging system for rgc patients were evaluated. web-based prediction models based on independent prognostic factors were developed to predict the survival of the rgc patients. external validation was performed using a cohort of chinese patients. result: the mean number of retrieved lymph nodes was . , and in . % of patients, the number was = . the predictive abilities of the ajcc th and trm staging systems were no better than those of the ajcc th staging system (c-index: ajcc th vs. ajcc th vs. trm, . vs. . vs. . ; p [ . ). within each staging system, the survival of the two adjacent stages was not well discriminated (p [ . ). multivariate analysis showed that age, tumor size, t stage and n stage were independent prognostic factors for overall survival (os), disease-specific survival (dss) and disease-specific survival (dfs). based on the above variables, we developed web-based prediction models, the huang os model, the huang dss model and the huang dfs model, which were superior to the ajcc th staging system in their discriminatory ability (cindex), predictive homogeneity (likelihood ratio chi-square), predictive accuracy (aic, bic), and model stability (time-dependent roc curves). the stratified analysis showed that regardless of whether more or fewer than lymph nodes were retrieved, the predictive performances of the web-based prediction models were still better than those of the other three staging systems. a decision curve analysis showed that the huang model provided better net benefits than the other three staging systems. external validation showed predictable accuracies of . , . and . , respectively, in predicting os, dss and dfs. conclusion: the ajcc tnm staging system and the trm staging system did not enable good distinction among the rgc patients. we have developed and validated visual web-based prediction models that are superior to these staging systems. objective: to perform competing risk analysis and evaluate cancer-and noncancer-specific mortality in patients with gastric cancer after radical surgery. methods: a total of patients from our department (as training set) and a total of patients from the surveillance, epidemiology, and end results (seer) database (as validation set) were enrolled in the study. the cumulative incidence of cancer and noncancer-specific mortality was determined by univariate and multivariate competing risk analysis. results: the five-year cancer-and noncancer-specific cumulative incidence of death (cid) in the training set were . % and . %, respectively, which were significantly lower than that in the validation set ( . % and . %, respectively). multivariable analysis showed that age, tumor site, tumor size and ptnm stage were independent predictors of gastric cancer-specific mortality and overall survival, whereas age was an independent predictor of gastric noncancer-specific mortality. noncancer-specific cid surpassed cancer-specific cid for ptnm stage i patients after approximately years of surgery, but never for stage ii and iii patients. moreover, for stage i patients, the time point when noncancer-specific cid surpassed cancer-specific cid become earlier as age increasing, with only . years after surgery for patients more than years of age. conclusions: age is an independent predictor of gastric cancer-and noncancer specific mortality and overall survival for patients after radical surgery. for patients with stage i gastric cancer, noncancer-specific mortality is a significant competing event, with an increasing impact as age increases. aim: of the study was to analyse the possibility of function preserving gastrectomy based on the sentinel lymph node (sln) concept. methods: during last years in two clinics odessa national medical university we used mapping procedures in the patients with early gastric cancer. there were men and women, age to years, mean age . ± . years. blue dye was injected into quadrants of the submucosal layer surrounding the primary lesion using an endoscopic puncture needle in patients. blue lymphatic vessels and blue-stained lymph nodes can be identified by laparoscopy within min. of the blue dye injection. we used . % indocyanine green in patients, which we injected by intraoperative endoscopy. new technology indocyanine green (icg) fluorescent imaging was used for sln mapping in this patients. results: amany patients, in which we used blue dye for mapping sln, positive sln was in patients, negative-in patients. in all patients distal gastrectomy (dg) was performed with d lymphdissection. from patients with negative sln in patients metastasis in other lymph nodes were detected.among patients in whom we used icg fluorescent mapping positive sln were detected in patients. laparoscopic-assisted distal gastrectomy with d lymph node dissection was performed in these patients. in patients with negative sln partial wedge resection was performed in patients, segmental pylorus preserving gastrectomy was performed in patients. during follow-up period from to months no recurrences or metastasis were detected in these group of patients. qol in this group of patients was much better, than in patients with conventional distal gastrectomy. conclusions: icg fluorescent method is highly effective for detection of sln. in the patients with early gastric cancer function preserving gastrectomy based on sln navigation may be promising strategy to achieve better results. laparoscopic procedure taking advantage of robotic gastrectomy for gastric cancer to prevent pancreatic fistula gastrointestinal surgery and surgical oncology, ehime university, toon-city, japan backgrounds and aims: analysis of japanese national clinical database (ncd) showed that laparoscopic gastrectomy(lg) had rather increased pancreatic fistula (pf) compared with open gastrectomy. on the other hand, last year, multicenter collaborative research result of robotic gastric cancer surgery(rg)was shown that the complications including pf were significantly decreased as compared with lg. in this study, we have employed a new easy to use device in lg to minimize pf during suprapancreatic lymph nodes dissection requiring pancreatic retraction and compared with conventional lg and rg. materials and methods: internal organ retractor (aesculapÒ) to grasp the gastropancreatic fold and the suprapancreatic peritoneum to imitate davinci's forceps was guided with a thread outside the body. patients(jan. * nov. ) were divided into three groups as follows, group lg- (n = ), lg using the standard devices, group lg- (n = ), lg using organ retractor, group rg (n = ). amylase value in drain(d-amylase) and the volume in drainage, intraoperative bleeding, postoperative hospital stay, incidence of cd (] grade iii) were compared among three groups. results: data are indicated as lg- /lg- /rg(mean ± sd), respectively. on the day and third day after surgery, d-amylase were ± / ± / ± and ± / ± , ± (iu/l). d-amylase was significantly lower in lg- and rg group than in lg- the day after surgery. the operation time was significantly longer in rg, ± / ± / ± (min). bleeding volume and hospital stay did not differ among groups. pancreatic fistula (cd ] grade iii)was observed only in lg- group at (%) . discussion: pf(grade]cdiii), which may lead to mortality, occurred in lg- group. a significant elevation of d-amylase on the st postoperative day was prevented in lg- just like rg, which seemed to lead to prevent pf afterwards. the multijoint forceps is known to be an advantage of rg but it cannot be reproduced by lg using a linear forceps. however, another advantage such as vertical grasping and lifting of the gastropancreatic fold at rest could be mimicked by lg using this device, which seemed to enable a safe lymph node dissection and lead to reduce the pancreatic damage. conclusion: this inexpensive and easy to use method taking the advantage of rg seems to reduce surgeon's fatigue and tissue damage(pf). the study presents comparison of perioperative outcome between different surgical approaches for gastric adenocarcinoma (ac). methods: retrospective cohort of patients that underwent gastrectomy for (ac) at rambam hospital during - . patients data was collected based on demographic characteristics, bmi, operating room time (ort), number of lymph nodes (ln), length of hospitalization (loh), and perioperative complications. results: study population included patients after total gastrectomies, of them robotic and partial gastrectomies, of them robotic. age, gender and bmi were similar between patients who underwent any type of procedures. median length of hospitalization (loh) for robotic total gastrectomy was . days and it was significantly shorter than both laparoscopic total gastrectomy (ltg) . days (p = . ) and open total gastrectomy (otg) . days (p \ . ). similar significant differences in (loh) between the groups were observed among patients who underwent partial gastrectomy, but the comparison between robotic and laparoscopic procedures was limited due to small numbers of (lpg). median(ort) was significantly longer among robotic gastrectomies compared to open, the difference was min in total gastrectomy group and min in partial gastrectomy group (p \ . for both differences), but the difference in(ort) between laparoscopic and robotic procedures were smaller and non-significant. the number of dissected (ln) was similar between the procedures in total gasrectomies. in partial gastrectomies, the number of dissected (ln) was even higher among both laparoscopic and robotic gastrectomies compared to open (p \ . ).) conclusions: robotic total and partial gastrectomies for gastric (ac) are associated with oncologically adequate lymphadenectomy and faster patient recovery, but longer ort. objectives: during esophagojejunostomy using a circular stapler after latg, placement of the anvil head via the transabdominal approach proved difficult. the authors report on a method modified for laparoscopy-assisted, esophagojejunostomy performed by placing the pretilted anvil head(orvil) via the transoral approach. methods: between january and november , esophagojejunostomy was performed using orvil in patients after latg. the anesthesiologist introduced the anvil while observing its passage through the pharynx. during the anastomosis, we kept the jejunum fixed in position with a silicone band lig-a-loops, thereby preventing the intestine from slipping off the shaft of the stapler. results: esophagojejunostomy using the orvil was achieved successfully in all patients. no other complications, such as hypopharyngeal perforation and/or esophageal mucosal injury, occurred during passage. the postoperative complications of anastomosis were leakage in two patients and stenosis in patients, in whom mild relief was achieved using a bougie. conclusions: esophagojejunostomy using the orvil is a simple and safe technique. gastrointestinal tract surgery, fukushima medical university, fukushima-shi, japan; surgery, ohara general hospital, fukushima-shi, japan background: juvenile polyposis of the stomach is a very rare disease, and its malignant potential has been reported previously and total gastrectomy has been recommended as a standard treatment. recently, the usefulness of laparoscopic surgery for this case has been reported, however this type of surgery is thought that maintaining the surgical space is difficult because of distended and thickening stomach. case presentation: eight years ago, a -year-old woman who had no family history of gastrointestinal polyposis had been diagnosed with gastric polyposis and polyp-related anemia and received twice endoscopic submucosal dissection to early gastric cancer in another hospital. she had received an annual upper gastrointestinal endoscopy and she had taken iron supplements for anemia caused from the occasional bleeding from the polyps. however, the number of the polyps had increased over time. because she had a loss of appetite, she admitted to our hospital. enhanced computed tomography showed gastric wall thickening and multiple gastric polyps without lymphadenopathy or distant metastasis. colonoscopy showed no specific findings. she was diagnosed as the juvenile polyposis of the stomach, and she received laparoscopic total gastrectomy with roux-en y esophagojejunostomy. in operative findings, although there were the excessive distention and congestion of the stomach, standard laparoscopic surgery could be performed. the resected specimen revealed multiple variously sized polyps throughout the stomach except for lesser curvature and fundus and the histopathological examination revealed that all polyps were hyperplastic polyps without containing cancer. she was discharged on postoperative day . we successfully performed laparoscopic surgery to treat a rare case of juvenile gastric polyposis. introduction: we report a novel technique for combined use of laparo and thoracoscopy for faradvanced adenocarcinoma of esophagogastric junction (aeg). case presentation: a 's years old man presented with far-advanced aeg. an esophagogastroduodenoscopy revealed a type lesion with the entire circumference around esophagogastric junction (egj). contrast radiography revealed a severe stenosis in the egj and wall irregularity from egj to cardia. computed tomography revealed a stenosis of egj, suspected invasion into the left side diaphragm and some lymph nodes metastases at the abdomen. we diagnosed siewert type ii aeg (ct an m , cstage iiia : japanese classification of gastric carcinoma ver. ). surgical technique :the patient was placed in the reverse-trendelenburg position with the left upper body lifted and legs spread, under general anesthesia. the tumor was huge, exposed from the serous membrane and invaded the left crus. first we performed from laparoscopic proximal gastrectomy using five ports. then, three ports were added in the th, th, and th intercostal spaces with the patient in the same body position, and performed thoracoscopic lower esophagectomy under artificial pneumothorax with intrathoracic pressure of - mmhg, which allows the ventilation of both lungs. the lower esophagus was resected under the thoracoscopic view to ensure an adequate margin. following this resection, intrathoracic esophagojejunostomy was performed by using the laparo-and thoracoscopic techniques. the operative time was min, and the blood loss was g. he was discharged on the th day after the operation without any postoperative morbidity. the histopathological diagnosis was pt bn am , p , pstage iv. after adjuvant chemotherapy with capecitabine and oxaliplatin, ramcilumab monotherapy is undertaken now. ct revealed solitary lung metastasis in months after the operation. conclusion: malta for locally advanced aeg invading the surroundings could be performed safely. introduction: despite being the pioneer in laparoscopic surgery, europe did not have similar surgical experience compared to east asia due to decreased exposure to gastric cancer. several studies on minimally invasive gastrectomy for gastric cancer have been conducted in europe. however, some of them did not analyse total gastrectomy as a distinct entity combining both distal and total gastrectomies; moreover, most of them do not provide data on full five-year follow up for each patient. baltic countries stand in between east and west in terms of gastric cancer incidence: incidence rate per , is . in united kingdom, . in lithuania and . in japan. this exposure to gastric cancer provides unique opportunity to investigate the role of laparoscopic gastrectomy. therefore, a case-control study was designed to evaluate laparoscopic (ltg) versus open total gastrectomy (otg), comparing short-term surgical and long-term oncologic outcomes. surgery, jeju national university, school of medicine, jeju, korea; surgery, chosun university, school of medicine, gwangju, korea objective: although mcv (mean corpuscular volume) levels are known to be associated with the prognosis of various diseases, few study investigated mcv as prognostic factor after gastric cancer surgery. the aim of this study is to address the prognostic value of mcv in gastric cancer who underwent curative gastric cancer surgery. methods: patients (june -december ) with stage i, ii, and iii cancer were consecutively included in this study. all patients underwent curative gastric cancer surgery including subtotal gastrectomy or total gastrectomy. overall survival (os), disease-free survival (dfs) and postoperative complications rate were compared between mcv [ group and = group. results: of all patients, the mean mcv was fl (normal range, to fl). the dfs was significantly higher in the high-mcv ([ ) than low-mcv group(= ) (p \ . ) group. there was no significant difference in postoperative complications when compared with clavien-dindo scale. the survival rate of the high mcv group was higher but there was no significant difference. conclusions: mcv may be a predictive factor after gastric cancer surgery. unlike previous studies, patients with low mcv group showed lower dfs. more research is needed on the significance of mcv in variety of disease. methods: and materials. for years we observed cases with gist of stomach and duodenum. seven patients were brought to clinic with the bleeding and two patients were brought to clinic with vomiting and compensate stenosis. in all circumstances we done the ct, mrt and endoscopic examinations of stomach and duodenum with biopsy . in two circumstances we performed endoscopic operation. in one circumstance we successfully take off the gist from the duodenum endoscopically. during the operation we use the endoscopic instruments. in another circumstances,after endoscopic excision the tumor appear the bleeding which was stopped by endoscopic local heamostasis, by putting clipps on the vessels. in circumstances the tumors were in stomach. in circumstances we performed laparoscopic wedge resection the tumors by staplers. in circumstances when the tumor was very big and situated in the fundus of stomach, we performed laparoscopic resection of the fundal part of stomach by using laparoscopic staplers and 'liga sure' sealing. in circumstance we took off the tumor by putting laparoscopic trocars inside the stomach for instruments and for visualization tumor. after excision the tumor and took it of the stomach we sutured the holes in the stomach. we have no mortality after laparoscopic operation. there were no malignisation in all circumstances. we have cases morbidity. in circumstance the bleeding from the stomach that was stopped endoscopically. in circumstance there was wound infection. the aim of the study to decrease the morbidity in the patients with perforated ulcers of the stomach and duodenum. we observed patients with perforated ulcers of stomach and duodenum. women were , men were . average age about years. patients had perforation ulcer of stomach and duodenum. patients had perforations with bleeding. all patients were divided in two groups. the first groups patient operated laporocopically, in the second group patients operated traditionally. results: there were no mortality in the group that operated laparoscopically. in the group that were operated traditionally one patient died after rebleeding. the average stay in hospital in the group that were operated laporoscopically about days. in the groups with traditional operations, were about days. the morbidity in the first group were in cases. pneumonia in cases, suppuration of the troacar points were in cases. in the second group pneumonia were in cases, suppuration of the operation wound were in cases, subdiaphragmatic abscess was in cases. conclusion: laporoscopic operation in during treatment decrease the mortality, morbidity and hospital staying in the patients the perforated ulcer of stomach and duodenum . of the patients of the third group ( . %) were operated about ulcer rebleeding in the hospital, and ( . %)-about the profuse bleeding ulcer. noonr patient had recurrent bleeding. the average treatment time for patients in group was . ± . days. conclusions: the development of hemorrhagic shock in patients with peptic ulcer bleeding significantly increases the risk of rebleeding and mortality. the application of endoscopic hemostasis allows to reduce the risk of rebleeding and mortality compared with conservative antiulcer therapy. surgical treatment can achieve reliable hemostasis, but accompanied by higher mortality and longer duration of hospital treatment. tan tock seng hospital is second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. minimally invasive surgery for both benign and malignant diseases of upper gastrointestinal tract becomes more and more popular nowadays. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them with better understanding of the procedures. the viewing of videos help them with the importance of steps, standardization of steps. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start doing the procedure themselves after the graduation from the residency programme. this is the step by step instructional video of laparoscopic repair of perforated duodena ulcer for surgeons-in-training rotated to our department. in general duplication cysts are rare developmental congenital disorders of the gi tract. three morphological criteria should be met in order to confirm the pathological diagnosis: . they should be attached to the stomach's wall and should be the continuation of it, . at least one of the muscle layers of the stomach's wall should be included and .it should have normal gastric mucosa. the treatment is either enucleation or partial gastrectomy. aim: present our minimally invasive approach to a rare prepyloric submucosal cystic lesion causing gastric outlet obstruction. case report: a -year-old female with vomiting, weight loss and in bad general condition was diagnosed after a full work-up (blood tests, endoscopies, eus, ct and mri) with a submucosal cystic tumor. this cyst first was thought to be a duplication cyst. since the patient was young, our intention was to offer the least invasive surgical technique in order to spare gastrectomy and billroth anastomosis. results: the procedure was completed laparoscopically with enucleation of the cyst through a gastrotomy on the anterior wall of the stomach. after the enucleation of the cyst the gastric mucosa was sutured back and then the gastrotomy was closed with continuous sutures. the result of the pathological report confirmed a rare case of a heterotopic pancreatic cystic lesion. the postoperative course of the patient was uneventful and was discharged with instruction for her diet the th postoperative day. the patient months post-operative has no symptoms. conclusion: in such benign conditions and especially in young patients, gastrectomies could be avoided if possible and give their place to less invasive approaches in order to reduce lifelong risks and morbidity. trangastric enucleation of the cyst although a demanding approach is safe and could be considered as a 'gentler' technique with reduced morbidity. background: pancreatoduodenectomy is considered to be very invasive for early superficial duodenal tumors (sdts), which have a lower risk of lymph node metastasis. partial duodenal resection with endoscopic submucosal dissection for sdts is an attractive technique but it is associated with a high risk of complications. the full-thickness resection of the duodenum wall including laparoscopic and endoscopic cooperative surgery has risk of spreading tumor cells and digestive juices into the abdominal cavity. we have developed novel technique for sdts to decrease the risk of exposure to abdominal cavity of tumor cells and digestive juices, called nonexposed duodenum laparoscopic and endoscopic cooperative surgery (neo-dlecs). aim: the aim of this study is to evaluate the feasibility and safety of neo-dlecs for sdts. surgical procedure: the attachment of the transverse mesocolon was freed from the head of the pancreas and retroperitoneal tissues under laparoscopy. the duodenum and the head of the pancreas were mobilized from the retroperitoneum using the kocher maneuver. a standard esd was performed for the sdt using endoscope. the serosa of the esd ulcer bed was reinforced using the laparoscopic hand-sewn suturing technique in the seromuscular layer around the resected area. after completing the procedure, the endoscope was inserted and passed over the resected area to confirm that there was no stenosis or leakage. methods: ten consecutive patients with sdt underwent neo-dlecs in our institute between march and march . the clinicopathological features of the patients and surgical outcomes were prospectively collected and retrospectively analyzed. results: pathological diagnosis was adenocarcinoma for six patients, adenoma for three patients, and neuroendocrine tumor grade for one patient. the median tumor size was ( - ) mm. the median operative time was . ( - ) min. the median blood loss was ( - ) g. there were no conversions to open surgery in this series. intraoperative perforation was found in two cases during the esd procedure. however, all perforations were closed and reinforced using hand-sewn sutures. no postoperative complications were above grade in the clavien-dindo classification system. conclusions: neo-dlecs is safe and feasible and can be an option for surgical sdt resection. aims: wilkie's syndrome is caused by the entrapment of the rd part of the duodenum between the aorta and the superior mesenteric artery (sma). surgery is indicated for chronic cases and failure of conservative management, being reported a laparoscopic duodenojejunostomy as a minimally invasive option. methods: all cases treated by laparoscopic duodenojejunostomy in our centre because of chronic wilkie's syndrome were recorded. results: females and male underwent a laparoscopic duodenojejunostomy, with a mean age of years (range - ). all patients presented abdominal pain, and weight loss was identified in most of them. a reduced aortomesenteric angle measured by ct scan was the key for the diagnosis (mean angle . degrees, range - ). conventional laparoscopic approach was performed in two patients, the other two patients underwent a sils port approach. mean time of surgery was . min (range - ) and length of stay was days (range - ). after a mean follow-up of . months (range - ), patients improved their symptoms. conclusions: surgery is the mainstay in complicated or refractory cases of sma. laparoscopic duodenojejunostomy has the advantages of the laparoscopic approach (including rapid recovery time, reduced post-operative pain and shorter hospital stay) and it is feasible, safe and effective. in mexico in , gastric cancer represented the rd cause of death; it may manifest in a variety of histologic, anatomic, and genetic patterns, which influences the surgical approach. until now gastrectomy with curative intent is the only treatment that offers potential cure in gastric cancer. in recent years, laparoscopy has emerged as an important modality in the surgical management. in multiple trials no significant difference in recurrence, long-term survival and disease-free survival was observed when compared to the standard open gastrectomy. we present the case of a year old man. with a smoking history of pack years, suspended years earlier. he presented unspecific upper gastrointestinal symptoms; an upper endoscopy was made observing a suspicious depressed lesion of cm located in the greater curvature between the body and the antrum, the biopsy resulted in a poorly diferentiated signet-ring cell carcinoma of the stomach. an endoscopic ultrasound and a thoracoabdominal ct scan showed no evidence of enlarged adenopaties or metastatic disease. initially a diagnostic laparoscopy was made, there was no evidence of carcinomatosis, nor free intraperitoneal fluid; so the greater omentum was dissected towards the splenic and hepatic flexure; a d lymph node dissection was performed, and a subtotal gastrectomy with reconstruction of roux en y was done; intraoperative endoscopy was done to identify the lesion, so adequate margins could be obtained. the patient had a good post operative evolution and was discharged home at th day tolerating oral intake. minimally invasive techniques have proved equivalency of oncologic results when compared to the conventional approach; these techniques are becoming the preferred approach in the treatment of well-selected patients with gastric cancer and have a role in definitive staging, curative resection, and lymphadenectomy. appropriate selection of patients and optimal technical approach are paramount for good outcomes. most data of laparoscopic gastrectomy come from eastern countries, where the prevalence is higher; however western experience is growing along with evolution and development in surgical instruments and new technology. wilkie syndrome is a rare cause of high intestinal obstruction, resulting from the compression of the duodenum between the abdominal aorta and the superior mesenteric artery. the main symptoms are nausea and vomiting, weight loss, early satiety, abdominal distension and epigastric pain. historically, the barium study and arteriography were the diagnostic tests used; more recently the angiotac has shown greater sensitivity. the diagnostic criteria are: dilated duodenum, duodenal compression by the superior mesenteric artery and aortomesenteric angle less than degrees. patients with an acute condition usually respond to conservative treatment (decompression, correction of hydroelectrolyte alterations, nutritional support…). however, those with chronic symptoms usually require surgery preferably with laparoscopic approaches of duodenojejunostomy or the strong's procedure. the strong procedure mobilizes the duodenum by dividing the ligament of treitz. once the duodenal-jejunal junction is mobilized, the duodenum is positioned to the right of the superior mesenteric artery and it is preferred because it provides less morbidity due of the maintaining of the integrity of the gastrointestinal tract, but it has a failure rate of %. gastrojejunostomy allows gastric decompression, but does not relieve duodenal compression, so digestive symptoms may persist, leading to the appearance of a blind loop syndrome or recurrent peptic ulcers. on the other hand, the duodenojejunostomy, which according to some series may be the procedure of choice, may obtain a success rate higher than %. we advocate to initiate the surgical approach with the strong procedure and if it fails to perform to a duodenojejunostomy. during this procedure, gastro-esophageal reflux was evaluated and assigned to severe, moderate and slight category. if the reflux was observed slightly up to cervical esophagus, the case was assigned to moderate category. if the reflux was observed intensely up to cervical esophagus, the position was returned to head high position for the safety and the case was assigned to severe category. the anti-reflux surgery was considered in the moderate and severe categories. results: we have performed laparoscopic nissen procedure in cases. the outcome was assessed by reflux test performed on - postoperative day, and the results showed the reflux was disappeared in every cases. median follow-up period of this study was months ( - months) . in cases ( . %) ppi was restarted before months after the anti-reflux surgery. in cases ( . %) ppi was restarted after the anti-reflux surgery during the whole follow-up period of this study. the bmi of the patients had no relationship to the needed restart of ppi. to evaluate the degree of esophagitis objectively before and after the anti-reflux surgery we designed 'the esophagitis score'. in this scoring method, a number from - was assigned according to the degree of esophagitis along with the la classification. the results of the study have shown that the reflux esophagitis was improved obviously after the anti-reflux surgery even in the ppi restarted group (p \ . ). discussion: to extract the gerd patients who really need anti-reflux surgery is important. reflux test is feasible because of its convenience and visual effects for the patients. the results of the laparoscopic nissen fundoplication were good. background: laparoscopic paraesophageal hernia repair with fundoplication has become more and more popular nowadays due to less morbdity and mortality with shorter length of hospital stay. discussion: tan tock seng hospital is the second largest hospital in singapore. it is affiliated to two medical schools in singapore and it is a training hospital for both undergraduates and postgraduates. in our department, all the residents have to view the step by step instructional videos of mininally invasive surgeries before they can assist in the cases or perform on their own under the supervision of consultant surgeons. the viewing of the instructional videos help them understand the procedures better. the videos can also help them recognize the important steps and standardized safe approach. with the help of instructional video, they can not only assist better in the surgery but also reduce the learning curve when they start performing the procedure themselves during their training period. this is the step by step instructional video of laparoscopic paraesophageal hernia repair with fundoplication for surgeons-in-training who are posted to our department. conclusion: the step by step instructional video on laparoscopic paraesophageal hernia repair with fundoplication can help the surgeons in training reduce their learning curve and improve their surgical skills so that they can perform the procedure safely. the human immunodeficiency virus (hiv) is a neurotropic virus. there have been reports of patients with hiv who have esophageal motility problems, sometimes associated with opportunistic infections. the absence of contractility is defined as a major motility disorder according to the chicago v . classification, which is characterized by normal esophagogastric union relaxation and % peristalsis failure. we present the case of a -year-old male patient with a history of acquired immunodeficiency on treatment with efavirenz, emtricitabine and tenofovir. he presented progressive dysphagia, gastroesophageal reflux and pyrosis of months of evolution. physical examination showed no alterations. upper endoscopy is done reporting a normal esophagus and diffuse chronic gastritis. the esophagogram reported inadequate esophageal motility with contrast stasis and a delayed emptying. the esophageal manometry reported an upper esophageal sphincter with high resting pressure. the middle and distal esophagus showed absence of peristalsis with a pan-esophageal pressurization pattern. the lower esophageal sphincter presented normal resting pressure and borderline relaxation ( %). the integrated relaxation pressure was less than mmhg. the diagnostic impression was absence of contractility (chicago classification v . ).medical management was initiated with inhibitors of the proton pump, isosorbide dinitrate and injections of botulinum toxin without success. it was decided to program the patient for a heller myotomy with toupet fundoplication. a trans-surgical endoscopy revealed a complete myotomy with no leakage or obstruction. the patient went home on the second postoperative day tolerating a solid diet.heller myotomy by laparoscopy with partial fundoplication is safe in the treatment of patients with hiv and esophageal motility disorders, reporting a mortality of . %. the effect of endoscopic treatments prior to surgery is controversy aims: epiphrenic diverticulum represents an infrequent entity and it is usually associated with esophageal motility disorders, such as achalasia, distal esophageal spasm, nutcracker esophagus or hypertensive lower esophageal sphincter. nowadays, epiphrenic diverticulectomy, esophageal myotomy and partial fundoplication is the gold standard technique; although it supposes a challenging procedure and it may provoke lots of complications. approach for diverticulectomy usually depends on the distance from the upper border of the diverticulum's neck to gastroesophageal junction, considering that thoracoscopy should be carried out when this distance is more than cm. methods: we presentthecase of a -year-old male patient, with a bodymass index of anda medical history of diabetes, smoking and alcoholism. his symptoms were mainly regurgitation and dysphagia. upper endoscopy showed esophageal dilatation and the presence of a diverticulum with its neck cm over the gastroesophageal junction. ct scan confirmed these findings and manometry showed achalasia. in the video we can see how we perform a laparoscopic diverticulectomy with esophageal myotomy and dor fundoplication. results: patient was discharged home on the second postoperative day with no complication. after more than two years of follow-up, he has not suffered regurgitation, heartburn, dysphagia or chest pain. conclusions: we present a case with an epiphrenic diverticulum secondary to achalasia in which we performed a laparoscopic diverticulectomy, esophageal myotomy and dor fundoplication. some authors suggest that the correction of the underlying motility disorder is the key in the management of these patients and they do not recommend concomitant diverticulectomy for all cases. however, we consider that the complete procedure, adding diverticulectomy, supposes the gold standard and it is feasible to perform for teams which are skilled in esophageal and gastric laparoscopic surgery, despite its high morbidity rates. purpose: a laparoscopic wedge resection for a gastric submucosal tumor closed to gastroesophageal junction or involved to gastroesophageal junction is technically challenging and more aggressive compared with tumors in other sites of the stomach. a gastroesophageal reflux disease would be more prevalent after laparoscopic wedge resection of a gastric submucosal tumor in gastroesophageal junction because of the destruction to low esophageal sphincter. we hypothesized that a prophylactic anti-reflux surgery after this surgery would be less prevalent the gastroesophageal reflux disease (gerd) and more improve the quality of life of the patients. the aim of this study is to analyze our experience with prophylactic anti-reflux surgery after laparoscopic wedge resection for a gastric submucosal tumor of gastroesophageal junction materials and methods: we retrospectively collected data from patients who diagnosed with submucosal tumor of near the gastroesophageal junction underwent laparoscopic wedge resection between january and december . the patients were divided into groups according to operation with prophylactic anti-reflux surgery (group a) and without one (group b). results: there were no difference in the frequency of the preoperative gerd symptoms between the groups, whereas postoperative gerd symptoms and postoperative use of acid suppressive medications were more frequent in the group b (p = . , p = . ). however, there were no differences in the follow-up endoscopic findings in terms of reflux esophagitis and hill's grade between the groups. in group a, postoperative mean low esophageal sphincter (les) pressure was . ± . . the les pressure was dropped until mmhg in the only one patient. however, there was no reflux symptom in this patient. conclusions: the prophylactic anti-reflux surgery after laparoscopic gastric wedge resection of gastroesophageal junction is an effective method of prevent gastroesophageal reflux symptoms. background: the most critical obstacle is a pancreatic leakage(pl). the most cause of pl might be an activation of pancreatic juice by the mixing of pancreatic juice and intestinal fluid because of the anastomosis technique, the difference of anastomosis between pancreatic duct and caliber of jejunum, and the topple of jejunal mucosa. aim: in this study, we devised the new anastomotic method of pancreato-jejunostomy, so called ' pancreatic stent sliding guide' (pssg) method using a pancreatic duct stent. we would like to demonstrate its method and results. (operative procedure) the cases of hybrid laparoscopic pancreatico-duodenectomies (pd) were done by shuriken-shaped umbilicoplasty with pssg. the pancreatic duct stent, which is fit for a diameter of pancreatic duct, is used for the direct puncture without any incineration. the aims of direct puncture are both the avoidance of the enlargement of anastomotic opening and disturbance of blood flow. the contralateral of anastomotic opening is also punctured and the stent is pulled out of the jejunum. the - pds with the needles at both ends is used for anastomotic thread. firstly, the eversion anastomosis of posterior wall is done by sliding the needle on the stent. and then the anastomosis of anterior wall is done by the same way. the stent of contralateral side is cut and the hole is closed. materials and methods: the cased of pancreato-jejunostomy by pssg method were done by february . the average of patient's age was y.o. the disease of patients were pancreatic cancer (n = ), bile duct cancer (n = ), and papilla vater cancer(n = ). the pancreatic leakage by the isgpf were grade : ,a: ,b: ,c: respectively. in the same periods, we underwent the more ten cases of open pd by pssg method. the pl were only one case of grade a and there were none of clinical pl. conclusion: our new device of pancreato-jejunostomy by pssg might be very effective for the decrease of pl from the view point of machanisms of pl even for laparoscopic pd. year old, male patient presented with upper abdominal discomfort and pain, without nausea, vomiting or weight loss. an sub mucosal lesion was found on endoscopy examination in first part of the duodenum. endoscopic ultrasound has showed . cm sub mucosal lesion in first part of duodenum (anterior wall and close to pylorus). cytology examination from the lesion has showed neuroendocrine tumor. computed tomography of abdomen and chest were normal. his blood laboratory examinations were within normal limits. patient underwent da vinci robotic partial gastrectomy with intra corporeal billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology report confirmed diagnosis of carcinoid tumor with ki less than %. surg endosc ( ) :s -s p -robotics & new techniques-education integrated education for colorectal disease-a digital solution for a digital age united kingdom aims: surgical plume has problem in poor visibility of the operative field, inclusion of harmful chemical substances, and biological risk. it is desirable that plume should be removed appropriately to minimize these risks. we assessed whether these problems can be solved by using commercialized evacuator semi-quantification of residual chemicals in the abdominal cavity: was performed using industrial smoke tester by aspirating the intra-abdominal plume onto filter papers and digitizing the stains. ( ) detection of dna in the exhausted gas from the evacuator: the hepa filter, which was interposed at the inlet or outlet of the evacuator, was analyzed using pcr method to detect any dna derived from porcine tissues. results: ( ) laparoscopic visualization: judgement score were . vs. . for ec and . vs. . for us (evacuator: on vs. off, both p \ . ), indicating the visualization was significantly better in the use of the evacuator on both devices general surgery, royo villanova hospital general surgery minimally invasive surgery centre, jesús usón minimally invasive surgery centre methods: i report my experience at the american university of beirut medical center for laparoscopic adrenalectomy cases, left adrenalectomy and cases for right adrenalectomy. three out of the series are large adrenal of cm, and all of these were completed laparoscopically.the video will show the steps of this procedure.a large rt. adrenal mass measuring cm, wt. gm was removed laparoscopically using trocar techniques. the lateral position facilitated the exposure and ease of dissection. the mass was removed by extending one of the trocar site with muscle splitting using endocatch mm. results: patient was discharged home days after surgery. the operative time was hour. pathology revealed carcinoma with no involvement of the capsule or vascular invasion patients (male: n = ; female: n = ) underwent minimally invasive adrenalectomy (tp: n = ; rp: n = ) at our institute. mean patient age was . years ( - years). besides comparing operative (intraoperative blood loss, previous abdominal surgeries, conversion rate, operative time, tumor size) and perioperative factors (time of hospitalization, time to oral intake, histology, postoperative complications) in each group, perioperative outcomes of a learning curve (lc)-the first procedures in both groups-was also analyzed in terms of tumor size, significantly larger lesions were removed with tp (tp: . ± . mm vs rp: . ± . mm; p = . ). the number of asa (american society of anesthesiologists) ii patients were significantly higher in the tp group while there were significantly more asa iii patients in the rp group conversions ) showed no significant difference. the analysis of lc showed a significant difference in previous abdominal surgeries min vs rp: . ± . min; p = . ] all favoring the tp approach. conclusion: both methods proved to be feasible and safe in terms of minimally invasive adrenalectomy. based on our own experience the tp approach resulted in improved operative time and conversion rates to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the face of a massive splenomegaly or neoplastic pathology. material and method: clinical case: a -year-old man followed in the dept. of internal medicine for a hepatosplenomegaly of probable lymphoproliferative origin. additional explorations of interest are provided. result: intervention: complete laparoscopic approach, right lateral partial decubitus, massive splenomegaly, ? cm, splenuncle of - cm that is resected, section of short vessels, dissection of the splenic hilum, vascular section with endogias, splenectomy with full extraction in a pocket through reduced laparotomy in the left flank for anatomopathological study the aim of this video is to demonstrate the safety and efficacy of the laparoscopic approach in the treatment of large splenomegaly. currently, this approach is recognized as the one of choice in benign splenic pathology, being controversial in the case of a massive splenomegaly or neoplastic pathology it can transform into adenocarcinoma. patients and methods: between and we performed laparoscopic nissen fundoplication (lars) in cases of gerd. in cases of gerd patients be was proved by endoscopy and histological examination. the demeester score was higher ( . versus . , p \ . ), and bile re?ux was measured more frequently among the be patients on the other hand during the . years long endoscopic follow up early barrett carcinoma developed in patients, . months after the lars. both patients underwent a limited surgical resection of the distal esophagus and esophagogastric junction, regional lymphadenectomy, and reconstruction by interposition of an isoperistaltic jejunal segment. there were no complication. histological examination was shown pt n stage disease in both cases. oncological follow up was months long ( . y) and both patients are still disease free. conclusions: although lars can affect regression in a part of be patients, progression to adenenocarcinoma can also occur. endoscopic surveillance is important in the case of be to recognize early cancer, to perform limited surgical resection with low morbidity and long overall-and disease free survival gastric cancer development a nomogram for predicting the conditional probability of survival after d lymphadenectomy for gastric cancer this study aimed to devise a nomogram to predict the conditional probability of cancer-specific survival (cpcs) in gastric cancer (gc) patients after gastrectomy with d lymphadenectomy. methods: clinicopathological data for , gc patients who underwent d lymphadenectomy in a large-volume eastern institution (the training cohort) were analysed. cancer-specific survival (css) was predicted using cox regression models. a conditional survival nomogram was constructed to predict cpcs at and years post-gastrectomy. two external validations were performed using a cohort of , chinese patients and a cohort of italian patients. results: in the training cohort, the -year cpcs was . % immediately post-gastrectomy and increased to . %, . %, . % and . % at , , and years post-gastrectomy, respectively. multivariate cox regression analyses showed that age; tumour site, size and invasion depth; numbers of examined and metastatic lymph nodes; and surgical margins were independent prognostic factors of cancer-specific survival (all p \ . ) and formed the nomogram predictor variables. internal validation showed that the conditional nomogram exhibited good discrimination ability at and years post-gastrectomy (concordance index, . and . , respectively) gastric cancer does non-compliance in lymph node dissection affect oncological efficacy in gastric cancer patients undergoing radical gastrectomy? univariate and multivariate analyses revealed that non-compliance was an independent risk factor for os. logistic regression analysis demonstrated that the extent of gastrectomy, primary tumour site, history of intraperitoneal surgery, bmi and open gastrectomy were independent preoperative predictive factors for non-compliance. cox analysis demonstrated that age, pt, pn, and the extent of gastrectomy independently affected os in patients with noncomplaint lymphadenectomy. however, os was significantly better in the compliant group than in the non-compliant group regardless of the recommendation for chemotherapy. stratified analysis demonstrated that os was significantly better in chemotherapy patients than in patients without chemotherapy and stage ii patients (pt n /n m and pt n m ) in whom chemotherapy was not recommended. conclusion: non-compliance is an independent risk factor after radical gastrectomy for gc we prospectively collected and retrospectively analysed the medical records of patients with proximal gc who underwent lspsd. the data were split / , with one group used for model development and the other for validation testing. results: of the patients enrolled in this study, ( . %) required laparoscopic haemostasis treatment. a multivariate analysis determined the following preoperative adverse risk factors for the model group: gender, preoperative n stage, and terminal branches of the splenic artery (spa), and we developed a scoring system based on these findings. each of these factors contributed point to the risk score. the intraoperative laparoscopy hemostasis rates were . , . , . , and . % for the low-, intermediate-, high-, and extremely high-risk categories, respectively. there were statistically significant differences among groups (p \ . ). with the increase in risk, both blood loss volume (blv) and operative time (min) of lspsd increased significantly (p \ . ).the area under the receiver operating characteristic curve for the score of intraoperative laparoscopic haemostasis was . . the observed and predicted incidence rates for intraoperative laparoscopic haemostasis were parallel in the validation set. conclusions: this simple we compared the survival of src patients with that of tubular adenocarcinoma patients according to bmi. results: the -year survival of src was significantly worse than that of wmd (p \ . ) but superior to that of pd (p \ . ). bmi-stratified analysis showed that in the high-bmi group, the prognosis of src was similar to that of wmd (p [ . ) and better than that of pd (p \ . ). in normal-bmi patients, src had a worse prognosis than wmd (p \ . ) but a more favorable prognosis than pd (p \ . ). src among low-bmi patients displayed much poorer survival than did both wmd (p \ . ) and pd (p = . ). multivariate analysis indicated that the risk of death was lowest for src patients with a high bmi and highest for src patients with a low bmi baseline characteristics were compared in a -patient rspshl cohort and a -patient lspshl cohort. one-to-four propensity score matching was performed to determine between-group differences. result: in total, patients were matched, including patients who underwent rspshl and who underwent lspshl. no significant differences in baseline characteristics were observed between these groups after matching. significant differences in total operative time, estimated blood loss (ebl), splenic hilar blood loss (shbl), splenic hilar dissection time (shdt), and splenic trunk dissection time were detected between these groups (all p \ . ). furthermore, no significant differences were evident between rspshl and lspshl in the overall noncompliance rate of lymph node (ln) dissection ( the highest body temperature within week after operation was used to establish diagnostic thresholds for high body temperature and low body temperature, which was obtained by x-tile software. the study used cox regression to analyze the influence of high body temperature on -year dfs. results: a total of patients were included in the analysis. the diagnostic threshold for high body temperature was defined as °c; patients with a high postoperative body temperature were allocated to the high temperature group (htg), while another patients were allocated to the low temperature group (ltg) cao department of gastric surgery, fujian medical university union hospital, fuzhou, china background: laparoscopic surgery for remnant gastric cancer third step: baring of the right side of the esophagus. fourth step: exposure of left gastroepiploic vessels and lns dissection in the splenic hilar area. fifth step: baring of the left side of the esophagus. the above procedure was performed for rgc patients with stage ct - an /? disease. results: there was no conversion to open surgery. mean operation time was . ± . min, mean blood loss was . ± . ml, and mean times to first flatus p -upper gi-gastric cancer a novel prognosis prediction model after gastrectomy for remnant gastric cancer: development and validation using international multicenter databases fuzhou, china; department of gastrointestinal surgery the model calibration was accurate in predicting -year survival. dca showed that the model has a greater benefit. the results were also confirmed by bootstrap internal validation. in external validation, c-statistics and dca showed good prognostic performance in patient datasets from participating institutions. moreover, we verified reliability of the model in an analysis of patients with different eln counts p -upper gi-gastric cancer a novel abdominal negative pressure lavage-drainage system for anastomotic leakage after r resection for gastric cancer while risk of gastric cancer for ppi users was higher than non-ppi users when duration between - year, = year, = year and = year. the risk of gastric cancer when duration = year(rr = . )and duration = year(rr = . )are higher than risk of gastric cancer when duration between - year (rr = . ). according to location subgroups meta-analysis,risk of non-cardiac gastric cancer for ppi users higher than non-ppi users conclusion: based on a systematic review with meta-analysis, we found the correlation between long-term use of ppi and the risk of gastric cancer and long-term use of ppi may increase the risk of non-cardiac gastric cancer when duration = year p -upper gi-gastric cancer age-adjusted charlson comorbidity index (acci) is a significant factor for predicting survival results: there were patients included in the analysis. the high-acci and low-acci groups had significant differences in preoperative abdominal surgery history, asa grade, tumor size, tumor stage, histologic type, age and comorbidity (all p \ . ). the incidence of postoperative complications was . % in the high-acci group and was significantly higher than that in the low-acci group (p = . ). the overall survival rate (os) and cancer-specific survival (css) rate in the low-acci group were both higher than those in the high-acci group (p \ . ). univariate and multivariate analyses showed that the acci was an independent risk factor for os and css (p \ . ). furthermore, a combination of the tnm staging system and acci showed a trend toward higher prognostic value and higher auc for os and css than the tnm staging system alone (p \ . ). conclusions: the acci was an we aimed to investigate the clinicopathological features and prognosis of patients with mgc and the impact of postoperative adjuvant chemotherapy on long-term survival. methods: the clinical and pathological data of patients diagnosed with gastric adenocarcinoma and undergoing radical gastrectomy from stratified analysis showed that, in advanced gastric cancer (agc), the -year os rates of mgc without adjuvant chemotherapy and sgc without adjuvant chemotherapy were . % and . %, respectively, with a statistically significant difference (p = . ). the -year os rates of advanced mgc after adjuvant chemotherapy and of advanced sgc after adjuvant chemotherapy were . % and . %, respectively, and the difference was not statistically significant (p = . ). the -year os rate of advanced mgc after adjuvant chemotherapy was significantly higher than that of patients without adjuvant chemotherapy ( . % vs. . %, p = . ). conclusions: mgc is a poor prognostic factor after radical gastrectomy for gastric cancer background: whether the tumor-node-metastasis (tnm) staging system is suitable for patients with node-negative gc is still controversial. the modified staging system established by rpa showed good prognostic performance in a variety of cancers. the application of rpa has not been reported in the prognostic prediction of gc. methods: node-negative gc patients who underwent radical resection at fujian medical university union hospital (n = ) and sun yat-sen university cancer center (n = ) with an at least -year follow-up information were selected as the training set. rpa was used to develop a modified staging system. patients from the surveillance, epidemiology, and end results databases (n = ) were selected as the external validation set. results: the -year overall survival (os) rates of patients with th ajcc-tnm stage ia-iiia in the training set were ia %, ib %, iia %, iib % and iiia %. multivariate analysis (mva) showed that larger tumor size, older age, and deeper depth of invasion were independent risk factors for os in patients with node-negative gc (all p \ . ). patients were reclassified into rpa i, rpa ii, rpa iii, and rpa iv stage based on rpa, the -year os rates were %, %, %, and %, respectively, with significantly difference (p \ . ). two-step mva showed that the rpa staging system was an independent predictor for os (p \ . ) were retrospectively collected. patients were classified into two groups according to bmi of \ kg/m ( patients; high bmi group) and = kg/ m ( patients; low bmi group). for these patients, clinicopathological variables were analyzed using propensity score matching to mitigate the selection bias: sex, age, asa physical states, clinical stage, laparoscopy-assisted total gastrectomy (latg) or totally laparoscopic total gastrectomy (tltg), d lymph node dissection, combined resection of other organs, method of anastomosis, jejunal pouch reconstruction. the surgical results and postoperative outcomes were compared and examined between the two groups. results: a total of patients were matched for the analysis. contrary to our expectations, there were no differences in the surgical results about operative time and estimated blood loss (low bmi . ± . min, high bmi . ± . min; p = . , low bmi . ± . g, high bmi . ± . g; p = . , respectively). furthermore, there was no significant difference in postoperative outcome of complication (clavian-dindo [ iiia) and the length of postoperative hospital stays (low bmi cases, high bmi cases baiocchi general surgery, university of brescia-spedali civili, brescia, italy background and aim: recently indocyanine green (icg) was introduced in clinical practice as a fluorescent tracer. the use of icg for sentinel lymph node (ln) mapping was investigated in lots of fields such as breast methods: we conduced a single center prospective trial. we included patients with gastric cancer candidate to surgery. icg was injected intraoperative or the day before surgery, via submucosal or subserosal. total or subtotal gastrectomy was performed open, laparoscopic or video-assisted access. during gastric cancer standard lymphadenectomy we studied lymphatic flow and ln bright in vivo and ex vivo japan introduction: in japan, the number of elderly patients with gastric cancer has been increasing in correlation with the increase in average age of the population. the aim of this study is to assess the safety and efficacy of laparoscopic gastrectomy for cancer in elderly patients compared with the short-term outcome in the nonelderly. method: we reviewed patients who underwent laparoscopic gastrectomy (dital gastrectomy,proximal gastrectomy,total gastrectomy)between ).the incidence of advanced cancer(stageiior more)was higher in elderly patients there were no significant differences in the operating time,blood loss and postoperative hospital stay. there were no significant differences in the incidence of postoperative morbidity. conclusion: in elderly patients, there was a tendency of reduction surgery being selected according to individual condition, but there was no significant difference in the short-term outcome.hence,we conclude that laparoscopic gastrectomy is indicated even in elderly patients. p -upper gi-gastric cancer improved technique of vacuum therapy and carried out ltg . a patient factor (the gender, the age and bmi), an operation factor (operation time, the bleeding amount, lymph node dissection and conjurer), a coincidence related complication (clavien dindo classification, sutural insufficiency of grade more than , anastomotic stricture, anastomotic region bleeding and reflux esophagitis) and the post-operatively length of stay were considered . result: cs crowd met cases ( . %) and ls cluster ( . %) cases. years old of age medians ( - ), men and women were examples ( . %), examples ( . %) and bmi median . ( . - . ) by a patient factor, and a significant difference didn't admit by two groups for days, the post-operatively average length of stay was days by ls group by cs group. conclusion: operation time was short for a coincidence by linear stapler more than a coincidence by circular stapler in comparison of an esophagoenterostomy way in ltg on the day before the operation, we endoscopically clipped several points located cm proximal to the tumor edge to cover about half of the tumor. after lymph node dissection, we incised the stomach with an endoscopic linear stapling device,including the previously placed clips. reconstruction was performed in all patients who underwent billroth i or roux-en-y procedures. result: no complications were observed during pre-operative endoscopic clipping or intraoperatively p -upper gi-gastric cancer small intestinal tumors after laparoscopic surgery in our hospital small intestinal tumors are rarely observed, accounting for about - % (malignant cases: - %) of all gastrointestinal tumors. therefore, occasionally, their diagnoses can be difficult. however, recently, capsule and balloon endoscopes have been widely employed were examined regarding patient backgrounds, diagnostic methods, pathological findings, postoperative courses, and prognoses. results: the subjects consisted of males and females, with a mean age of years. their chief complaints were black stools the median distance from the treitz ligament or bauhin valve was cm ( - ) postoperative complications were abdominal abscess ( cases; . %) and surgical site infection (ssi), hemorrhage, and paralytic ileus ( case each; . %). pathological diagnoses were lymphoma metastatic small intestinal tumor ( cases; . %), and granuloma, lipoma, peutz-jeghers polyp, clear cell sarcoma, malignant mesothelioma, and ectopic pancreas most patients were diagnosed in bleeding, complicated by anemia and black stools. however, as most tumors were relatively close to the treitz ligament and bauhin valve, almost a half could be diagnosed with a small intestine endoscope before surgery patients were classified as popf and no-popf according to their grade b or c popf status. popf was diagnosed according to international study group of pancreatic fistula (isgpf) criteria or clinical findings. patient characteristics, intraoperative parameters, electrosurgical device type, pathological findings, and early postoperative outcomes were compared. electrosurgical devices were classified asthunderbeat (tb) or laparosonic coagulating shears (lcs) based on energy sources. results: eighteen patients developed grade b or c popf. among them, ( . %) and ( . %) were diagnosed with popf according to isgpf criteria and clinical findings ), operation time (p = . ) and electrosurgical device type (p = . ) were significant risk factors for popf following lag ) and tb device (or, . ; % ci ) were independent risk factors for popf following lag. conclusions: operation time and tb use significantly affect the risk of popf and should be considered in future clinical studies. p -upper gi-gastric cancer feasibility and nutritional benefits of double flap with no-knife stapler reconstruction after laparoscopic proximal gastrectomy for gastric cancer were analyzed. receiver operating characteristic curves were generated, and by calculating the areas under the curve(auc) and the c-index, the discriminative ability of crps during different periods were compared, including pre-crp, postoperative days , , and postoperative maximum crp (post-crp max ). a decision curve analysis was performed to evaluate the clinical utility. result: ultimately, patients were included this study and the median follow-up time was ( - ) months. for postoperative recurrence, the auc and c-index of pre-crp were . and . , respectively, significantly higher than the other crps, all p \ . . among = ''''''the = '''' post-crps = '''' post-crp = '''' sub = similar findings were observed for overall survival. conclusion: both pre-crp and post-crp max , cheap and easily obtained, are independent predictors of recurrence for gc. act significantly prolonged the rfs for stage ii/iii gc patients with high-prep p -upper gi-gastric cancer robot-assisted gastroduodenal surgery: a single center experience robot-assisted gastroduodenal surgery (ras) was introduced to overcome the technical limitations of conventional laparoscopy. it provides a d-amplified view to the surgeons and an increased ability to control the operative field by manipulating optics, as well as enhanced mobility and precision of instruments. the aim of the present study is to evaluate the main outcome of a single center experience in gastroduodenal robotic surgery. materials and methods: we report a case series of patients who underwent robot-assisted gastroduodenal surgery at sanchinarro university hospital between conclusions: robot-assisted gastroduodenal surgery is a safe and feasible technique in experienced centers with advanced robotic skills. in the literature, there are only few reports of robotic assisted gastroduodenal resection. further studies are necessary to better confirme our results. p -upper gi-gastric cancer atypical methods: retrospective review of ogd reports before and after the introduction of the new guidelines. inclusion criteria: all elective ogds. exclusion criteria: emergency ogds and elective therapeutic ogds. data recorded: patient demographics, endoscopist, indication, number of photos, anatomical site photographed, pathology identified and whether pathology photographed or not. results: ogds reviewed, before the guidelines (group ) and afterwards (group ). the most common indication was reflux ( %) in group and anaemia ( %) in group clinical utility of systematic pre treatment staging laparoscopic exploration methods: all locally advanced gastric adenocarcinoma managed in surgical oncological unit between st january and th november were prospectively enrolled in the study. in the absence of emergency surgery or preoperative contraindications, all patients with curative intent underwent either preoperative chemotherapy followed by surgical exploration in the intent of curative gastrectomy (g)or systematic pretreatment laparoscopic exploration (l) benkabbou surgical department the patient background (age, gender, bmi) and c-stage of the preoperative factor were matched using propensity score matching method, and the surgical results were compared and examined. results: thirty rg groups matched rag cases. the operation time (rag / lg) was significantly longer in the rag group as . ± . min / . ± . min (p \ . ). amount of blood loss was not significantly different each other; ml / ml (p = . ). pathologically t a case was involved in cases in rag and cases in lg. the extent of lymph node dissection (d ? / d ) was / cases in both groups conclusions: rag in our clinical experiences can be safely introduced and short-term results are comparable to those of lg. verification of superiority of robotic surgery including long-term results seems to influence the future of robotic surgery conclusions: totally laparoscopic gastrectomy is feasible method in terms of surgical outcomes. furthermore, totally laparoscopic total gastrectomy is not technically difficult in advanced gastric cancer such as early gastric cancer and safety method. key words: gastrectomy, reconstruction, laparoscopic surgery, stomach neoplasm aims: meckel's diverticulum (md) is one of the most common congenital anomalies of the small intestine caused by an obliteration defect of omphalomesenteric duct. the objective of this study was to review surgical treatment and clinical outcomes of md, and evaluate the safety and feasibility of minimal invasive surgery (mis) in md. methods: we performed a retrospective analysis of medical record for patients who underwent meckel's diverticulectomy at six hallym-university-affiliated hospitals between d), as well as the average of drainage stay. patients who underwent laparoscopic repair required significantly less parenteral analgesics than the open group.the mean postoperative stay was significantly shorter for laparoscopic group (mean, . d) than the open one.morbidity of medical and surgical complication was higher in open groups ( vs ). the most common complication in both groups was medical complication.more case of pneumonia was occurred in open groups compared to laparoscopic groups methods: a retrospective study using our prospective database was designed to analyse all the resected md in our centre. epidemiological data, clinical setting, diagnostic test and histological results were reported. results: md was resected in patients, males and females, with a mean age of . years ( . - . ). in cases, a laparoscopic approach was chosen. eighty-seven percent of the patients had a presurgical imaging test (ultrasounds, ct-scan or meckel's scan) background: perforated peptic ulcer (ppu) is a substantial health problem with significant postoperative morbidity up to % and mortality up to % worldwide. aims: this study aimed to estimate the sensitivity scoring systems for prognosis morbidity of patients operated for ppu with diffuse peritonitis. methods: a total of patients were underwent emergency repair for ppu with diffuse peritonitis in pirogov russian national research medical university's surgical clinics during - years. different scoring systems used to predict outcome in ppu patients were identified: boey score, peptic ulcer perforation (pulp) score, asa, mannheim peritonitis index (mpi), world society of emergency surgery sepsis severity score (wses score). to quantify the strength of the concatenation of prognostic score and morbidity we use odds ratio (or) with % ci ), respectively. pulp score and asa score have good prognostic value in relation to morbidity, but less than boey, mpi and wses sss. patients with pulp [ had or with % ci of p -upper gi-gastroduodenal diseases gastrostomy tube placed by laparoscopy as a new therapeutic option for continuous intestinal infusion treatment with levodopa/ carbidopa we present year outcomes of our initial consecutive patient cohort. methods: patients were identified in a prospectively maintained irb-approved database ( - ). post-operative eckardt scores and a -point validated system questionnaires were obtained via telephone interviews one patient required reoperation for failed myotomy. the mean eckardt score at years was . (± . ), with all fourteen patients having an eckardt score \ . all patients reported significant improvement in their quality of life. classic gerd symptoms (heartburn and regurgitation) were present in ( . %) patients. proton-pump inhibitors are being used by % of with patients with excellent symptom control. seven patients returned for a repeat egd (median . yrs) with patients having normal anatomy and having la grade a esophagitis ( patient on ppi). barrett's esophagus was not detected. conclusion: long-term results from our early experience with lhm are excellent and durable with only one patient requiring re-intervention in years until recently the esophagectomy was the only choice in treatment of patients with end-stage achalasia. developing of minimally invasive techniques such as a laparoscopic heller miotomy and peroral endoscopic myotomy (poem) allowed to use them as a treatment options. aim: to present an experience of treatment of patents with end-stage cardiac achalasia. materials and methods: since . till the laparoscopic heller myotomy was performed in , and esophagectomies were performed in patients with failed previously myotomy made in other clinics. gastric tube was used to replace the esophagus in patients underwent esophagectomy after skeletization of crura posteriorly to esophagus, two separated rectangular patches of parietene progrip mesh (covidien) measuring x . - cm were attached to the posterior surfaces of the crura. the patches were fixated themselves due to special hooks. than continious twodirections suture was placed through both crura along with the patches using self-gripping v-loc - suture (covidien). the same suture was used for construction of nissen fundoplication wrap . cm long. aditional anchoring stich through the wrap and esophageal wall was placed using ti-cron - suture (covidien). d laparoscopy was used while suturing using richard wolf epic system. results: all the procedures were performed successfully. there were no cases of bleeding from the suturing points either from the crura and the fundus wall. there were no crural dehiscence while suturing, even if the distance between crura was more than cm. the mean duration of suturing facilitated by d laparoscopy was min (range, - min) for crural repair, and min (range, - min) for fundoplication. there were no excessive postoperative pain in all the patients. there were no disphagia month postop in every patient. conclusions: . the new technique of posterior buttress of crural repair using small patches of parietene progrip mesh and v-loc suture showed feasibility and safety. . the use of d in such case most commonly manifested symptoms are cough, sore throat, hoarseness, dysphonia, globus and only % patients with lpr have typical gerd symptoms. also ppi therapy are less effective in patients with lpr in comparison with patients which have typical features of gerd. purpose: to compare the outcomes between surgical treatment and conservative therapy in patients with laryngopharyngeal reflux. materials and methods: for the period chesarev faculty surgery # , federal state autonomous educational institution of higher education i.m. sechen, moscow, russia p -upper gi-reflux-achalasia a case of a primary parahiatal hernia associated with a type i hiatal hernia emergency county hospital parahiatal hernia is a rare disease that occurs when an abdominal organ protrudes through an opening adjacent to an anatomically intact esophageal hiatus. the herniated organ is usually the stomach, although cases of omental and colonic herniation exist we report the case of a -year-old woman which accused epigastric pain, starting years prior, pseudo-angina, heartburn and bloating. based on imagistic findings the patient was diagnosed with a parahiatal hernia and an associated type i hiatal hernia. patient underwent surgery and a cm diameter defect in the diaphragm lateral to the left crus was discovered, through which - % of the stomach had herniated. the hiatal orifice was slightly enlarged but anatomically intact, with an associated small sliding hiatal hernia. we performed closure of the defect, hiatoplasty and a floppy-nissen fundoplication pneumatic dilation with mm balloon was performed under general anesthesia. radiological contrast control and endoscopy reevaluation revealed a perforation just above the squamo-columnar junction. a minimally invasive approach was decided. an fully covered esophageal stent was inserted. radiological control after days reveals left pleurisy and migration of the stent. the same day was performed an endoscopic repositioning of the stent with clip fixation. left pleural puncture was performed and clear fluid was extracted. the condition of the patient got worse and she was transferred on icu ( . ). we performed left pleurostomy and initial exploratory laparoscopy-no intraperitoneal lesions. due to difficult transhiatal access to the inferior mediastinum the surgery was converted to open-perisophageal mediastinal abscess was found, evacuated and drainage and jejunostomy were performed. after a week, the patient presented progressive altered condition, febrile syndrome. thoraco-abdominal ct-scan showed left pleural effusions. left pleurostomy was performed, with extraction of fetid fluid. continuous lavage was instituted. on st of august, the pleurostomy tube drained gastric content, and the clinical examination revealed signs of generalized peritonitis. laparotomy was performed with lavage, drainage and posterior decompression gastrostomy. results: postoperative evolution was favorable, with the suppression of pleural drainage in . and discharge in . with alimentation exclusive on jejunostomy. one month later, she had normal clinical and radiological examination evaluation of efficacy was performed with reflux symptom index (rsi) specific for extraesophageal symptoms, subjective satisfaction and occurrence of dysphagia and gas-bloat syndrome. a rsi score [ was considered as pathological. results: rsi significantly decreased after surgery ( and msa as compared with total fundoplication; , % of patients were satisfied with surgery: a comparison between techniques showed superiority of msa objective: to demonstrate the efficacy of hiatorraphy without the use of meshes in thegiant paraesophageal hiatus hernia, as well as the standardization of our technique, with thetechnical steps that we make successively. material and method: clinical cases: -year-old man,with symptomatic hiatal hernia with progressive intolerance and dysnea. egd: the stomach rotated in a giant hiatal hernia.gastroscopy not completed due to endoscope loop formation within giant hiatal hernia with gastric volvulation. ct: large hiatal hernia,combined volvulation (axial axial mesenteric organ), the stomach in a right subpulmonary situation. results: intervention: laparoscopic approach.hh of large paraesophageal size,double organoaxial-and-mesenteric volvular component,gastric walls very thickened and adhered to the mediastinum.reduction of all content and the sac,is adhered to the pleura, extended med-iastinal esophageal disection, up to vein pulmonary and get enough abdominal esophagus and rule out the presence of an short esophagus,posterior-anterior and left tutorized modified hiatorraphy with stitches in ''u'' with non-absorbable suture on teflon reinforcement patches.nissen fixed to both pillars, intramediastinal drainage.egd at the st day with esophageal stenosis due to inflamation of the nissen, resolved with medical treatment. dischage at th day.asymptomatic and without radiological recurrence after months of follow-up. conclusions: in giant and paraesophageal hiatus hernias, modified primary hiatorraphy together with mediastinal esophageal dissection extended can be an effective and safe alternative, and can be advised as a technical gesture prior to a collis nissen and-or placement of a hiatal-hiatoplasty mesh united states of america aim/background: prescribed opioids for pain control have been implicated as major contributors to addiction through their illicit use. efforts to reduce opioid prescriptions and measure their impact on outcomes are novel. we analyzed how patient outcomes are affected with reduced opioid prescriptions following laparoscopic foregut surgery narcr: %), length of hospital stay (narcs: . days vs. narcr: . days), -day readmission rates (narcs: % vs. narcr: %) and perioperative complication rates. additionally, no significant qol outcome differences between the groups were reported at one month postoperatively. conclusion: our study supports reducing opioid prescriptions as a strategy to counter illicit drug use and addiction patients who underwent paraesophageal hernia repair at a tertiary referral center were analyzed retrospectively. demographic data, asa classification, characteristics of peh, onset of symptom, dysphagia severity score, characteristics of fundoplication (partial vs. total; laparotomy vs. laparoscopy; emergency vs. elective) and surgical outcome (length of stay, complication and -day mortality) were recorded and reviewed. results: patients were included; % were female (mean age of . years old and mean body mass index of . ). mean onset of symptom was . weeks after peh repair, dysphagia severity scores were changed . from . . conclusion: in our series, the dysphagia severity scores reduced after surgery upper gi surgery, the catholic university of korea mary's hospital, incheon city, korea p -upper gi-gastric cancer general surgery biopsy from the mass has showed poorly differentiated signet ring cell adenocarcinoma. chest computed tomography revealed mm thoracic aortic aneurism. abdominal computed tomography showed mm infra renal aortic aneurism and no evidence of metastatic disease general surgery year old, female patient presented with upper abdominal pain, weight loss ( kg during last three month), without nausea or vomiting biopsy was done and pathology result showed intestinal type, her -negative adenocarcinoma of the stomach. chest and abdominal computed tomography (ct) were normal. endoscopic ultrasound (eus) revealed cm lesion with invasion to the muscularis propria (mp) she was treated by neo adjuvant chemotherapy ( cycles carboplatine ? fu). patient underwent laparoscopic partial gastrectomy with modified d lymphadenectomy and billroth ii gastrojejunostomy. total operating time (ort) was min. three day after operation patient started regular diet and was discharged home on day fife. final pathology result confirmed intestinal type, modified differentiated adenocarcinoma of the stomach economou st surgical department general, visceral and transplant surgery, section minimally invasive surgery, heidelberg university hospital, heidelberg, germany; department of surgery, iuliu hatieganu university, cluj-napoca, romania; general and visceral surgery, klinikum mittelbaden, baden-baden, germany surgery, toyonaka municipal hospital, toyonaka city, osaka, japan; gastroenterological surgery, osaka university, osaka, japan; next generation endoscopic intervention, osaka university, osaka, japan aims: uncomplicated healing of anastomoses in colorectal surgery is the basis for early adjuvant oncology therapy. the basis for proper healing is good blood flow. we use by robotic surgery foor control the firefly by intuitive. since january , icg has used for blood flow in laparoscopic bowel surgery for the d-light system of storz. method: use of icg wants to accurately determine the resection line for free colon operations based on good blood circulation. we use icg pulse in two batches and color detection using d light from storz to verify blood flow. the first dose is given after the skeletalisation of the intestines intraabdominally and the second after the colic anastomosis to verify its vitality. results: in the period under review we performed laparoscopic operations on the free colon, % of the operations were elective. we had . % leakage across the set, however, in the subset of elective operations, we had only a leak of . %. conclusion: in an unselected set of colorectal operations, leakage was . %, but only . for elective operations. in our group there was a clear effect of using icg in elective laparoscopic resections with an intracorporal anastomosis, the effect was not shown in others, probably due to leakage were factors other than blood flow. objective: right hemicoloctomy (rhce) is the first choice in treating the right colon cancer. complete mesocolic excision with extended lymph node dissection at the roots of superior mesenteric artery (sma) branches enables removal of all lymphatic tissue and prevents local recurrence. previously variability of sma branches was demonstrated. the aim of presented study was to compare the distribution of sma branches in two ethnically different cohorts methods: preoperativect scans with vascular d reconstruction were assessed in patients ( - years) from russia and patients ( - years) from turkey with right colon cancer operated in - . the distribution of ileocolic artery (ica), right colic artery (rca) and middle colic artery (mca) was investigated. results: ica and mca could be found on ct scans in all patients, whereas rca had significantly different distribution between patient cohorts: it was visible in ( %) of turkish patients and only in ( %) of russian patients (p = . ). conclusion: these results suggest that there might be ethnical differences in sma branches distribution. in turkish patients all named sma branches ate visible on ct scans in %, whereas in russian patients only in %. the majority of patients from russia don't have rca. ica and mca could be found in all patients regardless ethnicity. knowing the variant of sma branching before the operation can help plan extended lymph node dissection. the national training programme in laparoscopic colorectal surgery (s-micras-lapserb) in serbia was set up to introduce standardized and structured training in laparoscopic colorectal surgery. method: an assessment based structured training programme (lapserb) started in . series of hands on supervised workshops were conducted for four different hospitals using the structured training by single trainer. this study aims at retrospective analysis of prospectively collected data for patients undergoing colorectal resections. we look at short-term clinical and pathological outcome of patients within laparoscopic colorectal resections performed in national training program. results: during the period november until november , laparoscopic colorectal resection was performed in ( male and female) patients. mean age of patients was . ( - ). the most common indication was colorectal cancer ( patients, . %), ( . %) patients were operated due to the colorectal polyps not suitable for endoscopic resection and ( . %) was operated due to ibd. there were ( . %) right colonic, ( . %) left colonic /tme and ( . %) other resections. average number of lymph node harvested in patients with colorectal carcinoma was . . there were / ( . %) r resections mean duration of hospital stay was . days ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . postoperative complications were encountered in / patients ( . %). overall, mortality rate was . % ( / ). conclusions: this study demonstrates successful and safe adoption of laparoscopic technique for colorectal resections. short term clinical and pathological outcomes are compared to published data and shows wider adoption at the national level. standardization of operative technique and structured training remains the key in success. introduction: femal adnexal tumor of probable wolffian origin (fatwo) was first described in . it is a tumor of mesonephiris wolffian duct origin. fatwo is rare tumor which is usualle benign. in the literature has been reported cases and only cases of recurrent disease. next rare tumor in pelvic localisation is sex cord-gonadal stromal tumor of sertoli cells. methods: we present case report of women who presented metastatic fatwo with duplicity of sertoli tumor. results: -year old women underwent years ago exstirpation of tumor in the left broad ligament. histologically there was rare benign fatwo. this year was indicated adnexectomy at gynecology department. during the operation was done bilateral adnexectomy and discovered tumor of anterior wall on upper rectum. microscopic examination showed sertoli tumor on the left ovary. afterward we completed next examinations. colonoscopy without any abnormality. on ct scan was tumor cm without contact to rectum wall, without distant metastasis. the same was described on rectal ultrasonography-normal wall of rectum, tumor probably from uterus. at diagnostic laparoscopy was tumor mass cm, with necrosis arising from anterior wall of the rectum. next small metastasis on pelvic peritoneum. we performed debulking of this big tumor and metastasectomy, there was no infiltration to muscularis propria of the rectum. patient did not have any postoperativ complications. microscopic examination of the rectal tumor and small peritoneal metastasis showed metastatic fatwo. after weeks she underwent laparoscopic second look operation. there were small metastasis on pelvis peritoneum. we removed two biggest metastasis and the rest was destroyed wit j plazma. microscopic examination showed in this metastasis sertoli tumor. conclusion: our patient has metastatic fatwo and sertoli tumor. fatwo is so rare, that in the literaure is not enough information for observation or adjuvant therapy. in one case was described imatinib mesylae (gleevec) therapy with good results. surgeons must be ready to meet new diagnosis. bochdalek hernia is a type of congenital diaphragmatic hernia. in most cases, it is diagnosed during the neonatal period. we present a case of laparoscopically treated congenital bochdalek hernia that led to jejunal strangulation in an adult . case: an fifty eight year old obese(bmi = . ) female admitted for gradually worsening right flank pain, vomiting and respiratory distress for one day. there was no trauma history to chest or abdomen. the past medical history were old cva, well controlled hypertension and dm. she was hemodynamically stable. her right flank was very tender. there was no abdominal distension. initial cbc tests showed leukocytosis ( , /ul) , but electrolytes were normal. chest pa revealed right side diaphragmatic hernia. there was poorly enhanced, herniated small bowel in the right hemithorax on chest ct scan. the patient was taken for emergency operation. on laparoscopy, the normal liver was displaced leftward because of herniated bowel. there was incarcerated jejunum and omentum which could not be reduced. so we widened a cm sized posterolateral diaphragmatic defect first, and then we could reduce the strangulated jejunum( cm in length) and omentum. there was no hernial sac. the defect was closed with -o prolene. finally, the strangulated jejunum was resected and anastomosed extracorporeally. the hospital progress of the patient was not eventful. on post-operative day , the patient was allowed soft diet. the patient was discharged on post-operative day without any complication. conclusion: congenital diaphragmatic hernia is an uncommon condition in adults, but you should keep in mind the diaphragmatic hernia as a cause of intestinal obstruction and respiratory distress in an adult. prompt surgical intervention is required to a favorable outcome. laparoscopic repair of bochdalek hernia is a good management option. aims: intestinal obstruction is one of the most frequent abdominal conditions in the emergency department (ed). up to % of patients having undergone a laparotomy will have an episode throughout their lives, of which % up to % will respond to conservative management. the laparoscopic approach is widely accepted and supported by the studies published up todate. it is recommended in patients with suspected single band, who have less than one previous laparotomy and less than h of clinical evolution. our objective is to validate in our experience that these premises are the appropriate ones in the election of candidates for a minimally invasive approach. methods: we present a series of cases admitted with symptoms compatible with adhesive intestinal obstruction in the ed of a third level hospital during months. all patients underwent abdominal ct to rule out the possible causes of obstruction. emergency surgery was indicated because of failure of a conservative medical treatment or for the findings of the complementary tests. results: a initial laparoscopic approach was performed in the patients, with a conversion rate of % of the cases (resection and anastomosis was required in patients, due to loop suffering or intestinal tumor not seen in the ct). among the patients who required laparotomy, % had more han h of clinical evolution before de surgery and % had free fluid in the tc. as surgical complications: intestinal perforations were produced secondary to the manipulation. there was recurrences of obstruction in the following months. conclusion: the laparoscopic approach is feasible in selected cases and experienced hands. acording to our results it is recommended to perform it in only in patients with less than h evolution and with a single band image in the ct without free fluid. the intestine should be explored avoiding the manipulation of the most extensive loops to prevent complications and keep in mind the possible conversion to laparotomy in case of complications. aim: the aim of the study was to evaluate whether physiologic and operative severity score for the enumeration of mortality and morbidity (possum) is useful to predict the risk of complications in patients older than years. methods: we performed a retrospective study of patients older than years old diagnosed with acute abdomen who were admitted to the department of general, minimally invasive and elderly surgery in olsztyn between may and october . results: the most common disagnosis was ileus. the mortality rate in surgery department was %.after relocation to the intensive care unit, the overall mortality rate was . %. the patients who died a short time after surgery had mortality rates greater than % and morbidity rates greater than % according to possum. conclusions: this study shows that possum seems to be a valuable scale to predict the risk of death after surgery in older patients. patients with higher mortality and morbidity scores should be very carefully selected for surgery. aims: diaphragmatic hernia in adulthood is rare. the most common causes are blunt and penetrating trauma. we present an intraoperative video of the laparoscopic repair of an adult onset, non-traumatic, diaphragmatic hernia in a patient with splenomegaly. method: a year old woman was referred to upper gastrointestinal surgery with epigastric burning and pain in the left side of her chest, radiating to the left shoulder, for one year. there was no recent or distant history of trauma. she has a past medical history of treated hepatitis c, cirrhotic liver disease, splenomegaly, thrombocytopenia and iron deficiency anaemia. gastroscopy was interpreted as a fundal diverticulum. ct abdomen/pelvis with intravenous (iv) contrast showed this to be a left diaphragmatic defect with herniated stomach causing a volvulus, which lay immediately above an enlarged spleen. a ct three years prior to this showed no diaphragmatic hernia. the patient had some symptomatic relief with a proton pump inhibitor and oral antacids, however due to her persistent symptoms surgery was undertaken. the patient had laparoscopic repair of the diaphragmatic hernia. ports were as follows: mm umbilical, mm left upper quadrant, mm right upper quadrant, mm left iliac fossa. a left posterior diaphragmatic defect was found, just above the enlarged spleen, containing the incarcerated fundus of the stomach. the hernia was reduced by gradual dissection of the sac. a cm nonabsorbable polypropylene mesh (proceed) was used to cover the defect a cm margin. this was tacked in place with protack. a single french robinson drain was left in situ. results: the procedure was uncomplicated. oral diet was introduced on post-operative day and the drain was removed and patient discharged on day . there were no post-operative complications. conclusions: the video shows an effective dissection of a left sided diaphragmatic hernia and mesh repair, overcoming multiple technical challenges secondary to splenomegaly and portal hypertension. aims: creating laparoscopic anastomosis is a challenging surgical skill with high clinical relevance. to assure efficient training and enhanced learning curves, constructive and objective feedback is essential. currently there is no appropriate instrument to assess the surgical performance while creating laparoscopic anastomosis. the aim of this study is to develop and validate the anastomosis-objective structured assessment of technical skill (a-osats) score. methods: to obtain an international expert consensus for a procedure specific checklist (psc) for laparoscopic anastomosis, a modified delphi survey with an integrated analytic hierarchy process is currently being performed. each a-osats sub step is assigned a specific weight to determine its importance to the final outcome of the anastomosis. to validate the a-osats score, a laparoscopic side-to-side small bowl anastomosis with a linear stapler and hand-sewn closure of the enterotomy was chosen and is performed by surgeons with varying degrees of laparoscopic experience on a live porcine model. all performances are recorded and rated twice using the a-osats by two blinded experts. results: the final a-osats score includes a weighted psc developed by the modified delphi survey and the already validated global rating scale of previously published osats scores. four key steps (bowel placement, creation of enterotomies, stapling, closure of enterotomy) and sub steps, as well as their definitions, were established during the delphi survey. to validate the a-osats, surgeons ( experts, intermediates, novices) have participated in the study so far. preliminary results showed significant differences between all three levels of laparoscopic experience (novices: . ± . ; intermediates: . ± . , experts: . ± . ; p \ . ) for the overall a-osats score as well as the psc itself (novices: . ± . , intermediates: . ± . , experts: . ± . ; p = . ). conclusions: the a-osats is a weighted score that objectively assesses surgical skill during the creation of laparoscopic anastomosis. preliminary results confirm construct validity of the proposed score. furthermore, by offering the possibility to differentiate single aspects during the procedure, the a-osats allows focused feedback to enhance one's performance. minor changes in weights are expected after the last round of the delphi survey. interand intrarater reliability will be assessed after final inclusion of all participants.aims: to apply augmented reality technology from three-dimensional colon models as preoperative planning method in colorectal surgery. method: from three-dimensional anatomical models of the colon we have developed holograms of augmented reality. the models were obtained from ct images (siemens somatom perspective Ò) with abdominal image cuts with mm thick. the recovery of the images was in dicom format and the processing to achieve the three-dimensional reconstruction was performed with the programs osirixÒ and horosÒ, which made a complete segmentation of the colon surface, and a modification of the image density. in this way models d were obtained of the isolated colon, and in relationship with the bone structure. the application colon d ar was designed (increased hyper experience-visualizer with slam technology) creating a hologram of augmented reality to scale : from each three dimensional model to make a projection of it on the abdomen of the patient by modifying the position in height of the reconstruction, using the bone pelvis as anatomic reference point to calibrate the placement of the hologram. results: in the preliminary phase (from october to december ) holograms of augmented reality were developed in patients with colorectal cancer (right colon, left colon, transverse colon and rectum) to complement the radiological reconstruction with the virtual model. in the application phase (from january ) the holograms developed are going to be applied as a method to improve preoperative study. conclusions: three dimensional reconstruction of the tumor in the preoperative plan of colorrectal surgery combined with hyperreality technology allows to develop models of augmented reality in order to improve colon anatomy knowledge and to plan the surgical technique.aims: laparoscopic adrenalectomy has become the standard of care for most adrenal masses. we report a case of laparoscopic adrenalectomy for left adrenal adenoma. methods: we present the case of a -year-old caucasian female patient with an asymptomatic, left-sided adenoma, that was incidentally detected during abdominal ultrasound. no headaches, palpitations, tachycardia, tremor, dizziness or vomiting were reported. pre-operative blood tests confirmed that the tumor was a non-secreting one and a ct-scan revealed a . . cm left adrenal mass. laparoscopic surgical excision was proposed. the patient was placed in semilateral right-sided decubitus position. four trocars ( epigastric- mm & subcostals- mm & mm) were used, without the use of a liver retractor. the adrenal vessels were clipped not only with the standard laparoscopic clips, but also with the hem-o-lok ligation system. results: the operation lasted for h with minimal blood loss. the patient's post-operative course was uneventful and she was finally discharged four days post-operatively. histology report ensured that it was adenoma of the adrenal cortex. aims: since the first laparoscopic adrenalectomy in (gagner), the laparoscopic lateral transabdominal approach has proved to be the one of choice. it provides an easy anatomical orientation, overall the technique is similar to other traditional laparoscopic procedures. on the other hand, the posterior retroperitoneoscopic adrenalectomy (pra), described in (waltz), has proven to be a safe technique and effective for the surgical management of several adrenal pathologies. the advantages include direct access to the adrenal gland, without the need for visceral mobilization or lysis of adhesions from previous abdominal operations and the ability to perform a bilateral adrenalectomy without repositioning the patient. currently there is controversy about which is the approach of choice, having to take into account the learning curve necessary for the retroperitoneal approach and the reduced number of patients with adrenal pathology subsidiary of surgical management. the objetive is to demonstrate the safety and efficacy of the standardized laparoscopic approach of the left adrenal gland with trocars for selected cases. methods: clinical case: -year-old man, resistant hypertension despite concurrent use of three antihypertensive agents, with biochemical and radiological diagnosis of left adrenal adenoma with primary hyperaldosteronism. demonstrative video of the technical steps in a standardized way that we propose for laparoscopic left adrenalectomy only using trocars. results: full laparoscopic surgical approach in right lateral decubitus position: trocars-lateral transabdominal approach. steps: . laparoscopic liberation of the splenic flexure of the colon for the colo-spleen-pancreato-gastric en block mobilization until identification of the left pillar, . dissection of the medial border of the gland, identification of left renal and diaphragmatic vein, as well as the adrenal vein which is dissected and clipped, . dissection of the lateral edge of the adrenal gland, . lower pole dissection of the gland completing the resection with ligasureÒ. the patient presented a successful postoperative recovery, being discharged h after the intervention. asymptomatic, the patient does not need antihypertensive drugs at year follow-up. conclusion(s): the standardization of the procedure allows reducing the number of trocars, maintaining the safety and effectiveness of the minimally invasive approach. aims: cortical-sparing adrenalectomy is a suitable treatment for hereditary and sporadic bilateral pheochromocytoma, in cases of low risk of malignancy, to reduce the possibility of adrenal insufficiency assuming the chance of local recurrence. the aim of the study is to analyze the functional results of partial adrenalectomy by retroperitoneal endoscopic approach in singleadrenal patients or patients requiring bilateral adrenalectomy. methods: prospective study between january and october including pheochromocytoma patients diagnosed with low risk of malignant mutations. all patients agreed to be included in the study. experienced endocrine surgeons who have been trained in minimally invasive endocrine surgery performed the procedure using the same surgical technique. demographic variables and clinical characteristics were collected, subsequently carrying out the descriptive analysis of the data. results: a total of eight patients were registered, five associated with men type syndrome and three in the context of vhl syndrome. retroperitoneoscopic resection was performed without laparoscopic or open conversion and no postoperative complications; the average hospital stay was . days. preservation of the functional cortex without corticosteroids was achieved in ( . %) of out cases with a follow-up of . ± months. today, these seven patients have a preserved adrenal function without hormone replacement. conclusions: cortical-sparing adrenalectomy by the retroperitoneal endoscopic approach, in expert hands, is safe and feasible for the treatment of hereditary and sporadic pheochromocytoma in a context of low malignancy, making it possible to avoid the need for corticoid replacement in most cases. biomedical sciences, university of west attica, athens, greece partial adrenalectomy has been suggested for patients benign adrenal tumors especially in the case of hereditary syndromes, like multiple endocrine neoplasia type , von hippel-lindau disease and neurofibromatosis type i. aims: this systematic review aimed to investigate the role of partial adrenalectomy in the treatment of hereditary pheochromocytoma. methods: electronic databases were searched with the search terms 'men ii', 'von hippel lindau', 'neurofibromatosis', 'laparoscopic partial adrenalectomy', 'robotic assisted partial adrenalectomy' for the time period up to and including december . full publications, including clinical trials randomized or not, retrospective studies, case series, case reports that provided relevant data met inclusion criteria results: thirty five possibly relevant studies were identified. abstracts were reviewed and fourteen articles were excluded as they were review articles or articles presenting data on open partial adrenalectomy. twenty one studies, that met inclusion criteria were retrieved in full text and included in the systematic review. eight studies presented data on partial adrenalectomy in patients with von hippel lindau including two case series with median follow up ranging from to . years and six case reports. thirteen studies presented data on partial adrenalectomy in patients with men ii, including two case series and eleven case reports. recurrence rate was estimated at about % for pheochromocytoma. overall steroid dependence rate was estimated at %. conclusion: minimally invasive partial adrenalectomy is a therapeutic option especially in patients with heritable pheochromocytoma, given that tumors are often bilateral, tumors are commonly benign and severe morbidity and mortality may be associated with life-long steroid replacement therapy such as the possibly lethal addisonian crisis . however, data are limited, follow up is not standardized and not appropriately reported and rcts are difficult to be done due to the rarity of the disease. a multinational registry on the short term and long term outcomes of partial adrenalectomy in hereditary pheochromocytoma would be a significant source of knowledge. results: patients were operated on after an average of months with complaints. in both groups, the leading symptoms were severe dysphagia and severe regurgitation. no intraoperative complication was detected. in the transoral group, one patient had to be reoperated on for bleeding, another patient developed pneumonia in the transcervical group. the average duration of the surgeries ( . vs. min, p \ , ), the time to oral feeding ( . vs. . days, p \ , ) and the mean hospital stay ( . vs. . days, p \ , ) were significantly shorter in the transoral group than the transcervical group. patients were completely symptomless postoperatively. after transcervical treatment, complaints were developed in cases (moderate dysphagia and hoarseness). after transoral surgery, recurrent symptoms were observed in patients, had to be reoperated transcervically due to severe regurgitation. conclusion: transoral stapler diverticulostomy is a fast procedure and offers short hospital stay especially in comorbid, aged patients and intermedium diverticulum size. in the long term, some of the patients may require reintervention due to persistent regurgitation. the transcervical approach has higher perioperative morbidity, which can be performed in patients with less than cm or large diverticulum size. aims: complex hiatal hernias, either implicating large hiatal defects or concerning cases of recurrence, often need apart from the primary closure of the hiatal gap, the re-enforcement of the crura with the use of meshes. our aim is to demonstrate the surgical technique for the on-lay placement of the absorbable mesh (phasix tm st mesh /bard) in challenging cases, presenting both the laparoscopic and the robotic approach. methods: we present video fragments from procedures of laparoscopic and robotic reconstruction of complex hiatal hernias, performed by our team, in which an absorbable mesh was utilized in an on-lay fashion. results: patients having undergone a minimally invasive surgical approach (laparoscopic or robotic) for the treatment of complex hiatal hernias with the use of an absorbable mesh, had an uneventful post-operative course and very short hospital stay and recovery time. the -month follow up revealed no recurrences or late complications. conclusions: treating complex cases of hiatal hernias with a minimally invasive approach can be proven quite challenging, with high recurrences and possible complications rate. a proper surgical technique, either laparoscopic or better (based in our primary experience) robotic, by experienced surgical teams and the use of meshes with the right strategy, minimizes the complications, offers all the benefits of minimally invasive surgery and reduces the recurrence rates. aims: several flexible endoscopic techniques for symptomatic zenker's diverticulum have been developed during the last decade. thulium laser has limited tissue penetration and may decrease the risk of perforation. this study reports the first use of thulium laser through flexible endoscopy for cricopharyngeal (cp) myotomy. aims were safety and efficacy of flexible endoscopic thulium laser myotomy and quality of life (qol) changes after treatment. methods: a retrospective review of a prospectively collected database of patients who underwent thulium laser septum division for symptomatic zenker's diverticulum was done. demographic data, presenting symptoms, diverticulum characteristics, and intraoperative data were analyzed. functional outcome swallowing scale (foss) and m.d. anderson dysphagia inventory (mdadi) questionnaires were administered to determine severity of dysphagia and its effect on qol, both preoperatively and during follow-up visits. all the operations were carried out under general anesthesia. a continuous laser configuration and an emissionpower of w was used in non-contact mode. once the mucosa was opened, the fibers of the cricopharyngeal muscle were divided until the buccopharyngeal fascia was visibile. results: between march and september , patients ( males) underwent flexible endoscopic cp myotomy with thulium laser. mean age was ± . , mostly males ( . %). seven patients ( . %) presented with recurrent diverticulum after previous transoral or open treatment. mean diverticulum size was . ± . cm. preoperative main symptoms were dysphagia ( . %), regurgitation ( . %), and cough ( . %). foss score was = in patients ( . %). mean mdadi global and composite score were . ± . and . ± . . complete division of the septum was achieved in all patients. mean hospital stay was . ± . days. there was only one perforation treated conservatively. no -days mortality was observed. at median follow-up of months, foss was = in ( . %) patient and mdadi global and composite score were . ± . and . ± . . all main symptoms were significantly reduced and qol significantly increased. conclusions: flexible endoscopic approach with thulium laser is a safe and effective treatment option for zenker's diverticulum either as a primary treatment or as a rescue therapy. objective: this study sought to explore prognostic factors for patients with borrmann type iv gastric cancer and to establish a predictive model for survival benefit of postoperative adjuvant chemotherapy in such patients. method: this study reviewed the clinical data of patients who underwent curative surgery at fujian medical university union hospital from to for borrmann type iv gastric cancer using a prospective database. cox regression analyses were performed to identify prognostic factors that formed the basis for a nomogram and risk groups. establishment of risk groups to identify patients with borrmann type iv gastric cancer who would benefit from adjuvant chemotherapy. results: patients who underwent r resection were included in this study.multivariate analysis showed that bmi, tumour differentiation, pt stage, pn stage, and asa score were independent prognostic factors. patients in the act-group had longer os than patients in the sagroup, although the p-value for this difference was marginally above the threshold for statistical significance ( . % vs. . %, p = . ). stratified analysis showed that there was no significant difference in os between the act-group and the sa-group for each ajcc stage (stage ii: . % vs. . %, p = . ; stage iii: . % vs. . %, p = . ).a nomogram was established based on these independent risk factors, and nomogram scores were used to divide all patients into a high-risk group (score [ ), an intermediate-risk group ( \ score = ) and a low-risk group (score = ).further stratified analysis based on ajcc stage showed that the -year survival rate was higher in the adjuvant chemotherapy group than in the surgery alone group for low-and intermediate-risk patients in each ajcc stage, while high-risk patients in stage iii did not significantly differ. objective: this study sought to explore the prognostic factors for smoking patients with gastric cancer and to establish a predictive model for the survival benefit of postoperative adjuvant chemotherapy in such patients. methods: we studied patients who were diagnosed from september to september at union hospital of fujian medical university. cox regression analyses were performed to identify prognostic factors. the kaplan-meier method was used to assess the effect of smoking history on the benefit of adjuvant chemotherapy after gastric cancer surgery. a decision tree algorithm was used to identify smoking patients who benefited from postoperative adjuvant chemotherapy. results: the median follow-up time for the whole group was . months, and the average age of all the included patients was . years.multivariate analysis showed that age (p \ . ), bmi (p \ . ), degree of tumor cell differentiation (p \ . ), and ajcc stage (p \ . ) were independent risk factors for the prognosis of smoking patients. based on these independent risk factors, a decision tree model for the benefit of adjuvant chemotherapy for smokers with gastric cancer was established, and the smoking patients were divided into the low-risk patients . %), medium-risk patients ( - year os, . %) and high-risk patients ( - year os, . %) (p \ . ). conclusion: cigarette smoking may reduce the efficacy of adjuvant chemotherapy after gastric cancer surgery. our decision tree model is simple and effective for identifying smokers who would benefit from adjuvant chemotherapy. objective: our study investigated the effect of lymph node (ln) noncompliance on the longterm prognosis of patients after laparoscopic total gastrectomy (ltg) and explored the risk factors of ln noncompliance. methods: the clinicopathological data of gastric cancer (gc) patients who underwent ltg with d lymphadenectomy from june to december were prospectively collected and retrospectively analyzed. the effects of ln noncompliance on the long-term prognosis of patients with gc after ltg were explored. results: the overall ln noncompliance rate was . %. ln noncompliance was significantly correlated with age, bmi, asa score, tumor size, macroscopic tumor type and tnm staging (p values \ . ). the survival rate of patients after ltg with ln compliance was significantly superior to that of patients with ln noncompliance (p = . ). the stratified analysis of tnm stage indicated that there was no difference between the os of stage i patients with ln compliance and those with ln noncompliance; os of stage ii/iii patients with ln compliance was significantly better than that of those with ln noncompliance. cox regression analyses showed that ln noncompliance was an independent risk factor for os. logistic regression analysis showed that high bmi ([ kg/m ) was an independent risk factor for preoperative prediction of ln noncompliance in cstage ii/iii patients. compared with patients with a low bmi (bmi \ kg/m ), those with a high bmi were more likely to show ln noncompliance during surgery, especially during the dissections of # , # a and # a ln stations. conclusion: ln noncompliance was an independent risk factor for poor prognosis in patients with advanced gastric cancer (agc) after ltg. patients with high bmi were more likely to have ln noncompliance, especially during the dissections of # , # a and # a ln stations. ln tracing was recommended for these patients to reduce the rate of ln noncompliance. aim: to study the differences in pathology, survival, and recurrence between special remnant gastric cancer (srgc) and nonspecial rgc (nrgc). method: a total of rgc patients were analyzed in hospitals in china from january to july .we compared the -year overall survival (os) disease-free survival (dfs) rates and used two-step regression explore the influence of the rgc categories on patient outcomes. results: all of the patients divided into srgc group (group s) (n = ) and nrgc group (group n) (n = ). the r resection rate and lymph node (ln) dissection number of group s were significantly higher than group n (p \ . ). the difference in -year os was not significant (p = . ), but the -year dfs of group s was worse than group n (p = . ). twostep multivariate analyses showed nrgc was an independent risk factor for poor dfs. of the patients who had undergone r resection, patients ( . %),suffered recurrence, and the recurrence rate of group s was significantly higher than group n (p = . ), moreover, the ln recurrence rate of group s was significantly higher than group n (p = . ). cox regression analysis showed that age, ca level, n stage and category of rgc were independent risk factors for rgc recurrence. conclusion: srgc has a higher r resection rate and ln dissection number than nrgc, but among patients who had undergone radical gastrectomy, srgc patients had worse dfs and a higher tendency for ln recurrence; thus, they should be treated differently in the clinic. objective: the aim of this study was to report our institution's experience with a novel abdominal negative pressure lavage-drainage system (anplds) for anastomotic leakage (al) after radical gastrectomy (rg) for gastric cancer (gc). background: al is a severe complication associated with high morbidity and mortality after rg for gc. the optimal creation of drainage in al patients after rg remains controversial. methods: the study enrolled patients who underwent r resection for gc at our institution between and . anplds was routinely used for patients with al after january . al rates and postoperative outcome were compared before and after the anplds therapy. we used multivariate analyses to evaluate clinicopathological and perioperative factors for associations with al and failure-to-rescue (ftr) after al. results: al occurred in patients ( / , %), leading to deaths. the al rate was similar before ( - , period ) and after ( - , period ) the implementation of anplds ( . % vs . %, p = . ). age and malnourished were independently associated with al. the ftr rate and abdominal bleeding rate after al occurred were respectively . % and . % for the entire period, but compared with period , it significantly decreased at period ( . % vs . %, p = . ; . % vs . %, p = . , respectively). what's more, only anplds therapy was an independent protective factor for ftr after al. conclusion: our experience demonstrates that anplds is feasible and cost-effective for the management of al after rg for gc. objective: to apply the principles of the 'metro-ticket' paradigm to develop a novel tnm staging system (ntnm) for gastric cancer (gc). background: the 'metro-ticket' prognostic tool for hepatocellular carcinoma has been proven to predict outcome, but a similar concept has not been investigated for gc. methods: the ntnm considered the distance from the origin on a cartesian plane incorporating the pn (x-axis) and pt (y-axis) stages. gc patients undergoing radical resection at fujian medical university union hospital (fmuuh) (n = ) were included. the ntnm was validated using external cohorts from the sun yat-sen university cancer center (sysucc) (n = ) and surveillance, epidemiology, and end results (seer) (n = ) databases. results: ntnm classes with the same distance from the origin have same stage; the stage increases with this distance. among all patients, . % (n = ) were restaged in the ntnm compared with the th edition of the ajcc-tnm classification; . % (n = ) were downstaged in the ntnm compared with the th edition. the ntnm provides significant survival differences between stages (all p \ . ). the survival difference between stages ib and iia was especially large for the ntnm (p \ . ) compared to the th and th editions (p = . ). the concordance index and hazard ratio increased successively with the ntnm stage. similar findings were observed in both external cohorts. conclusion: compared with the ajcc-tnm classification, the 'metro-ticket' ntnm for gc is easier to remember and provides some improvements; therefore, the ntnm may be considered for adoption in future editions of the ajcc-tnm classification. objective: to investigate the prognostic value of complete blood count (cbc)-based biomarkers for patients with resectable gastric cancer (gc). methods: patients with gc who underwent curative resection between december to december were included. estimated area under the curve (auc) and multivariate cox regression models were used to identify the best cbc-based biomarker. time-dependent receiver operating characteristics (t-roc) analysis was used to compare the prognostic impact. results: based on multivariate analysis, the lymphocyte-monocyte ratio (lmr) and hemoglobin (hb) level were the independent prognostic factors (both p \ . ). based on the lmr and hb level, we established the cbc-based inflammatory score (cbcs). higher cbcs was associated with older age, female sex, higher american society of anesthesiologists (asa) score, proximal tumor location, larger tumor size, later stage and vascular involvement (all p \ . ). univariate analyses showed that higher cbcs was also associated with poorer overall survival (os), which was consistent in each stage (all p \ . ). multivariate analysis revealed that the cbcs was a significant independent biomarker (p \ . ). furthermore, t-roc curve of the cbcs was superior to that of the prognostic nutritional index (pni), systemic immune-inflammation index (sii), modified glasgow prognostic score (mgps) and c-reactive protein/albumin ratio (crp/ alb) throughout the observation period. conclusion: preoperative lmr and hb were optimal cbc-based biomarkers for predicting os in gc patients after curative resection. based on the lmr and hb, we developed a novel and easily obtainable prognostic score called the cbcs, which may improve the prediction of clinical outcomes. purpose: the aim of this study was to evaluate the prognostic value of the eighth ajcc tnm staging classification for patients with gastric cancer who had already survived for years. patients and methods: patients who underwent radical gastrectomy at a large eastern center were considered. the prognostic value of staging systems were assessed and compared. additional external validation was performed using a dataset from the surveillance, epidemiology, and end result (seer) database. results: the -year overall survival (os) rate for patients in the training set was . %. with the prolongation of the survival time after surgery, the -year os improved significantly (p \ . ). however, there were no significant differences in survival curves among patients who have survived years after surgery. the auc and c of the eighth ajcc classification for predicting of -year os decreased gradually after surgery and appeared stable after years. for patients who survived years after surgery, we constructed a new tnm staging system (ntnm) according to the survival curves of t stage and n stage. a -step multivariate analysis showed that ntnm, age and sex were independent prognostic factors. the ntnm demonstrated superior prognostic stratification, with higher c-statistic and likelihood ratio chi-square scores and lower aic values than those of the ajcc classification. similar results were observed in the external validation set. conclusion: the ntnm predicted an additional survival more accurately than did the ajcc classification for patients who have survived years after surgery; this may guide decisions regarding surveillance. objective: to investigate the relationship between preoperative sarcopenia and systemic inflammation and evaluate the prognostic impact of these factors on patients with resectable gastric cancer (gc). methods: patients with gc who underwent radical gastrectomy between december and december were included. a multivariate cox regression analysis was performed to identify the prognostic factors. a novel prognostic score (slmr) was developed based on preoperative sarcopenia and the lymphocyte-monocyte ratio (lmr), and its prognostic value was evaluated. results: in total, patients with resectable gc were included in the study. on multivariate analysis, preoperative sarcopenia and the lmr were shown to be independent prognostic factors (both p \ . ). a low lmr was an independent predictor from sarcopenia (p \ . ). based on preoperative sarcopenia and the lmr, we established the slmr. an elevated slmr was associated with older age, higher asa scores, larger tumor size, advanced stages and vascular invasion (all p \ . ). multivariate analysis revealed that the slmr was a significant independent predictor (p \ . ). we incorporated the slmr into a prognostic model that included tumor size and tnm stage and generated a nomogram, which accurately predicted -and -year survival for gc patients. objective: to explore whether adjuvant chemotherapy is still needed in patients aged less than years with pt n - and pt / n gastric cancer. methods: multi-center cohort data of patients with gastric cancer who underwent radical gastrectomy were analyzed. kaplan-meier curves and cox regression were used to analyze the relationships between chemotherapy and prognosis. additionally, nomograms to predict the benefit of chemotherapy were established. results: in total, , patients with pt n - and pt / n gastric cancer were included. patients ( . %) were aged \ years. the -year overall survival (os) was not significantly different between the \ years of age group and = years of age group ( . % vs. . %, respectively; p = . ). lymph node (ln) metastases (hr . ; p = . ) and ln dissection number \ (hr . ; p \ . ) were independent risk factors for the os of patients aged \ years. adjuvant chemotherapy did not improve the -year os for patients aged \ years with pt n - and pt / n gastric cancer (p = . ). however, chemotherapy showed a significant benefit (p = . ) when there were ln metastases and/or ln dissection number was \ . two nomograms were constructed, and the calculated difference was the potential benefit of adjuvant chemotherapy for the patients aged \ years. conclusions: ln metastases and ln dissection number \ were independent prognostic risk factors of patients aged \ years with pt n - and pt / n gastric cancer. patients with these risk factors may benefit from the addition of adjuvant chemotherapy. objective: the choice of reconstruction after distal gastrectomy remains controversial. we have performed roux-en-y (r-y) method after laparoscopic distal gastrectomy(ldg) as a standard since , but we have performed billroth ii (b-ii) method in an increasing number of cases, depending on the patient. we retrospectively investigated the outcomes of patients with b-ii method after laparoscopic distal gastrectomy in our hospital. methods: patients who underwent b-ii and r-y reconstruction after ldg from january to december were included. the patient characteristics, surgical outcomes, and postoperative outcomes between the procedures were retrospectively analyzed. we also compared extend of gastritis on endoscopy and loss of body weight after surgery at year. results: b-ii / r-y : / . b-ii was selected in the elderly patients with poor asa-ps (p \ . ). in surgical outcomes, operative time was shorter for b-ii than r-y (p \ . ), and blood loss was also smaller (p = . ). in postoperative outcomes, there were significant differences in complications (?grade ) (b-ii vs. r-y: . vs. . %, p = . ) and length of stay (b-ii vs. r-y: median . vs. -day, p \ . ). there was significant difference in presence of gastritis between b-ii ( . %) and r-y ( . %) (p \ . ), but no significant difference in loss of body weight (p = . ). conclusion: b-ii reconstruction may be an adequate procedure for high-risk cases because of its shorter operative time and the absence of severe complications. background: numerous studies have shown that the short-term efficacy of three-dimensional ( d) laparoscopic radical gastrectomy (lg) is comparable to that of two-dimensional ( d)-lg. whether d-lg affects the recurrence pattern after surgery has not been investigated. using data from a prospective clinical trial, the present study compares the recurrence patterns between d-lg and d-lg. methods: from january to april , a total of patients were recruited for the clinical trial (nct ). the recurrence types, the first recurrence time and recurrence-free survival (rfs) were compared between the two groups. multivariate analyses of factors associated with rfs were performed to identify whether d-lg affects the recurrence patterns. results: ultimately, patients were analyzed ( in the d-lg group and in the d-lg group), and there were no differences in the clinicopathological data between the two groups. distant metastasis was the most common type of recurrence. there were no significant differences between the two groups in the recurrence types, the first recurrence time or rfs (all p [ . ). according to the th american joint committee on cancer tumor-node-metastasis (tnm) staging system, both groups were stratified into pathological (p) i, ii, and iii stages. the stratified analysis showed that there were no statistically significant differences in rfs between the d group and the d group among patients in each subgroup (all p [ . ). the multivariate analysis of rfs showed that pathological tnm (ptnm) stage and lymphovascular invasion were independent risk factors (all p \ . ). the multivariate analysis of post-recurrence survival (prs) showed that adjuvant chemotherapy was an independent protective factor (p = . ). conclusions: distant metastasis was the most common type of recurrence after lg. the postoperative recurrence patterns, rfs and prs after d-lg were similar to those after d-lg. purpose: the aim of this study is to evaluate the efficacy of delta-shaped anastomosis compared to circular stapler anastomosis in laparoscopic distal gastrectomy with billroth i reconstruction (ladg-bi). method: this is a single-center randomized controlled study. eligibility criteria included histologically proven gastric adenocarcinoma in the lower third of the stomach, clinical stage i tumor. patients were preoperatively randomized to circular stapler anastomosis or delta-shaped anastomosis. the primary endpoint is the number of analgesics use during days after surgery. we compared the surgical outcomes of the two groups. postoperative qol was evaluated using the postgastrectomy syndrome assessment scale- . this trial was registered at the umin clinical trials registry as umin . results: between december and september , patients (delta-shaped anastomosis , circular stapler anastomosis ) were enrolled. there was no difference in the number of analgesics use during day after surgery (median : delta-shaped anastomosis vs. : circular stapler anastomosis, p = . ). there was no difference in the overall proportion with in-hospital grade ii-iiib surgical complications ( %: delta-shaped anastomosis, %: circular stapler anastomosis). there was no operation-related death in either arm. regarding postoperative qol evaluated month after surgery, diarrhea subscale was significantly worse in delta-shaped anastomosis than in circular stapler anastomosis. conclusion: we did not demonstrate the advantage of delta-shaped anastomosis in terms of postoperative pain. since delta-shaped anastomosis tended to cause postoperative abdominal symptoms related to diarrhea, we should carefully apply the delta-shaped anastomosis to ladg. introduction: the use of a three-dimensional( d) camera for laparoscopic surgery has been reported in literature. however, there are only few comparative studies demonstrating its benefits, and no reports on the application of d vision to single-incision laparoscopic surgery. this study aims to compare d vision to the previous two-dimensional( d) system in solo single-incision laparoscopic distal gastrectomy(sidg). methods: medical charts of gastric cancer patients who underwent solo sidg from february to december were retrospectively reviewed. patients were grouped into either d group or d group depending on the type of camera used. all the operations were performed by a single surgeon using a flexible camera(olympus, japan), fixed onto a passive scope holder without the use of a scopist or an assistant. operative data, postoperative outcome, and early complication were analyzed. results: ninety had their operations under d vision and used the d scope. in both groups, there was no difference in age, body mass index, staging, and other demographic or histopathologic criteria. operative time was significantly faster in the d group( . ± . vs. . ± . mins., p = . ) and ebl was also less( . ± . vs. . ± . ml, p = . ). patients in the d group started small fluid diet faster( . ± . vs. . ± . postoperative days, p = . ), and were discharged faster( . ± . vs. . ± . postoperative days, p = . ). early complication was also less in the d group( . % vs. . %) but there was no statistical significance(p = . ). conclusion: the use of the d camera improves operative outcome and hospital stay in patients undergoing solo sidg. the frequency of anastomotic leakage after gastrectomyreaches - %. at the same time, mortality in this group of patients reaches %, and the use of aggressive methods of surgical treatment for the treatment of anastomotic leakage increases the mortality rate from to %. since , vacuum-assisted closure has been used to treat anastomotic leakage of various localizations. the essence of this method is based on the creating a local negative pressure, which is transmitted to the drip cavity through a special porous spongy system. the negative pressure created in the closed cavity, allows you to remove exudate, helps to reduce tissue swelling, improvesmicrocirculation, which in turn contributes to the development of granulations and wound healing with separation of the fistulous course. failures in using the method of vacuum therapy in anastomotic leakage are associated with the great difficulty of delivering a polyurethane sponge with a drainage tube to the leakage zone. in this regard, we have developed an improved method of endoscopic local vacuum therapy, in which the delivery of a polyurethane sponge was carried out with the help of a thread through a pharyngeal ring, a leakage zone and brought out through a drainage tube. this technique has been successfully used in the treatment of four patients with anastomotic leakage after operations on the upper part of the digestive tract. for complete healing of the cavity of the leakage and defect of the organ wall, it took , , and sessions of replacing the vac system, respectively (average . ± . ). there were no complications during the endoscopic local vacuum therapy. when the control endoscopic studies after months after the completion of the treatment at the site of defects of the seams of the anastomoses formed tender scar tissue without signs of narrowing of the organ. aims: enhanced recovery after surgery pathways are safe and effective for patients undergoing gastrectomy. this study aimed to identify perioperative factors influencing the adherence to the protocol, the postoperative course, and the consequent length of stay. methods: between and , patients were referred to our institution for gastric cancer. among these, patients underwent atypical gastric resection and were excluded from this analysis. were assigned to either total or distal gastrectomy and represent the study population. all patients were managed with a standardised perioperative pathway according to eras principles. according to data from the literature and based on our clinical experience, patients with optimal adherence to eras protocol may fit the criteria for discharge within ninth postoperative day, that was considered our ideal threshold for hospital discharge. data were retrospectively collected and analysed from a prospectively maintained database. statistical analyses were performed using spss version for macintosh. the v test, with a significance level of . , was used to investigate the association between the outcome and perioperative categorical variables. when parametric assumptions were met, student's two-tailed t-test was used to compare the means of continuous variables; otherwise, the mann-whitney test was performed. a significance level of . was chosen. logistic binary regression with a backward selection procedure and selection criteria of p-value \ . were exploited to determine significant predictors. results: preoperative, intraoperative and early postoperative variables were considered. among all, multivariate regression analysis revealed that incomplete preoperative immunonutrition, failure to extubate the patient at the end of surgery, intraoperative crystalloids infusions [ ml and blood transfusion [ ml, surgery duration [ min, and failure to mobilise patients within h from surgery were associated with delayed discharge. the logistic regression model was statistically significant (p \ . ) and correctly classified . % of cases. sensitivity and specificity were . % and . %, respectively. conclusions: results seem to be clinically rational and focus the attention on the importance of some perioperative clinical issues for the management of postoperative course. these variables could be considered as clinical goals to be reached in order to get an early discharge. objectives: the purpose of this study is to confirm the safety of laparoscopic gastrectomy with intraperitoneal cisplatin administration as a treatment for advanced gastric cancer with potential for peritoneal seeding. methods: from july to august , patients with advanced gastric cancer who underwent ip chemotherapy after diagnostic laparoscopy were retrospectively studied. all patients underwent laparoscopic gastrectomy with ip chemotherapy or ip chemotherapy alone after a diagnostic laparoscopy. gastrectomy was performed for palliative purposes even with seeding. results: the average age of the patients was years. eight patients ( . %) had preop chemotherapy. curative resection (r ) was performed in patients ( . %). in diagnostic laparoscopy, cytology was performed in patients ( . %) and cy was ( . %). peritoneal metastasis was detected in patients ( . %). of the total cohort, the year os rate was . % and the median survival time was months. in the case of stage iiib and below, the -year os rate was %, but it was % in stage iiic-iv group. when the r resection group and the r - resection group were compared, the -year os rates were . % and . %, respectively. hematological toxicity such as neutropenia was not seen in all patients. the mean hospital stay was . days and adjuvant chemotherapy was performed in patients ( . %). background: radical proximal gastrectomy (pg) and lymph nodes dissection are indicated for selected gastric cancers at the upper third of the stomach. with the advent of laparoscopic surgeries, more and more pg were performed by laparoscopic apporaches. in the past years, our team has accomplished and reported the oncological outcome of laparoscopic distal gastrectomies in cases of clinical stage i gastric cancer in taiwan. through the evolution of surgical trechniques and team work, we have cruised the learning curve of laparoscopic gastrectomy and reconstruction. materials and methods: in this report,we would like to present our surgical experience of laparoscopic proximal gastrectomy for gastric cancer patients. from to , pateints with gastric cancer underwent laparoscopic gastrectomies by the same surgical team at the national taiwan university. among them,six consecutive pateints (male:female = : ) with gastric adenocarcinoma of the upper stomach underwent laparoscopic pg in . the demographics, dissection, reconstruction methods and peri-operative outcome are presented. all six patients tolerated the procedure well, onepatient had mild anastomotic stenosis and improved with one session of endoscopic dilatation. one patient needed temporary proton pump inhibitor for controlloing acid reflux. four of the patients were pathological stage i, and the rest two pateint were stage iia and iiia disease. there was no tumor recurrence until now. summary: laparoscopic proximal gastrectomy is technically safe for treating upper third gastric cancers. the long term oncological outcome deserve further observation. introduction: open gastrectomy (og) has long been the preferred surgical approach worldwide for treatment of gastric cancer (gc). nowadays, several randomized, prospective trials have confirmed improvements in postoperative outcomes for laparoscopic gastrectomy (lg) compared to open procedures, with similar oncologic outcomes. however, most part of these studies comes from the eastern countries. material and methods: a prospective non randomized study was conducted with all patients operated of gc at ramón y cajal university hospital from january to december . over patients enrolled, patients underwent lg and og. textbook outcome was defined as the percentage of patients who underwent a complete tumour resection with at least lymph nodes in the resected specimen and an uneventful postoperative course, without hospital readmission. results: a textbook outcome was achieved in . % of patients operated of gc. the outcome parameter 'no severe postoperative complication' had the greatest negative impact on the textbook outcome. a statistically higher number of patients with early cancer ( % vs. . %) and subtotal gastrectomy ( . % vs. . %) were found in the laparoscopic group. no statistically differences were found between open and laparoscopic approach regarding operating time, rate of microscopic margin positivity, hospital stay, number of retrieved lymph nodes, complications, reinterventions, mortality and readmissions. no statistical differences in textbook outcome were found between both groups ( . % vs. %; p = . ). conclusions: laparoscopic gastrectomy for treatment of gastric cancer seems to be safe and feasible with similar textbook outcomes compared to open gastrectomy. introduction: laparoscopic surgery has been increasing for treatment of gastric cancer. however, standardization of this minimally invasive approach has not been reached yet because of its technical difficulties and the concern about oncological safety. the aim of the study was to analyze the outcomes of our learning curve in this complex surgical technique. material and methods: the first consecutive cases of laparoscopic gastrectomy (lg) performed at our hospital from november to february were enrolled. patients were divided into two groups based on the period they were operated. training phase (tp) was considered between and ( cases) and more-developed phase (mdp) between and ( cases). conversion, lymphadenectomy and retrieved lymph nodes (ln), hospital length of stay, mean operative time, complications, reintervention and mortality rates were compared between the two phases of learning curve. results: the number of retrieved ln was higher in the mdp ( ± , vs. , ± , ; p = , ). furthermore, we have also found less complications ( , % vs. , %; p = , ), a decreased reintervention rate ( , % vs. , %; p = , ) and overall mortality ( , % vs. %; p = , ) in the mdp. there were no significant differences in conversion rate, mean operative time, and hospital length of stay between phases. conclusion: although we consider that our learning curve is not completed yet because the average of monitored parameters have not reached a steady state, the improvement on surgical parameters and postoperative course in the last two years have showed our results are near to the best results published in the literature. aims: lymph node (ln) dissection proves to be essential for oncological gastrectomy, given that the presence of ln metastases is very high, even for early gastric cancer ( . % for t a and . % for t b). this way, d dissection for advanced gastric cancer and d ? for early gastric cancer are the gold standard procedures. some teams are using indocyanine green (icg) lymphography to improve their ln dissections, claiming that this technique facilitates the harvesting of small fluorescent ln that, otherwise, would be difficult to identify by conventional laparoscopic methods. methods: we herein present the case of a -year-old man with a t b distal gastric cancer. endoscopic ultrasound discarded the presence of metastatic ln and ct scan showed no distant metastases. icg was administrated endoscopically the day before the surgery, an amount of mg was injected along the submucosal layer around the tumour. in the video we can see how we perform a laparoscopic distal gastrectomy with d ? ln dissection and roux-en-y reconstruction. icg lymphography helped us to complete our expected ln harvesting, especially for groups (infrapyloric) and (left gastric artery). thanks to this technique, we could resect ln that we might have obviate during a usual laparoscopic procedure. results: patient was discharged home on the sixth postoperative day without complications and with adequate oral tolerance. conclusions: we present a case in which we have performed a laparoscopic distal gastrectomy with d ? dissection and roux-en-y reconstruction. we used icg lymphography to help us to improve our ln harvesting. although it is soon to assess if this technique may increase the number of retrieved ln and in which stations might be more useful, we consider this is a harmless method that may help gastric teams to complete their expected ln dissections. introduction: gastrointestinal stromal tumor (gist) represents around . % to % of gastrointestinal neoplasms, with the mesenchymal tumor being more frequent than the digestive one.the gist can be produced from the esophagus to the anus, at any point, being the stomach of ( to %) and the small intestine ( to %) more frequent sites.it is characterized by the expression of the tyrosine kinase growth factor receptor,cd ,differentiating it from other mesenchymal tumors,which do not express it.it is accepted that its origin corresponds to the interstitial cells of ramón y cajal,which act as a pacemaker for intestinal motility.they are very heterogeneous tumors, which vary in size,morphology and biological behavior,being neoplasms with uncertain malignant potential.the incidence is between the fourth and sixth decades,being the distribution by gender similar. clinical case: female patient of years,who goes to the general surgery service,as interconsultation,after a veda,by dyspepsia.it is reported stomach:ceiling mucosa without alterations,at the level of the greater curvature is seen a tumor of cm,hard to the touch with the biopsy forceps,slightly irregular covered with mucosa of normal appearance. computed tomography: stomach body:rounded image of nodular aspect which does not present heterogeneous enhancement after administration intravenous iodine contrast extending to peritoneal region, measures x x mm liver:hypodense image without heterogeneous enhancement adjacent to this,a mm rounded image that is suggested to be studied with nmr. gadolinium nmr liver hypodense image with well-defined limits without heterogeneous enhancement of cystic aspect. gastric roof,heterogeneous formation,which enhances with gadolinium mmx m-mx mm,having to discard a gist. surgical technique laparoscopic partial gastrectomy. pathological anatomy and immunohistochemistry . cm injury with net edges.uncertain malignant fusocellular nodule, cd ??? actin-dog ??? s -no mitosis or invasion of the mucosa is observed. conclusion: a case of stomach gist is presented,which,the main symptom was dyspepsia,being the clinical presentation very variable,in relation to the place in which it is located. there is fletcher criteria for the risk of malignancy,this being less than . cm,very low risk,less than cm, the patient evolved favorably,without surgical complications.aims: to present the surgical procedure of resection of the lesser gastric curvature and its pedicle with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: an -year-old man with prostate cancer treated with complete hormonal block and epoc, who consults for rectal bleeding of week of evolution. it is diagnosed of gist in gastric lesser curvature, x x cm, very vascularized and infiltrates the wall producing marked imprint on the fundus. it is tributary of left gastric artery. precise blood transfusion and presents hemodynamic stability, is decided surgical resection scheduled. results: the surgery is performed by laparoscopy, with a tumor of approximately cm, which is dependent on the lesser curvature. the esophageal hiatus and the lesser curvature are dissected with section of the left gastric pedicle. atypical gastrectomy of the lesser curvature including gist, making a gastric sleeve dependent on the greater curvature. the anatomopathological study reports pt pn with lymph nodes without adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require re-entry. conclusions: the laparoscopy surgery for atypical gastrectomy of lesser curvature is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. aims: to present the surgical procedure of total gastrectomy with d lymphadenectomy with laparoscopic surgery, fulfilling oncological criteria, carried out in the general surgery service of the hospital of torrecárdenas. methods: a -year-old male with a tobacco habit who consults due to epigastric pain and constitutional syndrome of months of evolution. it is diagnosed of gastric adenocarcinoma t n m . neoadjuvant qt is decided, after weeks of its completion, scheduled surgery is performed. results: the surgery is performed by laparoscopy, showing a stenosing tumor in at gastric antrum of approximately cm. dissection of the greater curvature with section of the right gastroepiploic at its birth and duodenal section is performed. dissection of the lesser curvature with d lymphadenectomy, section of the pedicle of the left gastric and the distal esophagus. the transit is restored with latero-lateral esophageal-jejunal anastomosis and jejunojejunostomy. the anatomopathological study reports ypt a and pn with / adenopathies, and disease-free surgical margins. he was discharged without complications on the th day and did not require reentry. conclusions: the laparoscopy surgery for total gastrectomy with complete d lymphadenectomy is safe and meets oncological criteria in selected patients and performed by an experienced esophagogastric unit. background: in gastric cancer surgery, to secure surgical margin, it is necessary to accurately judge the position of the tumor. however, with conventional marking clips, it is difficult to identify the exact location of the tumor during laparoscopic surgery. purpose: we investigate whether icg (indocyanine green) fluorescence navigation method is effective and safe for determination of cutting line in laparoscopic gastrectomy. patients and methods: subjects underwent laparoscopic gastrectomy (including robot-assisted surgery) based on the icg method for gastric cancer in the period from april to december . the day before surgery, icg diluted times ( . ml of reagent ? . ml of distilled water) was injected at cm from the tumor edge and . ml at the four submucosal layers around. then clip to the same part. gastrectomy based on standard surgery is performed, and the position of the tumor and spread of icg are confirmed by icg fluorescence navigation during operation, and a cutting line is determined. the extent of icg from the tumor is again measured with the excised specimen and compared with the pathological margin. results: among the patients who underwent intraoperative pathological examination, they were negative in all cases except one. the spread of icg was . cm on average, and considering the marking position ( cm) from the tumor edge, securing of . cm or more was possible. the operation time was . ± . min and the estimated bleeding loss was . ± . ml. conclusion: laparoscopic gastrectomy with icg method can evaluate tumor position and spread easily and in real time during operation and it was effective for determining the cutting line in laparoscopic gastrectomy. epstein-barr virus (ebv) has been known as one of causal virus of gastric cancer. ebv-related gastric cancer considered to be about % of the entire gastric cancer, and it is rare that ebvrelated gastric cancer has multiple lesions. the patient was years old female. she was diagnosed with upper gastrointestinal endoscopy with lesion in the lower major stomach body, lower anterior wall of the stomach body, rear wall in the middle part of the stomach, rear wall in the middle part of the stomach, and lesser curvature of the stomach angle, as a result of biopsy, adenocarcinoma was observed from the former four. the patient underwent a robot-assisted total gastrectomy. adding a newly found lesion, the histopathological diagnosis was pt b in the lower major stomach body, pt b in the lower anterior wall of the stomach body, pt b on rear wall in the middle part of the stomach, pt b on rear wall in the middle part of the stomach, and pt a in lesser stomach body, pn , pstageia. pathological examination results showed that the four lesions were positive for tumor cells in eber in situ hybridization and were considered to be ebv-related gastric cancers. she was discharged on the th day after the operation without any postoperative morbidities.there has been no sign of recurrence without postoperative therapy for months. results: a -year-old female with no medical history of interest or allergies to medications, who consulted for palpable mass at mesogastric level to the left of the midline associated with abdominal pain of - months of evolution, without concomitants or relationship with the intake, valsalva or physical efforts, without change in the depositional habit or toxic syndrome. the abdominal ct (computed tomography) revealed a cystic mass in jejunum mesentery, defined edges, about cm in diameter and that does not capture contrast; likewise, there is no ascites, retroperitoneal adenopathies or other intra-abdominal or pelvic masses, radiology recommends completing the study with abdominal mri (magnetic resonance imaging) that informs of possible lymphangioma at the level of the jejune mesentery. surgical exeresis was decided, which was carried out by laparoscopic approach, with emptying of the lesion and enucleation of the lesion without incidents, the postoperative evolution was favorable being discharged at h. the pathological anatomy reported fibro-adipose tissue with presence of lymphatic dilatations associated with a cystic lesion without epithelial lining, with serous fluid and abundant macrophagic reaction compatible with mesenteric lymphangioma. conclusions: the mesenteric cyst is a rare pathology with an incidence ranging from / , to / , , predominating in the fourth decade of life. it is defined as any cystic lesion in the mesentery, and is subdivided according to its origin into lymphatic, mesothelial, urogenital, dermoid, and enteric and pseudocysts. most of the time they are asymptomatic although they can (as in our case) present with abdominal pain and even produce complications such as intestinal obstruction, volvulus, intracystic hemorrhage, infection, rupture, and even malignant transformation. for the diagnosis, the palpation can be of great help, showing mass of well-defined limits and partially mobile. the imaging test of choice is abdominal ultrasound / abdominal ct, supplemented by magnetic resonance imaging. the recommended treatment is surgical exeresis, considering laparoscopy as the first option; if it is complete, it can be considered as a curative treatment. purpose: gastrostomy(og) is an alternative method for nutrition support, especial for the patients with oral-esophagus route obstruction or dysfunction. the most operation were conducted by young surgeon or residents. laparoscopic gastrostomy(lg) was a new coming procedure and the skillful suture techniques were needed. the most the residents can't be qualified for this operation. we designed the method for laparoscopic gastrostomy to provide the traning opportunity of suture skill training and guarantee the patient's safety. material and method: laparoscopic gastrostomy procedure was done with two mm trocar. the lower body of stomach was chose. four point around gastrostomy wound were chose for subcutaneous fixation. the straight needle with - prolene was inserted into peritoneal cavity from upper point, then punctured the sero-muscular layer of stomach. the needle was retrieved out from the same point by guidance of gauge needle. the same way was used for other three points. one purse string around gastrostomy was created by one hand suture method and fastened by köckerling knot tier after insertion of fr foley tube. finally, the peritonization was finished by hand tie externally and knot were keep in subcutaneous layer material-method: we present the case of a year old woman who presented with melena, hematemesis, anemia (ht . %) and being haemodynamically unstable. after the stabilization of the patient, a gastroscopic examination followed, where it revealed a tumor of the fundus (adenocarcinoma). the patient was submitted to laparoscopic total gastrectomy and oesophagojejunal anastomosis, omega type (o), and intestinal anastomosis braun, with the usage of trocars (umbilical mm as inserted in laparoscopic surgery of a single incision, and two mm in the midclavicular line bilaterally). the oesophagojejunal anastomosis was conducted with the use of a linear stapler for the posterior wall and the convergence of the anterior wall with laparoscopic sutures in two layers. patient remains in well condition, months after the operation. conclusion: laparoscopic approach seems to be safe for treatment of gastric cancer of the fundus and of the gastroesophangeal junction, as it offers better surgical field view and less postoperative complications. the restoration of the continuity of the gastrointestinal tube with anastomosis of w type is considered safe alternative to the classic roux-en-y anastomosis. git and bariatric surgery, faculty of medicine, alexandria university, alexandria, egypt; git surgery, faculty of medicine, alexandria, egypt background: superior mesenteric artery syndrome is best described as compression of the third part of duodenum by the superior mesenteric artery, resulting in obstruction. the study of this rare medical condition was carried out since decades yet remain obscure. this study aimed to analyze different clinical presentations, diagnostic modalities, treatment approaches and outcomes, as well as to emphasize the importance of long term follow up. methods: thirty-five superior mesenteric artery syndrome cases were collected retrospectively from a facebook group called 'superior mesenteric artery syndrome awareness & support'. a questionnaire was designed using google form to obtain the demographics, presenting symptoms, risk factors and co-morbidities, investigations, means of treatment and the outcomes. data was entered into microsoft office excel for statistical analysis. results: the median age at diagnosis was years. the median body mass index was . kg/ m ;. the median time interval from symptom onset to initial diagnosis was months. the major presenting symptoms were abdominal pain ( . %), nausea ( . %), and vomiting ( . %). abdominal computed tomography scan with contrast ( . %) was commonly used for confirmation of diagnosis. thirteen cases ( . %) were congenital. thirty patients ( . %) had received treatment. the overall management success was only . %. surgical management ( . %) was the most used regimen. conclusion: diagnosis of superior mesenteric artery syndrome is established after a thorough assessment of the clinical presentations and confirmation with suitable imaging modalities. the choice of treatment should be dependent on the causes and severity as different patients respond differently to therapy. recurrence is possible in all patients thus a long-term follow up is required. aims: in the last hundred years much has been written on peptic ulcer disease and the treatment options for one of its most common complications: perforation. laparoscopic repair of perforated peptic ulcer has been gaining popularity in recent years. treatment for perforated ulcer can be performed laparoscopically in % of cases, making it possible to avoid a median laparotomy which can lead to wound infection and late eventration. methods: a -year-old male presented to emergency room with a three-hour history of progressively worsening epigastric pain and nausea. physical examination revealed rebound tenderness compatible with an acute abdomen. a ct scan showed: important pneumoperitoneum unable to define the drilling point; distended stomach with plenty of fluid inside and dense content fundus / body suggestive of active arterial bleeding . results: the patient was emergently taken to the operating room for diagnostic laparoscopy . perforation shown in greater gastric curvature associated blood remnants. gastrotomy for clot removal is done without observing active bleeding. the gastrotomy was repaired using standard stitches. all exudate was aspirated and the peritoneal cavity was irrigated with warm saline solution the patient had an uncomplicated post-operative course. jp drain was removed and he was discharged one week after surgery. conclusion: the role of laparoscopic surgery in emergencies is well documented. laparoscopic approach is indicated in any case of suspected gastroduodenal perforation and seems to offer the same advantages as for the vast majority of laparoscopic procedures. laparoscopic surgery may therefore have a real place in the treatment of perforated peptic ulcer. the aim: of our study was to evaluate of effectiveness of local injection of platelet-rich plasma for treatment of peptic ulcer bleeding with hemorrhagic shock in experiment. methods: the study was performed on wistar rats according to local and international rules for working with experimental animals. the average weight of animals was ± grams. in all animals our modification of type acetic acid ulcer (susumu okabe, ) was modeled. we randomly divide all animals in groups. rats with only modeled ulcer were included in group . rats with modeled ulcer and hemorrhagic shock after - . ml blood sampling were included in group . in group we included rats with modeled ulcer and hemorrhagic shock and performed local injection of platelet-rich plasma (local periulcelar injection of . ml of autologous platelet-rich plasma). on st, th and th day measurement of the ulcers square and morphological study were performed. results: the data we have received demonstrate a tendency of decrease of ulcers' square in all groups with time flow. we also compared sizes of ulcerative defects in all groups at every point of the study. on the st day of investigation there were no differences (p [ . ) between ulcers' square in all groups. on the th day we found out more rapid decrease of size in group (p [ . ). however, this tendency had no statistical significance. on the th day difference was larger and it was statistically significant this time (p \ . ). also the better ability to stimulate the activity of fibroblasts and revascularization in the young connective tissue with improving oxygenation in the ulcers and enhancing of cell proliferation, differentiation and accelerating of maturation of connective tissue and healing of ulcers was demonstrated in group . conclusion: platelet rich plasma reduces inflammatory response and stimulates proliferation of gastric epithelial cells on th day with the restoration of secretory activity and epithelialization of ulcers in . % of experimental animals on th day, the activation of the fibroblastic reaction during the all experiment and decreasing of ulcers' square. h. fujii, depat. of surgery, japanese red cross fukui hospital, fukui, japan introduction: in conjunction with charmant, a local eyeglass frame manufacturer, we developed novel devices called the fj (free jaw) clip to grasp organs in the abdominal cavity and the f (free) loop plus to pull thread extracorporeally from within the abdominal cavity. product summary: the fj clip is used to grasp organs in the abdominal cavity, a stainless steel, removable forceps for use in laparoscopic surgery. it provides a strong grip but rarely crushes organ tissue. the clip comes in two sizes, one for use in a -mm port and the other for use in a -mm port, and in two lengths, . mm and . mm, respectively. to pull out thread tied to the fj clip, we developed the f loop plus, which is a g by -mm-long special stainless needle with f . -mm niti alloy thread which is used pull suture threads from inside the abdominal cavity to outside the body. case: we performed cases of reduced port laparoscopic and endoscopic cooperative surgery (lecs). we performed reduced port surgery (rps) by making a . -cm incision at the umbilicus, inserting trocars ( mm and mm), and inserting another trocar ( mm) at the left side of the abdomen. we expanded the left hepatic lobe with a -mm fj clip for penrose drain placement, grasped the front wall of the gastric body with a -mm fj clip, applying traction toward the legs to pull up the tissues around the tumor, and resected all layers of the tumor via oral endoscopic submucosal dissection technique. the resected area was closed with a suturing device or interrupted sutures in the abdominal cavity. a year-old female was admitted to the emergency department with complaints of abdominal cramping pain, back pain and diarrhea for one day. she also had fever, ever up to °. in these two weeks, she felt occasionally epigastric pain. her past medical history included hypertension. on physical examination, she was conscious and alert. abdominal examination revealed diffuse tenderness and knocking pain over right flank. laboratory tests indicated an degraded white cell count of /cumm with % band forms, c-reactive protein of mg/dl and abdominal liver function tests (alanine aminotransferase: u/l, alkaline phosphatase: u/l, gammaglutamyl transferase: u/l) without hyperbilirubinemia. abdominal x-ray showed paralytic ileus. our presumptive diagnosis was acute peritonitis, based on the patient's symptoms. empirical antibiotics were administered immediately, and a computed tomography (ct) imaging study was performed. the ct scan showed a stick like foreign body noted between ventral side of pylorus and smv lumen, about . cm in length and associated with perifocal infiltration and segmental smv thrombus formation. (fig. ) however, there is no obvious pneumoperitoneum and no evident ascites is associated. an emergency exploratory laparotomy was performed, revealing stomach perforation at posterior wall with a cm fish bone thourgh pancreas into smv. localized inflammation and fibrosis were identified without obvious fluid accumulation( fig. - ) . removal of fish bone and simple closure of stomach perforation were performed. blood cultures revealed bacteroides thetaiotaomicron. three weeks later, she received a follow-up ct scan which showed smv obliteration with chronic pylephlebitis. aim: here we present a case report about the endoscopic treatment for iatrogenic gastric perforation secondary to a chest tube insertion. methods: a case report of a -year-old male with history of a road traffic accident. described injuries were severe brain injury with gcs \ at pre-hospital care arrival, thoracic injury with several rib fractures on the left hemithorax and hypoventilation on the left side. prior to hospital transfer a chest drain was inserted on the left side, and the patient was intubated. results: at hospital admission, the patient was hemodynamically stable and connected to a mechanical ventilator. thoracic exam showed persistent hypoventilation on the left chest. no other abdominal or pelvic injuries were found in the physical exam. a frontal chest x-ray revealed pneumothorax and the chest tube was not viewable. a further ct scan showed the chest drain placed in the abdominal cavity, into the stomach, besides a subdural hematoma, comminuted pelvic fracture of the pubic rami and a left sacroiliac fracture. during the first h in the icu, neurological worsening was observed, and a new cranial ct revealed enlargement of the subdural hematoma, for what the patient underwent decompressive craniectomy, with improvement thereafter. following a five-day period of stabilization after surgery, the patient was evolving satisfactorily, and the removal of the intragastric chest drain was considered. endoscopy was performed to confirm the placement of the drain, and it was removed under direct vision. approximately twenty five centimeters of the catheter were visualized in the gastric lumen, and then successfully removed. the patient recovered well and was discharged from icu to medical hospital ward after fourteen days, and a week later he was discharged home. conclusion: endoscopic management for gastric perforation after a chest drain insertion may result effective and can prevent open surgery morbidity. aims: intestinal infusion treatment with levodopa/carbidopa (duodopa) is a therapeutic option concerning the advanced parkinson disease cases with no response to the conventional treatment. the drug requires carrying out a gastrostomy either by percutaneous endoscopy way, or by laparoscopy -if the first one is not possible-. later, a duodenum-yeyunum tube is placed in order to infuse the duodopa gel continuously by a portable bomb. in this report, we explain the laparoscopic gastrostomy technique. method: sin this report, we include two patients with advanced parkinson disease: the first one is a year-old female patient suffering from an important gait disorder; and the second one is a year-old male patient with uncontrolled motor fluctuations. in both cases, a percutaneous endoscopic gastrostomy was proposed, but neither was feasible because of the non-traslumination between the gastric and the abdominal wall. under general anesthesia, neumoperitoneum by veress needle was performed. three main trocars and one accessory were placed. at the level of the gastric antrum, a cm incision was conducted to insert a gastrostomy tube, to be the guide for the drug infusion catheter. next, the gastrostomy is fixed to the abdominal wall by the stamm technique, externalizing the catheter through the accessory trocar in the medial line. results: on the first post-operative day, a duodenum-yeyunum tube is placed by endoscopic control through the gastric device. both patients got well satisfactorily, and no complications were described; and they develop a total normal life within the limitations of their underlying disease. conclusions: the duodopa intestinal infusion shows a significative improvement for the advanced parkinson disease symptoms, compared with oral medication; appreciating positive results referring to life quality. when the catheter placement by endoscopy way does not seem posible, gastrostomy by laparoscopy constitutes a valuable surgical option for the treatment of this kind of patients. peptic perforated ulcus (ppu) is a common surgical emergency and laparoscopic repair has been introduced as an alternative to open repair. it has shown good results and allows closure and peritoneal lavage, just like the open repair does but with the advantages of a minimally invasive surgery. the objective is to report the outcome of laparoscopic ppu in our hospital. methods: from january to october , patients with a clinical diagnosis of ppu were assigned to undergo laparoscopic repair. this retrospective study included all husm patients who underwent laparoscopic ppu repair by emergency surgeons. minimum follow-up of months is carried out. results: of the patients in this series, % were men and % were women, between and years of age at the time of surgery, average of years. the time between the manifestation of symptoms and surgery was [ to h in % of patients. in patients there was a history of previous ulcer or non-steroidal anti-inflammatories intake and up to % were smokers. a ct scan was performed in all cases to reach the diagnosis primary closure with simple suture plus omental patch was the elected technic ( %). the approach was performed with trocars in %, trocars in % and in case. cases ( %) were gastric ulcer, duodenal cases ( %) and in one case no perforation was found. the conversion rate was %, in two cases due to technical difficulty and in the other case because the level of the perforation was not found. the median postoperative stay was days although there were cases with intrabdominal complications. there was an exitus due to a metastasic pulmonary neoplasia diagnosed in the immediate postoperative period. there were no cases of recurrence in the follow-up time. conclusion: in most centers, including ours, the rate of laparoscopic management has gradually increased along with the improvement of technical skills. improvements in the outcome of laparoscopic ppu repair are to be expected with more experience surgeon and a good selection of the cases. general surgery, jzu hospital ,,sveti vracevi,, bijeljina, bosnia-herzegovina introduction: diverticulum is an outpouching of a hollow organ. gastric diverticulum is rare form od this disease. incidence of detection varies depending on investigation method. it has been reported in . % cases of autopsies, . % cases of gastroduodenal roentgenographies with contrast, and . - . % cases of upper endoscopies.small diverticula are usually asimptomatic, but bigger diverticula can cause variable symptoms such as abdominal pain, feeling of epigastric fullness right after meal, feeling of discomfort in upper parts of abdomen, and severe 'foetor ex ore' .diagnosis is usually established in procedures such as gastroduodenal roentgenographies with contrast, upper endoscopies and abdominal ct scan. case report: a -year-old woman came to our hospital because of feeling of discomfort and mild pain in upper abdomen that lasted for last year. diagnosis is established after ct scan of abdomen and upper endoscopy procedure. initially she has been prescribed conservative therapy (proton pump inhibitors). since the symptoms persisted, laparoscopic resection of the gastric divertuculum was performed using endogia stapler. considering the feeling of discomfort and abdominal pain dissapeared, the patient was discharged from hospital on the fourth postoperative day. conclusion: asymptomatic gastric diverticula doesn't require treatment. since gastric diverticulum can have complications such as bleeding, perforation and neoplasia, patient without symptoms should be monitored. initial therapy for symptomatic diverticula is conservative therapy (proton pump inhibitors). if conserative therapy doesn't procude expected results, laparoscopic resection of the diverticulum should be considered. introduction: the acute perforation of a gastric ulcer is a serious entity that requires urgent surgical treatment in most of the occasions, it is increasingly accepted that the approach of choice is laparoscopic, depending, above all, on the time of evolution of the process. objectives: to demonstrate the safety and efficacy of the laparoscopic approach in the perforation of a pyloric peptic ulcer, even in cases of severe peritonitis, by means of a standardized procedure, insisting on the sequential thorough washing of the cavity.material and methods: we present a video of the surgical intervention of a patient with acute abdomen, with a history of nsaid ingestion, exploration and ct-analysis compatible with perforation of hollow viscus, probably of gastric origin. results: intervention: complete laparoscopic approach, trocars. severe biliopurunitic peritonitis, by pyloric perforation 'acute', liquid culture, suture of the perforation, epipoplasty, sequential thorough washing of the cavity with physiological saline and placement of drainages.correct postoperative period, discharge from the hospital on the th day after completing antibiotic treatment. endoscopy and helycobacter test are performed on an outpatient basis with normal results. conclusion: the laparoscopic approach is safe and effective in acute and complicated gastric ulcer disease, even in cases of severe peritonitis. surgical procedure: the clean-net procedure involves the selective dissection of both the serosa and muscle layer using a laparoscopic monopolar endoscopic scissor. the preserved mucosal layer provides a mechanical barrier between the gastric lumen and peritoneal cavity that aids in the prevention of peritoneal cavity contamination with gastric contents. tumors are observed with an upper gastrointestinal endoscope with the injection of indocyanine green (icg) into peri-tumoral submucosal layers at points. selective seromuscular dissection is performed using a laparoscopic electrocautery monopolar scissor. the mucosa surrounding the gist is then resected using a endoscopic mechanical stapler to prevent exposure of the gastric lumen to the peritoneal cavity and peritoneal tumor cell seeding. results: there were males and female, and the average age was years. the operation time was min, the average bleeding volume was . ml, the postoperative hospital stay was . days. the mean tumor diameter was . mm, the final histopathological diagnosis was gist, schwannoma. there were no postoperative complications of clavien-dindo classification or more. conclusion: clean-net was found to be safe and useful for the treatment of gastric smt with ulceration. year outcomes: laparoscopic heller myotomy stands the test of time aims: laparoscopic cardiomyotomy leads to excellent relief of dysphagia in % of patients and avoids thoracotomy or laparotomy. methods: we present a video illustration of the procedure that was modified at the american university of beirut medical center. so far, patients underwent laparoscopic cardiomyotomy, age range of to years, with males and females. most of them have had previous balloon dilatation. results: all cases were successfully completed laparoscopically without complications. followup of months to years revealed excellent results with complete resolution of symptoms and no need for further medications. this will result in minimal post-operative pain and very short recovery period and is associated with low complication rate. conclusion: cardiomyotomy for achalasia is ideal for laparoscopic approach. magnification allows for precise division of muscle fibers. the new technique of hydro dissection and enseal for division of esophageal muscle allows for completion of the procedure without injury of the mucosa. therefore, adequate release of the obstructing segment followed by anti reflux procedure toupet will lead to excellent results with minimal morbidity and no mortality. aims: laparoscopic repair of huge hiatus hernia methods: twenty two cases of huge hiatus hernia presented to the american university of beirut medical center. patients underwent through trocars in the upper abdomen reduction of the hernial sac from the chest. special care was taken in the dissection of the mediastinum to keep the thoracic fascia and pleura intact. the defect was sutured primarily by -ethibond sutures reinforced by onlay prolene mesh u-shaped was fixed at the rt. and lt. crus and a floppy nissen fundoplication performed . results: the video presentation includes the technical aspects and the method of reducing and repair of huge hiatus hernia.aim: nowadays, there is little experience in the world of applying robotic surgical system (rss) in treatment of patients with hiatal hernia (hh) and reflux-esophagitis (re). the aim of study was to determine the possibility and feasibility of using rss in treatment of patients with hh. materials and methods: a total of patients underwent robot-assisted hh repair without mesh, followed by fundoplication with our original method ( °full symmetric wrap). the clinical and technical analysis did not reveal any advantages over similar laparoscopic procedures, so we abandoned the use of rss for hh type i, and these patients were excluded. there were ( %) patients with hh type iii and ( %) type iv. the surgeries were performed by experienced robotic upper gastrointestinal surgeon and conducted with the davinci si surgical system (intuitive surgical, sunnyvale, ca). results: average operation time was ± ( - ) min. the respondents' mean age was . ± . years (range - ) and bmi was . ± . (range . - . ) cm/kg . average blood loss was ± ( - ) ml. average hospital stay was ± . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) days. the average follow-up time was ± . ( - ) months. postoperative x-ray imaging and upper gi endoscopy was conducted in all ( %) patients. there was no hh recurrence diagnosed. we did not observe a relapse of hh or clinical manifestations of re in the early (less than days) and long-term (more than months) postoperative periods. conclusion: we can conclude that robot-assisted surgery is safe, appropriate and justified in patients with hh type iii and iv. all procedures performed to the patients with giant hh revealed clear technical advantages of rss over similar laparoscopic operations: an enlarged d hd image, bendable instruments with endowrist technology allowed for precise dissection of tissues (hernial sac, cicatricial adhesions) in a narrow anatomical space-posterior mediastinum without damage to pleura, pericardium and vagus nerves. we believe that use of rss in treatment of patients with reflux esophagitis and/or hh type i is unjustified, due to the lack of proven advantages over laparoscopy. introduction: the presence of major anatomical obstacles -such as massive caudate lobe-in the confined operative field of laparoscopic hiatal hernia repair (lhhr) poses significant challenge to the foregut surgeon. aim: to provide a safe alternative for lhhr using a laparoendoscopic approach. method: this patient is a year old female, with bmi of . . her past medical history includes diabetes, hypertension and hyperlipidemia. she had gerd for years. her egd showed x cm hiatal hernia and class b esophagitis. manometry showed ineffective esophageal motility. we used the classic five ports approach for lhhr. we found a massive caudate lobe which was comparable to the size of an already enlarged left lobe of the liver. the operative strategies: terminating the procedure proceeding with the standard approach and taking the risk of bleeding from the caudate lobe itself or the inferior vena cava (ivc) with possible catastrophic outcome. using the laparoendoscopic approach. the following three steps facilitated the performance of safe and effective surgery. additional liver retractor this improved exposure and minimized manipulation of the caudate lobe. extracorporeal sliding arthroscopic knots (esak) esak are similar to the knots used in endoloop. they are tied extracorporeally and require a single insertion of the knot pusher as they do not unravel. transoral incisionless fundoplication (tif) we performed tif to avoid a limited operative field and to prevent excessive tissue manipulations associated with laparoscopic fundoplication. tif also preserves the angle of his and produces partial fundoplication which has less side effects of dysphagia and gas bloat syndrome. results: the operative time was min (lhhr min and tif min). there were no complications. patient discontinued omeprazole which she used daily for years. at months follow up, her gerd related quality of life (hrql) and gerd symptom (gerss) scores were ( vs ) and ( vs ). conclusion: the laparoendoscopic repair of hiatal hernia in the presence of anatomical obstacles is safe and effective. longer follow up is needed to assess the durability of this repair. gastroesophageal reflux disease (gerd) is a condition that reduces the quality of life and can causedisorders associated with acid reflux, such as bronchial asthma, barrett's esophagus and esophagealadenocarcinoma. gerd is often caused by existing of hiatal hernia. nowadays, some surgeons haddifficulties with the laparoscopic approach to treatment of recurrent hiatal hernias.patient was a -year-old man. he requested medical assistance with dysphagia, nausea after eatingand heartburn getting worse in a horizontal position. conservative treatment was not effective.transthoracic nissen fundoplication was performed in . the main complaints of the patientpersisted during the postoperative period. the upper half of stomach and s-like curved esophagus werelocated in the mediastinum according to multislice computed tomography of the thorax in august.in our clinical center was performed laparoscopic cruroraphy, cardiomyotomy and nissen fundoplicationin november. during the surgery the normal anatomical position of stomach has been restored, s-like curve of esophagus has been removed; a gastric cuff (collis-nissen) has been created and anteriorand posterior cruroraphy has been performed. the patient was in intensive care during h. anasogastric tube feeding was continued during the first h. passage of the contrast through theesophagogastric junction was free within h after surgery. patients had been discharged within days after surgery.this case report shows that at the current stage of surgery laparoscopic approach can be useful not onlyfor treatment of primary hiatal hernias-but also for treatment of recurrent ones. aims: laparoscopic heller myotomy procedure, completed with an anti-reflux procedure is technically demanding. we report a case of laparoscopic heller myotomy followed by a dor anterior fundoplication. methods: this is the case of a -year-old caucasian woman with gradual dysphagia for solids and liquids, accompanied by severe regurgitation and chest pain. an initial diagnosis of achalasia was made in , with the use of manometry and barium swallow. endoscopic dilatations were attempted pre-operatively with no clinical improvement. decision was made to perform a laparoscopic heller myotomy, combined with a standard dor anterior fundoplication. a -ports operation took place (one intra-umbilical mm trocar-single incision laparoscopic surgery (sils) technique, two -mm subcostal trocars, and one another mm subcostal trocar for the use of liver retractor). the operation lasted h and min. results: no post-operative complications were noted. the post-operative swallow test showed improvement of the esophageal patency. the patient started a liquid diet three days later and was discharged six days post-operatively. two months later the patient presented no complications. conclusions: heller's myotomy has demonstrated good long-term results in the treatment of esophageal achalasia and the laparoscopic approach has been well established in the last two decades. it is a very demanding operation to perform and the disease is relatively rare, making the learning curve difficult to achieve. aims: achalasia is a type of motor disorder of the esophagus due to degeneration of ganglion cells in the myenteric plexus, leading to failure of relaxation of the lower esophageal sphincter, accompanied by a loss of peristalsis in the distal esophagus. the association of a long-term achalasia and a large size hiatus hernia is an infrequent entity. among the therapeutic options is medical treatment, endoscopic treatment and surgical treatment associated with an antireflux procedure. the laparoscopic approach being the more indicated due to its better results in terms of morbidity, mortality and recurrences. the aim of the video is to show the effectiveness and safety of the laparoscopic approach in this infrequent pathology, pointing out the importance of performing a standardized procedure. methods: -year-old male patient, with personal history of chronic ischemic heart disease and obesity, diagnosed with long-term achalasia with moderate dilatation of the esophagus associated with giant hiatus hernia. the complementary explorations and iconography of interest are exposed. results: intervention: complete endoscopic approach, trocars. reduction of hernial content into the abdominal cavity, dissection of the hernial sac and esophageal lipoma. extended mediastinal esophageal dissection. complete resection of both the sac and lipoma, respecting the posterior vagus. heller's myotomy of cm, including cm distal to the ueg, perforation of mm of the mucosa at the ueg level, suture and blue methylene verification of the sealing. hiatorraphy and dor-type anterior fundoplication as antireflux technique. correct postoperative, with egd control on the rd po day and discharge on the th. asymptomatic at months after surgery. conclusion(s): for achalasia laparoscopic heller myotomy with a partial fundoplication should be the treatment of choice in patients who are at low surgical risk. the length of the myotomy, especially distal to ueg is one of the most important aspects of the surgery, to achieve an effective disruption of the les. the presence of a giant hiatus hernia makes the procedure difficult, increasing the risk of complications, such as perforation. standardization is essential to increase safety and efficacy in these complex cases. purpose: there is evidence that the application of mesh-reinforced hiatal repair has resulted in a significant reduction in recurrence rates in comparison with primary suture repair, at least in short-term follow-up. however, and instead of this, the standard of care for repairing large paraesophageal hiatal hernias (lphh) remains controversial because no clear guidelines are given regarding indications, mesh type, shape and position. the aim of this study is to evaluate our short-term outcomes in management of lphh with a biosynthetic monofilament polypropylene mesh surrounded by a high-purity and adherent titanium dioxide surface coating to enhance the biocompatibility (tio mesh tm ). methods: a retrospective study was conducted on our institution between december and october . data were collected on patients with lphh greater than cm in which a laparoscopic repair was carried on by primary suture and additional reinforcement with a tio mesh tm . clinical and radiological recurrences, dysphagia and mesh-related complications were investigated. results: there were females and males with a mean age of years (range, - years). all operations were completed laparoscopically. median postoperative stay was days. after a mean follow-up of months, patients developed clinical recurrence of reflux symptoms ( . %) and radiological recurrences ( . %). there were no mesh-related complications. conclusions: the use of tio mesh tm for laparoscopic repair of lphh is suited and with a reasonably low recurrence rate in this short-term study. additional long-term studies with enormous numbers carried out for years will be necessary to affirm whether this mesh is convenient in the prevention of recurrences and mesh related complications. background: surgery for refractory gastroesophageal reflux disease (gerd) has a satisfactory outcome, however sometimes fundoplication fails and redo surgery is required. several publications have investigated the feasibility of performing reoperative fundoplications using laparoscopic techniques. the aim of this study was to describe our experience in laparoscopic redo fundoplications in the last years. material and methods: we retrospectively reviewed consecutive patients who required laparoscopic redo fundoplication from january to august .the indications were recurrent symptoms of gastroesophageal reflux disease (gerd) ( . %), recurrent symptomatic paraesophageal hernia ( . %), dysphagia ( . %) and acute volvulus ( . %). results: all redo fundoplications (basically toupet . % and nissen . %) were completed laparoscopically. the mean operative time was min (range, - . min). a mesh was placed in % of cases. intraoperative and postoperative complication rates were . % and . % respectively. the mean hospital stay was days (range, - days). one patient ( . %) from the laparoscopic group required a third operation-one for acute recurrent paraesophageal herniation of the redo wrap one month after surgery, which was repair laparoscopically again. symptomatic outcome was successful in . % without any kind of proton bomb inhibitors therapy. conclusion: laparoscopic redo fundoplication is technically feasible and clinically effective with a reasonable low rate of postoperative complications p -upper gi-reflux-achalasia objectives: in recent years, balloon dilatation (bd) for diseases requiring correction of the impaired patency of the sphincter zones of the esophagogastroduodenal region has become widespread. purpose: to assess the effectiveness of the use of the balloon dilatation in patients with impaired sphincter zones of the esophagogastroduodenal region. materials and methods: in the institute department of surgery for the period from to , bd was performed in patients. of them diagnosed with achalasia of cardia (ac): - stage, - stage, - stage, - stage. patients diagnosed with pylorospasm, patients had compensated stenosis and patients had subcompensated ulcerative pyloroduodenal stenosis. there were males, females, average age ( . ± . ). bd was performed under endoscopic and / or x-ray control by 'boston scientific' balloons with a diameter of - mm, mm and mm, a course of - sessions with an interval of - days and a cylinder exposure of - min. evaluation of bd was performed using esophagogastroscopy, balloon manometry and x-ray passage of barium. results: in the course of the study, the existing indications were refined and new indications were developed for performing an endoscopic bd in pyloroduodenal stenosis and in ac. in patients with stage - ac, a positive result was noted in . % of cases already after the first session of bd. recurrences of ac after bd for up to years were established in ( . %) patients: at stage -in . %, at stage -in . %, at stage -in . % and at stage -in . %. repeated bd courses in case of ac recurrence in ( . %) cases turned out to be ineffective. recurrence of pyloroduodenal subcompensated stenosis was diagnosed in . % of cases in the period of months after performing bd. conclusions: bd is an effective method for correcting the permeability of the sphincter zones caused by the pathology of the esophagogastroduodenal region. keywords: balloon dilatation, achalasia of cardia, pylorospasm, ulcerative pyloroduodenal stenosis, recurrences. introduction: the reoperation in antireflux surgery significantly increases morbidity and mortality up to - %, reaching rates of % in patients undergoing or more surgeries. the advantages of laparoscopic surgery used in this surgical technique have amplified its acceptance and use, resembling its results in terms of feasibility, safety and efficacy of laparoscopic surgery to open surgery.objective: :evaluate the currently literature about antireflux surgery reintervention, focusing on the main indications of re-intervention, type of approach and morbidity and mortality of laparoscopic antireflux surgery. material and methods: a literature search was conducted in two electronic databases, med-line and embase. the search was limited to the period to . terms were used in relation to the procedure or intervention and the underlying disease. we chose observational studies (cohort, cases and controls and series of cases), where the main indication for antireflux surgery would have been gastroesophageal reflux disease. results: a total of studies were selected, most of them were case series ( . %), cohort studies ( %) and case-control studies ( . %). a total of patients. the main indications were anatomical faults, of these failures, recurrent hiatus hernia and sliding occupy the highest percentage, while physiological failures, failure in esophageal and gastric motility occur more frequently. the main type of approach was laparoscopic in %, the conversion rate was . % and the open approach was reserved for complex cases with more than one re-intervention . %, for abdomen . % and chest . %, this last for cases with high esophageal lesions that can not be repaired via trans-abdominal.the main complications were injuries to hollow viscera, such as: esophagus and stomach among others. these complications are related to the complexity of the procedure. mortality has remained low up to . %, however, the cause of death was due to medical complications and not related to the procedure. conclusions: this systematic review on reoperation in reflux surgery has confirmed that morbidity after reoperation surgery is higher than after primary surgery and reoperation indications increase with the use of new technologies (manometry) and the laparoscopic approach continues on the rise, with great adaptation to its use and improvement in results. aims: eras protocol is not commonly used in acute emergency procedures. elective lc is commonly performed as one day surgery, while in an emergency setting of acute cholecystitis, the in hospital stay averages , days. the aim of this trial is the application of eras protocol in patients with acute cholecystitis, undergoing laparoscopic cholecystectomy. methods: a randomized prospective trial was conducted in first surgical department of sismanogleion g.h.a. the study included patients, who were admitted with acute cholecystitis and underwent lc into h from their admission. preoperatively, they all received crystalloid isotonic solutions and antibiotics. . % were submitted to ercp, preoperatively, due to choledocholithiasis. the postoperative care included early mobilization into h after surgery, early fluid intake (into h) and early liquid food intake (into h). they all received systematically antibiotics, analgesics and antiemetic on demand. asa score was not an exclusion criterion. results: conversion to open procedure was necessary in . % of patients, whom were excluded from the study. all the rest were discharged into h from the surgery with the guidance to receive oral antibiotics for more days. readmission was necessary for patients, one week after the operation. the first one presented with bile leak and submitted to ercp with stent placement and percutaneous drainage of the intrabdominal collection. the second one presented with choledocholithiasis and underwent ercp with balloon catheterization. conclusion: it is commonly accepted that eras protocol in elective procedure enhances the postoperative recovery while reduces the in hospital stay and cost. in emergency condition eras cannot be applicated preoperatively. however, a modified post surgery application seems to have advantages equal to those observed in elective procedures. aim: laparoscopic cholecystectomy is the gold standard for the treatment of symptomatic cholelithiasis. administration of one single dose of chemoprophylaxis before an elective laparoscopic cholecystectomy is a broadly accepted practice. however, its value is currently questioned, especially in low risk patients. method: this study was conducted in a high volume surgical department. one hundred and twelve patients submitted to elective laparoscopic cholecystectomy were included in this research. a written consent was acquired after thorough patient briefing. half the patients that underwent surgical operation received one dose of antibiotics min prior to the incision and the other half did not receive any chemoprophylaxis. results: the age ranges from to years old. commonest concomitant diseases were arterial hypertension, type ll diabetes, hypothyroidism and respiratory deficiency. approximately % of patients were smokers and % were obese (bmi [ ). the duration of the operations was between and min. intra-operative gallbladder rupture was observed in patients (rate %). all the patients were discharged the first post-operative day and their monitoring continued for more days. in the chemoprophylaxis group, no surgical site infection or other major complication was observed. from the group that did not receive any antibiotics, one patient developed surgical site infection and specifically infection of the surgical port in the epigastrium, which was treated with drainage of the abscess and oral antibiotics administration. no other complications were recorded. conclusion: our study concluded no statistically significant difference between the two patient groups, which depicts that chemoprophylaxis may not be necessary in elective cholecystectomy operations. on the contrary, antibiotics increase the cost of hospital stay and are often accompanied by multiple mild or severe side effects. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -fio cjj authors: nan title: peripheral nerve society meeting july – , sitges, barcelona, spain date: - - journal: j peripher nerv syst doi: . /jns. sha: doc_id: cord_uid: fio cjj nan the peripheral nerve society was founded in from two groups of academic investigators, peripheral nerve study group and peripheral neuropathy association of america, interested in the basic biology and function of the peripheral nervous system and its application to the clinic. their invite only biennial meetings involved - attendees in cloistered settings organized by shoestring and local initiative. from this, we have grown remarkably. we now have an annual meeting of over people including meetings within the meeting for the special interest groups in inflammatory, diabetic and hereditary neuropathy. with this substantial growth and the success of jpns, the journal of the peripheral nervous system the society continues to flourish. has proven to be a year full of exciting changes for the society. pns has transitioned from a biannual, to an annual meeting. next year, the meeting will be taking place at the renaissance baltimore harborplace hotel from - july in baltimore, maryland. the development of a new website has been completed, please visit www.pnsociety.com to see the new face of the society. finally, pns has adopted new executive staff. with their guidance and the leadership of an active and diverse board of prominent professionals in the field the peripheral nerve society continues to grow and anticipates more exciting changes in the year to come. the peripheral nerve society provides annual meetings, teaching courses, guidelines, and other resources to aid in the education of members. becoming a member of pns means collaborating with prominent global professionals in the field to develop and provide the best treatments for people with peripheral nerve diseases and setting standards of care within the field. please participate in our future by joining the pns, volunteering for a project aligned with your interests and sending your ideas for the future to the executive office, or board member. peles e . department of molecular cell biology, rehovot, israel. two schwann cell-dependent mechanisms control the presence of na + channels at the nodes of ranvier: i. clustering of the nodal complex by glia-derived proteins and ii. restriction of nodal proteins within the nodal gap by the paranodal junctions. these mechanisms depend on specific cell adhesion molecules that mediate the contact between myelinating glia and their underlying axons at the forming nodes and the paranodal junction. during myelination, na + channels initially clustered at heminodes that border each myelin segment. this process requires gliomedin, nrcam and neurofascin (nf ), three cell adhesion molecules (cams) that mediate the interaction between schwann cell microvilli and the axon. na + channels clustering activity of gliomedin is tightly regulated by two distinct and functionally opposing proteolytic events. while the clustering activity of gliomedin is enhanced by its shedding from the surface of schwann cells by a furin protease, its activity is negatively regulated by bone morphogenetic protein /tolloid-like (bmp /tld), and tolloid-like (tll ) metalloproteinase. cleavage by these enzymes restricts the activity of gliomedin to the nodal area and prevents the formation of ectopic clusters along axons that are devoid of myelin segments, as well as below the myelin internodes. hence, proteolytic processing of gliomedin facilitates, yet limits, the clustering of na + channels to specific sites along the axon in a timely manner. furthermore, axon-glial contact mediated by gliomedin and nf at the nodes, not only plays a role in na + channel clustering during development, but also contributes to the long-term maintenance of na + channels at nodes of ranvier. in addition to clustering by gliomedin, the distribution of na + channels is restricted between two growing myelin segments by the flanking paranodal junction. at this site, axon-glia contact is mediated by a distinct set of cell adhesion molecules (i.e., caspr, nf and contactin) that also delineate the underlying axonal and glial cytoskeleton. this paranodal junction-dependent restriction of na + channels to the nodes is mediated by the spectrin-based paranodal axonal cytoskeleton. illa i . neuromuscular unit, neurology department, hospital santa creu i sant pau, universitat autònoma de barcelona, barcelona, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. currently, the diagnosis of cidp is based on clinical and electrophysiological criteria and does not take into consideration the presence of immune biomarkers. several autoantibodies against proteins of the node of ranvier in patients with cidp have now been described. these antibodies define specific cidp subtypes sometimes referred to as nodopathies and can have diagnostic and prognostic implications. anti-contactin (cntn ) antibodies. we have described the presence of antibodies to cntn in a small subset of patients with cidp. these patients shared a phenotype and have poor response to ivig. the anti-cntn antibodies are predominantly igg . pathological studies from skin and sural nerve biopsies of patients show morphological changes in the paranodes. experimental data supporting the pathogenicity of anti-cntn igg antibodies include: a) demonstration in vitro that the antibodies disrupt the binding of the cntn -caspr complex to neurofascin- (nf ); b) intraneural injections of antibodies progressively and specifically disrupt the paranodal axo-glial junction; and c) chronic infusion of antibodies induced clinical and electrophysiological worsening in animals with experimental autoimmune neuritis (ean). anti-nf antibodies. antibodies to neurofascins were first reported in patients with guillain-barré (gbs) and cidp and subsequently, antibodies specific to the nf isoform were found in a small group (< %) of patients with cidp. studies by us and confirmed by others have demonstrated that patients with cidp and anti-nf antibodies have a distinct phenotype that often includes a low-frequency tremor and poor responses to ivig. the autoantibodies are predominantly of the igg subtype. the passive transfer of monoclonal anti-neurofascin antibodies (which recognize all neurofascin isoforms) to mice with ean strongly exacerbated the severity of the pathology, but no studies have yet demonstrated that patient-derived anti-nf igg antibodies are pathogenic. a pathogenic role of the antibodies is however supported by sural nerve biopsies from patients with cidp and anti-nf antibodies that showed paranodal demyelination in the absence of inflammation, the loss of septate-like junctions and, the interposition of cellular processes between the paranodal loops and the axolemma. these alterations are reminiscent of those found in nfasc-null mice suggesting that anti-nf antibodies may specifically disrupt the nf -cntn -caspr complex at the paranodes. antibodies to other nodal proteins. recently neurofascin- and neurofascin- were reported as the main targets of autoantibodies in five patients with igg reactivity against the nodes of ranvier; the antibodies were predominantly igg . these patients presented with clinical features distinct from those in patients with anti-nf igg antibodies. four of these patients had subacute onset of sensory ataxia without tremor. the presence of anti-caspr antibodies has been reported in two patients with inflammatory neuropathies, one classified as cidp, the other as gbs. both patients had intense neuropathic pain. the skin biopsy from both patients showed paranodal disruption. some patients whose sera show nodal or paranodal reactivity in teased nerve fiber preparations have antibodies against other nodal proteins, such as gliomedin or neuronal cell adhesion molecule (nrcam) . the skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. indeed some mechanoreceptors can detect even nanometer-scale movements. movement is transformed into electrical signals via the gating of mechanically-activated ion channels at sensory endings in the skin. the sensitivity of piezo mechanically-gated ion channels are controlled by stomatin-like protein- (stoml ), which is required for normal mechanoreceptor function. under pathophysiological conditions following nerve injury or diabetic neuropathy the slightest touch can produce pain. it is at present unclear whether peripheral changes in sensory mechanotransduction may underlie hypersensitivity associated with neuropathic pain. here we have examined the role of the stoml modulation of piezo channels in mechanoreceptors and nociceptors to under pathophysiological conditions. we recently developed small molecules that act as inhibitors of stoml function. peripheral application of stoml inhibitors can alleviate hypersensitivity in models of neuropathic pain. our data strongly suggest that tactile evoked pain in models of peripheral neuropathy may be at least partly driven by sensitization of sensory mechanotransduction driven by stoml . coleman m . john van geest centre for brain repair, cambridge, uk. axons are lost early in many neurodegenerative disorders of peripheral and central nervous system. the degeneration of transected axons (wallerian degeneration) can be slowed tenfold by overexpression of a variety of nad-synthesizing enzymes, such as isoforms of nmnat or the related mutant fusion protein, wld s . wallerian degeneration is also delayed by deletion of tlr adapter protein sarm , a protein recently reported to promote nad degradation. it is important to understand fully the mechanism of wallerian degeneration because related mechanisms contribute to axon loss in a number of disease models, including models of peripheral neuropathies, parkinson's disease, multiple sclerosis and glaucoma. new data also suggest a role in hereditary spastic paraplegia. while depletion of nad is an attractive hypothesis for the mechanism of wallerian degeneration, especially as nad can be increased by dietary methods, it cannot explain a number of key observations. fk , an inhibitor of nampt, blocks the nad salvage pathway and strongly depletes nad, including within axons. however, instead of killing axons as the nad hypothesis would predict, it does precisely the opposite: it phenocopies the protective effect of wld s . moreover, ectopic expression of the bacterial enzyme nmn deamidase, a protein absent in mammals, protects injured axons both in transgenic mice and in primary neuronal cultures, but it has no effect on nad levels either under basal conditions or in degenerating nerves. these observations fit better with a proposed toxic role for the nad synthesis intermediate nmn, a model that can also explain the protective effect of wld s . a full understanding of the pathway should identify a number of points where intervention could be a treatment for multiple axonopathies. as with any medical discipline, expansoin of knowledge about the fundamental science behing a disorder of the human nervous system comes part and parcel with a change in our understanding of the epidemiology of any given disorder or groups of disorders. recent advances in our fundamental understanding of inflammatory neuropathies of the peripheral nervous system have been accompanied by drastic changes in our understanding of the neuroepidemiology of these disorders -the specific populationss affected by peripheral neuropathies, as well as the varying importance / contributions of select peripheral neuropathies to the overall burden of peripheral nervous system (pns) disease, and how this shift in epidemiological understanding influences the clinical approach to diagnosis and management of patients with pns disease. the past few decades have witnessed a paradigm shift in many aspects of pns disease diagnosis and treatment; from the association of human immunodeficiency virus (hiv)-associated neuropathies; to the increassing recognition of hereditary / familial peripheral neuropathies; to the increased recognition of specific neuropathies such as multifocal motor neuropathy with conduction block. in addition, timely events such as the recent, and increasingly irrefutalbe evidence for a link between zika virus and a guillain-barré syndrome, and the rather unexpected resurgence of peripheral neuropathies due to previously 'exotic' etiologies such as lepromatous neuropathy require prompt clinical attention. this plenary session aims to describe the evolving neuroepidemiology of peripheral nervous system disorders, and how these changes may influnece the clinical approach to the diagnosis, prognostication, and treatment of otherwise 'unusual' periphal nerve diseases. oxaliplatin chemotherapy for colorectal cancer is seriously limited by neurotoxic side effects which are not fully understood. oxaliplatin-induced peripheral neurotoxicity (oipn) comprises an acute syndrome and a chronic sensory neuropathy. the acute symptoms, notably cold hyperalgesia, have been attributed to transient ion channel dysfunction, and the worse they are the more severe the chronic neuropathy that ensues. we designed a combined in vitro and in vivo project, using neurophysiology to better understand the pathogenesis of oipn. in the in vitro study, differentiated f cells (rat drg neurons x mouse neuroblastoma n tg- cell line) were incubated for and hours in . m oxaliplatin, and their electrophysiological properties studied by patch-clamp. the treated f cells showed relatively depolarized resting membrane potentials, significantly decreased firing frequencies, and increased sodium current densities. moreover, a decrease in erg (ether-à-go-go-related gene) potassium current was also evident. in the in vivo study, we applied nerve excitability testing (net) to a wistar rat model of oipn. to investigate the acute syndrome, we compared behavioural and neurophysiological data of animal cohorts (controls and oipn rats, n= each) before and after oxaliplatin administration ( mg/kg, iv). twenty-four hours after the injection we observed differences between the groups in behaviour (cold plate test, p= . ) and in superxcitability of motor axons (p= . ). to investigate the chronic neuropathy, we compare a control group (n= ) with a treated group (n= , oxaliplatin mg/kg twice weekly x weeks, iv). both groups are studied with behavioural, neurophysiological (sensory and motor nerve conduction studies, net), and pathological (caudal and sciatic nerve, skin biopsy, drgs) methods. data are collected at baseline, end of treatment and weeks after treatment; to obtain a full net profile of all significant changes. in this highly translational approach to oipn, the in vivo net changes in the acute and chronic rat models can be matched on the one hand to findings from in vitro experiments, and on the other to clinical data, since net is also easily applied in humans. of these results. in denmark there is a unique situation to conduct epidemiological studies facilitated by the danish civil registration and the danish national hospital registry (dnhr). this enables us to identify all gbs patients in denmark in a given period. from the same period as the igos cohort was included we have identified all gbs patients admitted to or seen in outpatient's clinics of hospitals in denmark (september st to december st ). records from the population based danish cohort will be reviewed for demographic and clinical data and compared to the patients included in igos from denmark, as well as with the igos europe/america cohort. during this period patients from denmark have been included in igos. the danish group is comparable to the europe/america group not counting the danish patients (n= ) of the igos cohort in regard to sex and age at entry, gbs disability score at nadir, and percentage of patients needing mechanical ventilation. in the danish igos group % are males, the median age is ( - ) years, the mean(sd) gbs disability score at nadir . ( . ), and % of the danish group needed mechanical ventilation. in the europe/america group % are males, the median age is ( - ), mean(sd) gbs disability score at nadir . ( . ) and % of the group needed mechanical ventilation. at the meeting we will compare and present data from the danish population based cohort as well as epidemiological data. chronic inflammatory demyelinating polyneuropathy (cidp) is a common autoimmune disease of the peripheral nervous system (pns) that causes sensorimotor impairment. mice with a dominant autoimmune regulator gene (aire) g w mutation on the non-obese diabetic (nod) background (nod.aire gw/+ mice) develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. in sapp, demyelination is caused primarily by th t cells; however, the contributions of nerve-resident cells such as schwann cells are poorly understood. we identified a population of non-hematopoietic, integrin alpha + (itga +) cells in the pns that increases in frequency and number during sapp. these itga + cells coexpress numerous schwann cell markers including sox , p , s b, myelin protein zero, and peripheral myelin protein , suggesting that itga + cells are schwann cell-like. additionally, during sapp, these itga + cells upregulate the extracellular matrix protein periostin (postn), which has recently been shown to promote macrophage recruitment and activation in inflammatory disease and cancer. our data indicate that macrophages are pathogenic during sapp. therefore, we hypothesized that itg a+ cells promote macrophage recruitment during sapp via postn production. to test this hypothesis, we performed in vitro chemotaxis assays. conditioned media from nod.aire gw/+ nerve promoted significantly more macrophage chemotaxis than conditioned media from postn −/− nerve. furthermore, postn recombinant protein was sufficient to induce macrophage chemotaxis in vitro. our findings show that itg a+ schwann cell-like cells mediate macrophage chemotaxis by upregulating postn during sapp and suggests postn as a novel target for the treatment of cidp. "wear-off" frequency will be analyzed by assessing the proportion of subjects with any given degree of gs and rods intracycle fluctuation and the proportion of cycles in which gs and r-ods fluctuation occurs. to determine the extent of "wear-off" the degree of difference between maximum and minimum gs, r-ods, tugs, onls, and vas scores will be analyzed. currently subjects from different sites have been enrolled ( sites eligible for enrollment). this interim study report will provide preliminary representative data, demonstrating ivig "wear-off" effects on gs and other outcome measures. by better understanding the frequency and extent of ivig treatment-related fluctuations we expect that these results will help facilitate development of cidp treatment optimization strategies. we also expect that this information will be important in forming hypotheses to be tested in future studies (for example, comparing different dosage intervals, optimal ivig taper guidelines, or assessing the long-term outcome of short-term cycle to cycle clinical fluctuations). neuropathic pain is a frequent feature of peripheral neuropathy causing a significant impact on patients' quality of life and health care costs. resolving the genetic architecture of painful neuropathy will lead to better disease management strategies, risk stratification, and counselling. therefore, we aim to develop a reliable technique to rapidly and accurately re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. whole exome sequencing of thousands of samples remains expensive for clinical use. several targeted enrichment approaches are currently available to selectively enrich for genomic regions of interest. in this study, we compared the sensitivity, specificity, targeting efficiency, reproducibility of performance and cost effectiveness of truseq ® custom amplicon-next generation sequencing (tsca-ngs) and molecular inversion probes-next generation sequencing (mips-ngs) methods. for both methods, we constructed a targeted enrichment kit to capture the coding and exon-flanking intron sequences of nine sodium channel genes (scn a, scn a-scn a, and scn b- b) expressed in nociceptive neurons. probes were designed for the two methods using their respective informatics pipelines. in total, patients with diabetic and idiopathic neuropathy were tested by both methods. among the patients, patients were tested previously by sanger sequencing for scn a-scn a. approximately kb was captured and sequenced. % of the targeted regions showed an average coverage of ≥ x in tsca-ngs, and % in mips-ngs. we managed to identify potential pathogenic mutations and polymorphism variants by mips-ngs and tsca-ngs. moreover, we observed a perfect agreement ( %) between sanger sequencing data and those obtained using mips-ngs and tsca-ngs. both ngs approaches showed user-friendly software to design probes and exhibited a similar on-target efficiency. although the overall coverage per region varied across different dna samples, it was sufficient to detect any variant in these regions. mips-ngs has more versatile assay design, demonstrated a high degree of flexibility with probes re-placement and > x cheaper than tsca-ngs. mips-ngs is a reliable, flexible, and inexpensive method to detect genetic variations in thousands of patients. in our centers, this technology is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients. alonso-jiménez a , , belvis-nieto r , diaz-manera j , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; centro para la investigación biomédica en red para enfermedades raras, ciberer, madrid, spain; neurology department, hospital universitario dexeus, barcelona, spain. most acute demyelinating polyneuropathies have an immune-mediated pathogenesis and are included within the guillain-barré syndrome spectrum. occasionally, other mechanisms such as metabolic, infectious or toxic may lead to gbs-like presentations. thermatrim ® and pura alegria ® are different brands of the same illegal slimming product that is sold through online vendors and in which the exact composition is unknown. here we present a patient with an acute demyelinating polyneuropathy secondary to the intake of the slimming product "pura alegría". a year-old woman with no remarkable medical history reported days history of distal numbness in her feet that progressed in one week to her knees and her left hand. she had had an upper respiratory tract infection ten days prior to these symptoms. the neurological examination showed absent distal vibratory and arthrokinetic sensations and arreflexia in lower limbs, decreased vibratory sensation in her hands and a ataxic gate. the lumbar puncture showed . g/l of proteins with no cells. the emg fulfilled diagnostic criteria for acquired demyelination. intravenous immunoglobulin therapy was started but the symptoms kept worsening and corticosteroids were started. the patient mentioned then the slimming product. a brain mri showed diffuse leukoencephalopathy that was asymptomatic. steroids and the pura alegria slimming products were withdrawn and the patient recovered completely after one year of follow-up. "pura alegría", "thermatrim" and "thermatrim plus" are slimming products that were forbidden in spain after several cases of acute leukoencephalopathy and acute polyneuropathy. exact composition is unknown although the spanish drug agency detected the pesticide malonoben, a tyrosin kinase inhibitor, among the components. since nine cases of pura alegria/thermatrim neurological toxicity, including leukoencephalopathy and polyneuropathy have been described. all cases had good outcomes after treatment withdrawal, although recovery is slow and may be incomplete. our case highlights the need to carefully consider drug toxicity, including dietary supplements, in the differential diagnosis of gbs specially when evolution does not follow typical patterns. alvarez s , klein d , martini r , moldovan m , , krarup c , . center for neuroscience, university of copenhagen, denmark; department of neurology, developmental neurobiology, university hospital würzburg, germany; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. charcot-marie-tooth neuropathy type a (cmt a) resulting from peripheral myelin protein kda (pmp ) overexpression is the most common hereditary motor and sensory neuropathy in humans. the transgenic pmp (pmp tg) mouse line c carrying copies of the human pmp gene, has a slowly progressing neuropathy phenotypically like cmt a with thin and abnormally thick myelin profiles and supernumerary schwann cells. in addition, pmp tg nerves showed activated macrophages leading to axon-myelin compartment disruption and maldistribution of k+ channels (kohl b et al, am j pathol. ; : ) . the aim of the present study was to investigate the motor axon excitability in pmp tg versus wt littermates. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle and "threshold-tracking" the plantar compound muscle action potential (cmap). at age months, when the post-developmental maturation was nearly complete in the wt, the pmp tg cmap showed an increase in latency by %. the cmap amplitude was decreased by %, although the mean motor unit size (mscan method) appeared unchanged indicating a lack of collateral sprouting. furthermore, pmp tg showed abnormalities in both passive cable properties and voltage dependent parameters. at age month, the cmap latency of pmp tg was increased by % as compared to wt. in contrast to this marked conduction slowing along the tibial nerve from to months of age, the progression of excitability changes localized at ankle appeared modest. nevertheless, when pooling data from to months, the increase in pmp tg latency was correlated (spearman p< . ) with an increase in accommodation half-time during depolarizing electrotonus (+ % of threshold) from to ms and a reduction of the late subexcitable period of the recovery cycle from to % of threshold, both changes consistent with a redistribution of k+ currents consistent with the maldistribution of k+ channels. our data suggest that in the pmp tg cmt a model, a functional, thus potentially reversible abnormality in k+ channel distribution, accumulates along the nerve and aggravates the conduction impairment due to impaired myelin formation and maintenance. alvarez s , krarup c , , moldovan m , . center for neuroscience, university of copenhagen, denmark; department of clinical neurophysiology, rigshospitalet, copenhagen, denmark. mice heterozygously deficient of myelin protein p gene (p +/−) show a mild progressive dysmyelinating neuropathy, with conduction slowing and impaired excitability, phenotypically similar with charcot-marie-tooth disease type b (cmt b). we found that in p +/− the accumulating myelin abnormalities were paralleled by progressive changes in voltage-dependent motor axon function resulting in neurotoxic membrane depolarization (rosberg mr, et. al. neurobiol dis. : ) . the aim of this study was to investigate the relationship between demyelination and motor axon function in p +/−. demyelination of the right sciatic nerve by topic lysophosphatidylcholine (lpc) application was carried out in p +/− and wild-type (wt) mice, in year (mature) and years (aged) groups. multiple measures of motor axon excitability under anesthesia were carried out by stimulation of the tibial nerve at ankle (distal to lpc demyelination) and "threshold-tracking" the plantar cmap responses. live imaging studies by cellvizio (mauna kea technologies, paris, france) confocal laser endomicroscopy were carried out in transgenic mice expressing the fluorescent reporter yfp in peripheral nerve axons under the thy promoter. in mature wt the sciatic morphological and electrophysiological demyelinating features following lpc could be readily observed at hours but disappeared by weeks. no morphological changes could be observed at the tibial level. consistently, no conduction or excitability changes could be observed at the right tibial neve level as compared to the left tibial nerve in wt. in contrast, in p +/− the motor axon function was impaired at the tibial nerve level at weeks after sciatic lpc demyelination. in mature p +/−, although the cmap amplitude appeared preserved, the distal motor latency was prolonged whereas the excitability measures showed reduced deviations during threshold electrotonus and increased refractoriness at the expense of superexcitability of the recovery cycle, both consistent with membrane depolarization. furthermore, in aged p +/− the delayed tibial conduction was associated with a drop in cmap amplitude and a prolongation of the strength-duration time constant. taken together these data suggest that focal demyelination aggravates membrane dysfunction along the entire motor axon in p +/− providing a novel experimental model to explore the link between demyelination and axonal membrane dysfunction in cmt b. amass l , li h , gundapaneni b , schwartz j , keohane d . pfizer inc., new york, ny, usa; inventiv health inc., burlington, ma, usa. a number of factors can influence disease progression in transthyretin familial amyloid polyneuropathy (ttr-fap), a rare, fatal, hereditary amyloidosis. this analysis evaluated the specific role of baseline neurologic severity on neurologic disease progression in ttr-fap. a predictive model was created based on longitudinal data from val met patients who participated in the tafamidis (a selective ttr stabilizer) clinical development program. data from the intent-to-treat population of the double-blind, placebo-controlled registration study (tafamidis group, n= ; placebo group n= ) and its two consecutive open-label extension studies in which all patients received tafamidis were used. the second extension study is ongoing, but a formal, prospectively-planned interim analysis was conducted with the cut-off date of december , . this analysis focused on the first months of treatment for the overall study cohort analyzed. the neuropathy impairment score-lower limbs (nis-ll) was used to assess neurologic functioning at baseline and at subsequent study visits. a linear mixed-effects model for repeated-measures (mmrm) analysis, with baseline nis-ll, treatment, and their interactions with time as fixed effects, was used, and the slope and intercept for each patient were included as random effects. patients were primarily caucasian with early-stage neurologic disease (baseline nis-ll mean [standard deviation]: tafamidis, . [ . ]; placebo, . [ . ] ). across both groups, disease progression increased with increasing levels of baseline severity (nis-ll) (p< . ). however, the predicted magnitude of change from baseline to month for tafamidis was consistently less than that for placebo across a range of observed baseline nis-ll values, suggesting a disease-modifying effect of tafamidis. similar findings were observed for the nis-ll muscle weakness subscale. this mmrm analysis in patients with val met ttr-fap demonstrates that disease progression strongly depends on baseline neurologic impairment and highlights the disease-modifying effect of tafamidis across a range of baseline levels of neurologic severity. clinicaltrials.gov identifiers: nct , nct , nct . amino h , misawa s , sekiguchi y , shibuya k , watanabe k , suichi t , kuwabara s . department of neurology, chiba university, chiba, japan. guillain-barré syndrome (gbs) is a potential life threatening neurological disorder and respiratory insufficiency is one of the critical complications. eramus gbs respiratory insufficiency score (egris) is a method for predicting the chance of respiratory insufficiency in gbs. however, clinical characteristics and courses can vary for subtypes of gbs, whose occurrences differ for each region: acute inflammatory demyelinating polyneuropathy (aidp) is very common in european countries, whereas acute motor axonal neuropathy (aman) is frequently seen in asian countries. the aim of this study is to investigate the usefulness of egris in japan, where aman is more common than in the netherlands. clinical and electrophysiological profiles of consecutive gbs cases, who visited our hospital within days after symptoms onset between and , were reviewed. of the gbs patients, % were classified as aidp and % as aman according to the electrodiagnosis criteria by ho and colleagues. higher egris scores correspond to higher risk of respiratory insufficiency in total of the gbs patients, as well as in aidp patients. however, in patients with aman, egris scores did not always match the chances of respiratory insufficiency: up to % of the patients with low risk of egris showed respiratory failure, whereas only % of the patients with high risk of egris needs intubation/mechanical ventilation. in aman, associations with mechanical ventilation were seen for rapid progression (shorter duration between onset and hospital admission), more decreased vital capacity, and more frequent autonomic involvement. egris is useful also for japanese gbs patients. however, for aman patients, it should be used with discretion. another score to predict respiratory insufficiency might be required in asian countries. anandan c , litchy wj , laughlin rs , leep hunderfund an , naddaf e . mayo clinic, rochester, usa. in patients with suspected ulnar neuropathy, nerve conduction studies (ncs) are commonly requested to help with diagnosis and localization. however, routine ncs are often normal or not localizing. ulnar ncs recording from the first dorsal interosseous muscle (ncs-fdi) is thought to increase the diagnostic yield of electrodiagnostic testing, although not commonly considered. we developed a quality improvement strategy to routinely perform ulnar ncs recording from the abductor digiti minimi muscle (ncs-adm) as well as ulnar ncs-fdi in all patients referred for suspected ulnar neuropathy. we utilized the dmaic (define, measure, analyze, improve, control) model of process improvement to define our problem and create a map of the current process for ulnar neuropathy diagnosis in our electromyography laboratory. we determined baseline performance via review of distal sensorimotor polyneuropathy (dpn) is the most common complication of diabetes and risk factors beyond hyperglycemia have proven important particularly in type diabetes (t dm). only few prospective studies from early-stage t dm exist. we aimed to study the development of dpn during the first years after a screening-based diagnosis of t dm. from the addition-denmark study participants were eligible for this study. dpn was assessed by the michigan neuropathy screening instrument questionnaire (mnsi) at four time-points during follow-up. dpn was defined by a mnsi score ≥ . participants ( %) were positive in mnsi at baseline and thus excluded from this study. by kaplan-meier plot we evaluated the cumulative incidence of dpn and in cox proportional hazard models we calculated hazard ratios (hr) for the intervention groups in the addition trial and for various covariates proposed to influence the development of dpn. models were adjusted in steps for intervention group, age, sex, baseline mnsi, lipid-lowering and anti-hypertensive treatment. this study cohort consists of participants ( % men) with a median age of . years (p ;p : . ; . ) and median baseline hba c of . (p ;p : . ; . ). a cumulative incidence of % was seen during years of diabetes. there was no statistically significant difference in hr between the intervention groups or by sex but a significantly higher hr of . ( %ci: . ; . ) was seen for age (per year). the highest hr was found for a history of cardiovascular disease (myocardial infarction or stroke) up to ten years prior to the diabetes diagnosis with a hr of . ( . ; . ). weight, waist circumference, body-mass index and methylglyoxal (log transformed) showed modest but statistically significant associations with incident dpn with standardized hrs of . ( %ci: . ; . ), . ( %ci: . ; . ), . ( %ci: . ; . ) and . ( %ci: . ; . ) respectively. this study demonstrates a fairly low cumulative incidence of dpn in people with screen-detected t dm and provides evidence that macrovascular disease, obesity and oxidative stress are important risk factors for dpn even at the earliest stages of t dm. andermann syndrome, also known as agenesis of the corpus callosum and peripheral neuropathy (accpn), is an autosomal recessive disorder with a broad spectrum of mild to severe neuromuscular and psychiatric consequences. the gene variants causing disease were first identified in french-canadian families. in the present study, we intended to phenotype and genotype a series of non-french-canadian familial cases presenting with charcot marie tooth disorder associated with agenesis/dysgenesis of the corpus callosum. for this purpose, seven families, of consanguineous marriage, were studied. patients were clinically and para-clinically investigated using mri and electrophysiology (mncvs). for some, a sural nerve biopsy was taken. microsatellite markers around the accpn locus were used in two large families; followed by sanger sequencing of all the exons and intron-exons boundaries of the gene, in one patient from each of the families. the age at onset of the disease was at birth in the patients from the largest consanguineous family ( affected individuals). the biopsy from one patient showed a severe demyelinating neuropathy with many hypomyelinated fibres and mostly secondary axonal changes. these finding were compatible with electrophsiological data where the mncvs are of demyelinating range. dysgenesis of the corpus callosum in one patient and agenesis in another sib were revealed by mri. we identified one homozygous truncating mutation in this family. interestingly, no causative variant was found in a patient from another family and showing homozygous haplotype. two different heterozygous variants were identified at one hit in two patients from two non-consanguineous families. genetic investigations will be continued to identify the possible second hit. in the remaining families, no variant was found. the negative family cases will be subjected to ngs. at this stage, it is tempting to speculate on the genetic heterogeneity of accpn in our series. baba m , suzuki c , ogawa c , tomiyama m . department of neurology; diabetes center, aomori prefectural central hospital, aomori, japan. in we introduced a staging system of severity of diabetic polyneuropathy (dpn) by nerve conduction study (ncs) of the lower limb: sensory ncs of the sural nerve and motor ncs of the tibial nerve. the system consists of five stages; ncs- (normal): no abnormalities, ncs- (mildly abnormal): presence of delay of mcv, scv, minimal f-wave latency, or positive a-wave, ncs- (moderately abnormal): decrease in sural snap less than uv, ncs- (severely abnormal): decrease in plantar muscle-cmap to - mv, ncs- (ultimately abnormal): plantar muscle-cmap lost or less than mv with trace of sural-snap. to examine validity of the system, we conducted -year prospective observation on development of diabetic foot (df) by the ncs staging system. in addition, occurrence of ischemic heart disease (ihd) and stroke (is), and death of neuro-vascular events were also counted. n - , we carried out ncs in diabetics, and categorized them by the ncs staging system: % was ncs- , % was ncs- , another % was ncs- , % was ncs , and % was ncs- . we then followed them and prospectively counted the occurrence of df, ihd and is in patients (mean age ys). the occurrence of df during the following years was; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- : %. occurrence of any of df, ihd and/or is was as follows; ncs- : %, ncs- : %, ncs- : %, ncs- : %, ncs- %. there was no death from nsc- , − , and − groups (n= ), while two from ncs- group (n= ) were found dead in a bed or on a driver's seat, and other two from ncs- group (n= ) died of sudden cardiac arrest or infection after foot amputation. in summary, the present ncs grading system seems to work satisfactory not only for diagnosis of severity of the current dpn, but also for prognostic prediction of the dpn-related foot and vascular events. bacon c , feely sme , shy me . university of iowa hospital, iowa city, ia, usa. for patients with charcot-marie-tooth disease, also known as hereditary motor and sensory neuropathy, the rasch modified cmtnsv is a validated measurement of symptoms and impairment. the score is comprised of nine parameters of a clinical examination, including nerve conduction studies (ncs) . each parameter has an individual score ranging from zero to four, with the composite score having a maximum of prior to rasch modification. for patients who do not complete a ncs, the cmt exam score (cmtesv ) is used, with a maximum score of before rasch modification. one parameter of the cmtesv is the vibration sense. using a rydel tuning fork, a care-giver measures vibration sense in a patient's feet, ankles, and knees and a score is determined by the severity of reduced vibration sense. in cmt a patients, % received a score of out of , noted by a reduced vibration sense at the knee. in order to capture a more sensitive vibration measurement, we tested ways of taking the total raw score of the rydel tuning fork at each point of vibration sense, the toe, ankle, and knee bilaterally. scores range from zero to , with reflecting full vibration sense. in this modified measurement, vibration sense scores varied in wider distribution. for cmt a patients captured in this studied, a modified vibration sense score results in a potentially more sensitive total cmtesv score. bae dw , an jy . st. vincent's hospital, the catholic university of korea, suwon, korea. diabetic lumbosacral radiculoplexus neuropathies (dlrpn) are usually subacute painful, monophasic, asymmetrical lower limb neuropathies with incomplete recovery due to ischaemic injury and microvasculitis. the diagnosis relies mostly on clinical suspicion and characteristic electromyographic findings. however, in acute phase, neuroimaging has more important diagnostic significance than electrophysiological studies. here we describe mri and ultrasound findings in a -year-old woman with dlrpn who was diagnosed with diabetes three years ago, but has not received any other treatment. she had a -day history of acute-onset severe pain with weakness of muscles innervated by left femoral and obturator nerve and decreased sensation in left l -l dermatome. nerve conduction studies showed reduced amplitude in left femoral nerve and electromyography showed only increased insertional activity in left iliopsoas muscle and no volitional activity in muscles innervated by left femoral and obturator nerve. ultrasound revealed increased cross-sectional area (csa) of left femoral and lateral femoral cutaneous nerve. mri showed enhancement in left l , nerve roots and proximal femoral nerve and increased signal intensity in left iliacus and iliopsoas. we diagnosed her with dlrpn and started corticosteroid. nerve ultrasound has not been previously reported in a patient with cabin air in commercial airliners originates from aircraft engines or auxiliary power units. this bleed air may occasionally be contaminated with hydraulic fluids and engine oil that contains a number of potentially hazardous chemicals including tricresyl phosphate (tcp). over the last years reports are emerging about aircrew members that experience symptoms such as tingling or burning of extremities in addition to headache, and vertigo. this "aerotoxic syndrome" is controversially discussed in the literature and has been attributed to exposure to organophosphate contaminated cabin air. since tcp has been associated with peripheral neuropathy we aimed to determine the frequency of peripheral neuropathy among frequent flyers. civilian air crew members and frequent flying passengers (m:f = : , median age years, median exposition time in aircrafts of . hours) were examined at the university hospital of cologne or at the frankfurt airport (iata code fra) by a detailed questionnaire of past medical conditions, a standardized neurological examination and nerve conduction studies of sural, tibial, and ulnar nerves. we identified subjects with clinical and electrophysiological evidence for large fiber peripheral neuropathy. incidence of peripheral neuropathy was not correlated to exposition time in aircrafts. in addition subjects showed signs of ulnar neuropathy, subjects reported abnormal vibration sensation, subjects suffered from gait imbalance and individuals reported tingling of extremities. our study shows a . % prevalence of large fiber peripheral neuropathy among frequent flyers. comparison of these data with prevalence rate in an age-matched control group will reveal a possible association of chronic exposure to cabin air and risk for peripheral neuropathy. the high incidence of the symptom tingling in our cohort warrants further studies to determine the risk for small fiber neuropathy in this condition. intravenous immunoglobulin (ivig) are human igg derived from plasma pools of healthy donors. although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on bortezomib (btz)-induced peripheral neuropathies. female wistar rats were treated following a preventive schedule (btz and ivig co-treatment for and weeks) and a therapeutic schedule ( weeks of btz treatment followed by a -week ivig-btz co-treatment). caudal nerve conduction velocity (ncv), plantar and dynamic tests were performed at different time points. animals were sacrificed after ws (acute phase) or ws (chronic phase) and tissue samples (dorsal root ganglias -drg-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.in the preventive schedule, ivig was not able to rescue caudal ncv reduction caused by btz neither after nor after weeks of co-treatment. same results were observed in the therapeutic schedule. on the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that ivig injection protected from btz effect in both treatment schedules. morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from btz damage. this result is consistent with the morphological evaluation of the nerve. also, intra-epidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. finally, sciatic nerve and drg macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with ivig alone) levels in the preventive one. in conclusion, we were able to demonstrate for the first time that ivig treatment especially used as preventive treatment option may reduce btz-induced neuropathic painful pointing out the possible role of inflammation in the pathogenesis of this invalidating pathology. this work was supported by kedrion spa. we studied the prevalence, the molecular cause and clinical presentation of hereditary motor neuropathies in a large cohort of patients from the north of england. detailed neurological and electrophysiological assessments and next generation panel testing or whole exome sequencing were performed in patients with clinical symptoms of distal hereditary motor neuropathy (dhmn, patients), axonal motor neuropathy (motor cmt , patients) or complex neurological disease predominantly affecting the motor nerves (hmn plus, patients) . the prevalence of dhmn is . affected individuals per . inhabitants ( % confidence interval: . - . ) in the north of england. causative mutations were identified in out of index patients ( . %). the diagnostic rate in the dhmn subgroup was . %, which is higher than previously reported ( %). we detected a defect of neuromuscular transmission in cases and identified potentially causative mutations in patients with demyelinating multifocal motor neuropathy. many of the genes were shared between dhmn and motor cmt , indicating identical disease mechanisms therefore we suggest changing the classification and include dhmn also as a subcategory of cmt. abnormal neuromuscular transmission in some genetic forms provides a treatable target to develop therapies. barohn rj , gajewski b , pasnoor m , brown l , herbelin l , kimminau k , jawdat o , parks c , shlemon p , dimachkie mm and the pain-controls study team . the university of kansas medical center, kansas city, ks, usa. cryptogenic sensory polyneuropathy (cspn) is a common slowly progressive neuropathy that affects adults and presents with significant neuropathic pain for which multiple medications have been tried including antiepileptics, antidepressants, topicals and narcotics. a web based survey among neuromuscular experts suggested pregabalin as being more effective than other medications, however there are presently no comparative studies to assess the most effective medication. the objective of this study was to determine which of the pharmaceutical therapies (pregabalin, duloxetine, nortriptyline or mexiletine) is most effective for neuropathic pain and best tolerated in cspn. to achieve this objective we performed a prospective randomized open labelled comparative effectiveness adaptive design study of cspn patients through the patient centered outcomes research institute (pcori). cspn patients who fulfilled the inclusion and exclusion criteria were enrolled into this study. patients underwent a baseline neurological evaluation and randomly assigned to one of the neuropathic medications for months. the primary outcome is the change in likert-like pain scale. the secondary outcomes included nih pain interference scale, nih fatigue interference scale, nih sleep disturbance scale, sf- and adverse events. the outcome measures are performed at baseline, month , and . statistical analysis using bayesian adaptive design developed by berry consultant software will be performed to determine winner and losers (winner = greater than point improvement in pain). total number of patients to be enrolled is . recruitment has been challenging and a number of recruitment techniques have been used. to date, there have been patients screened, patients randomized from us sites. anticipated completion of enrollment by june and end of final patient assessment by september . interim analysis performed after first patients completed their months as part of bayesian adaptive design analysis and occurs every weeks. the distribution of randomization of patients to the medications at last adaptive design randomization was . % to medication , % to medication , . % to medication and . % to the th medication. this study may give physicians and patients evidence for future management of cspn patients. transthyretin amyloidosis (attr), which encompasses a group of disorders with significant clinical variability, is caused by transthyretin (ttr) derived amyloid deposition. the clinical aspects of autonomic nervous system involvement in attr are only partially known. the ongoing, multinational, longitudinal, observational transthyretin amyloidosis outcomes survey (thaos) provides the opportunity to expand our understanding of dysautonomia in attr. data from all symptomatic subjects enrolled in the thaos registry with a diagnosis of attr (cut-off date: january , ) were assessed for the presence and temporal course of autonomic symptoms, genotype and phenotype associations, and clinical burden according to the frequency and severity of symptoms. of symptomatic subjects enrolled in thaos, ( . %) had autonomic symptoms at enrollment including: gastrointestinal ( subjects, . %), urinary ( , . %), erectile dysfunction ( , . %), orthostatic hypotension ( , . %), xerophthalmia ( , . %) and dyshydrosis ( , . %). subjects with autonomic manifestations, compared with those without, were younger (mean age [standard deviation, sd] of . [ . ] vs . [ . ] years), with a longer duration of attr symptoms ( . [ . ] vs . [ . ] years). autonomic dysfunction was less common with wild-type attr ( of subjects, . %) than in mutation groups: val met ( / , . %); non-val met/non-cardiac ( / , . %); and "cardiac mutations" (val ile, leu met, thr ala, or ile leu mutations; / , . %). similarly, time (mean [sd] , years) from first attr symptoms to onset of autonomic symptoms, was longest for wild-type attr ( . [ . ] ) followed by "cardiac mutations" ( . [ . ]), non-val met/non-cardiac ( . [ . ]), and val met ( . [ . ] ). autonomic symptoms were present at disease onset in over a third of subjects ( , . %) . autonomic dysfunction was less frequent in subjects with cardiac phenotype ( of subjects, . %), than with mixed ( / , . %) or neurologic ( / , . %) phenotypes. the burden of autonomic symptoms (mean [sd] ) varied by genotype, val met ( . [ . ] , non-val met/non-cardiac ( . [ . ], "cardiac mutations" ( . [ . ]), wild-type attr ( . [ . ]), and by phenotype, mixed ( . [ . ]), neurologic ( . [ . ]), cardiac ( . [ . ]). dysautonomia is common, and a significant burden, in subjects with hereditary forms of attr. its prevalence is higher in val met than in other genotypes, and in the neurologic or mixed phenotypes. the objective of this study is to assess the usefulness of motor unit number index (munix) technique in charcot-marie-tooth type a (cmt a) disease and to test correlation between munix and clinical impairment. munix technique was performed in abductor pollicis brevis (apb), abductor digiti minimi (adm) and tibialis anterior (ta) muscles in the non-dominant side. a munix sum score was calculated by adding munix of these muscles. muscle strength was measured using the mrc (medical research council) scale. disability was evaluated with several functional scales including cmt neuropathy score version (cmtnsv ) and overall neuropathy limitation scale (onls). cmt a patients with known pmp gene duplication were enrolled. the munix of the adm, apb and ta muscles were correlated with the mrc of the corresponding muscle (p< . ). munix sum score was correlated with clinical scales: cmtnsv (r=− . , p< . ), onls (r=− . , p< . ). in conclusion, munix correlates with muscle strength and clinical measurements of disability in cmt a patients. the munix technique evaluates motor axonal loss and correlates with disability. the munix sum score may be a useful outcome measure of disease progression in cmt a. the objective of this study was to assess the usefulness of mri in charcot-marie-tooth type a (cmt a) disease and to test correlation between muscle fat fraction and clinical impairment. mri was performed in the non-dominant lower limb of cmt a patients and healthy controls. fat fraction of tibialis anterior muscle, cross section area and volume of sciatic nerve were determined. muscle strength of dorsiflexion was measured using a dynamometer. disability was evaluated with cmt neuropathy score version (cmtnsv ). cmt a patients with known pmp gene duplication were enrolled. fat fraction of tibialis anterior muscle was significantly increased in patients compared to healthy controls. it was correlated with muscle strength (r=− . , p< . ) and cmtnsv score (r=− . , p< . ). cross section area and volume of sciatic nerve were significantly increased in patients compared to healthy controls. in conclusion mri fat fraction correlates with muscle strength and clinical measurement of disability in cmt a patients. it may thus be a useful outcome measure of disease progression in cmt a. basta i , peric s , cobeljic m , bjelica b , bozovic i , kacar a , nikolic a , rakocevic stojanovic v , stevic z , lavrnic d . neurology clinic, clinical center of serbia, school of medicine, university of belgrade, belgrade, serbia. there is a complete lack of data about epidemiological and clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) in serbia and surrounding countries. furthermore, there is a striking scarcity of information about quality of life (qol) in cidp patients and all qol studies have been conducted in countries with high standards of health care. in august we have designed the iness (inflammatory neuropathy study of serbia) in order to comprise as many patients with cidp from serbia, republic of srpska (bosnia and herzegovina) and montenegro covering population of more than nine million people. our first aim is to analyze overall impact of cidp on physical, mental and social areas of life measured with generic, symptom specific and disease specific questionnaires -sf- , inqol and cap-pri, respectively. furthermore, we aim to analyze influence of the disease on patients' working status and presence of depressive mood measured by beck's inventory. following features of patients are included: sociodemographic data, clinical aspects of the disease, level of disability, severity of sensory symptoms, presence of comorbidities, electrophysiological characteristics, as well as fatigue, autonomic symptoms and neuropathic pain. we intend to define the most significant predictors of decreased qol in order to focus on patients with the highest risk and to improve care of cidp. we also want to see if cidp patients in complete remission as per clinical findings still have reduced quality of life. we have recruited patient so far and we expect to include around subjects overall. we will present the first data of the study at the pns meeting . beaudonnet g , prud'hon s , cauquil c , labeyrie c , not a , bouilleret v , adams d . neurophysiology chu bicêtre, le kremlin bicêtre, france; neurology chu bicêtre, le kremlin bicêtre, france. familial amyloid neuropathy (fap) is a life-threatening disease of autosomal dominant inheritance due to transthyretin (ttr) gene mutation, a liver-produced protein. current treatments slow down its natural course and are indicated from the very first objective symptoms. we aimed to evaluate two neurophysiological markers: sympathetic skin response (ssr) and heart rate variability (hrv) in the early detection of sympathetic damages due to fap. ssr and hrv were assessed in ttr gene mutated patients with neither clinic nor electroneuromyographic abnormalities and controls matched on gender and age. cases were recruited consecutively from current care in the french reference center for rare diseases of bicetre university hospital. ssr was recorded on the two palms and on the sole of the left foot with to stimulations between and ma. hrv was registered during three conditions of seconds each: normal breathing, deep breathing ( cycles of seconds of inspiration and seconds of expiration) and valsalva manoeuver during seconds. valsalva ratio, defined by the ratio between the longest and shortest rr intervals, was significantly higher in the control groups after bonferroni correction (means of . and . , respectively, p< . ). there was no significant difference between the two groups for any ssr parameter, although means of amplitudes were systematically higher in controls than among cases. our results confirm that autonomic nervous fibers are damaged early in both clinical and electroneuromyographic asymptomatic patients mutated on the ttr gene. valsalva ratio seemed to be the most discriminative marker. long-term follow-up with test repetition and confrontation with cardiologic assessment will help to precise how these tests could be used in current care. they might help to identify high risk patients to propose them an appropriate early treatment and could be used to follow treatment efficacy. familial amyloidotic polyneuropathy (fap) was originally characterized by andrade as an axonal neuropathy which subsequently was found to be associated with a number of mutations in the plasma protein transthyretin (previously named prealbumin). it is now recognized that cardiomyopathy may be a significant factor in a majority of patients with the hereditary form of transthyretin amyloidosis (fap) and many of the transthyretin (ttr) mutations are associated with cardiomyopathy with no or minimal signs of peripheral neuropathy. attr-wt also called senile cardiac amyloidosis and senile systemic amyloidosis is recognized as late-onset, usually in the th or th decade of life, and the fact that the majority of patients are males. transthyretin neuropathy proven by nerve biopsy has been rarely reported in this population. here we report our experience with patients having attr-wt characterized by cardiomyopathy but also with varying degrees of peripheral neuropathy. clinically, the neuropathy appears as typical axonal or mixed axonal/demyelinating neuropathy as is seen in fap. pathologically, two types of ttr deposition have been found, ( ) intraneural ttr amyloid deposits as seen in fap are present in some patients and ( ) other patients have extensive vascular deposition of amyloid in both perineural arteries and veins without deposits within nerve trunks. in conclusion, peripheral neuropathy may definitely be a part of the attr-wt clinical presentation and with the increase in numbers of attr-wt cardiomyopathy patients being identified, it is important to ascertain whether any evidence of peripheral neuropathy is due to the amyloidosis and not to compounding syndromes such as diabetes mellitus type ii. besora s , santos c , izquierdo c , martinez-villacampa m , simó m , bruna j , , velasco r , . neuro-oncology unit, hospital universitari de bellvitge-institut català d́oncologia, ĺhospitalet, barcelona, spain; medical oncology department, institut català d́oncologia, ĺhospitalet, barcelona, spain; department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. oxaliplatin (oxa) is the first-line chemotherapy agent in the treatment of colorectal cancer (crc). oxa-induced peripheral neuropathy is the most frequent long-term side-effect. retreatment with oxa is frequently considered in patients as salvage treatment. patients receiving oxa-based chemotherapy regimen at least twice at our institution between and were reviewed. the aim of this study was to investigate whether retreatment with oxa increases the risk of developing or worsening previous oxa-induced peripheral neuropathy. the severity of neuropathy was measured by national cancer institute-common toxicity criteria (nci), total neuropathy score (tns) © and nerve conduction studies. one hundred twenty-five crc patients were included. median age was years. after first-line oxa-based chemotherapy, . % of patients developed neuropathy according nci, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. severity of neuropathy was grade ( . %), grade ( %), and grade ( . %). median time to retreatment with oxa was months. frequencies of neuropathy before retreatment were as follows: . % grade , . % grade , and . % grade . after retreatment, severities of neuropathy were . % grade and . % grade . no patient developed grade . . % of patients did not develop neuropathy. peripheral neuropathy was the reason for stopping prematurely treatment after first-line and retreatment in . % and . % of patients, respectively. worsening of previous nci score was observed in . % of patients. the great majority of patients ( . %) remained within the same nci score than before retreatment after median [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients that did not develop neuropathy after first treatment (n= ), only and patients developed grade and , respectively, after a median of [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cycles. among those patients who initially developed grade and neuropathy, no differences in tnsc © scores just before and after finishing retreatment with oxa were identified ( [ - ] vs [ - ], p= . ). retreatment with oxa in crc patients is a feasible option even in patients who developed moderate or severe neuropathy previously. lack of worsening of previous neuropathy is observed in the great majority of patients. neurological monitoring of patients candidates to retreatment with oxa should be considered. bessaguet f , sturtz f , magy l , , desmouliere a , bourthoumieu s , demiot c . ea -myelin maintenance & peripheral neuropathy, faculties of medicine and pharmacy, university of limoges, limoges, france; department of neurology, reference center for rare peripheral neuropathies, university hospital of limoges, limoges, france. prolonged pressure and the resulting local ischemia are widely accepted as the primary etiology of skin pressure ulcers (pus) but precise mechanisms of their formation remain unclear. in this study, we wanted to study the potential role of sensory small nerve fibers in regulation of inflammation during pus formation. to achieve this goal, we developed a mouse model of a purely sensory neuropathy and this was induced by resiniferatoxin (rtx). in this model, seven days after a single injection of rtx ( g/kg; ip), mice present a thermal and mechanical hypoalgesia associated with large substance p (sp) and calcitonin gene-related peptide (cgrp) depletion without neurodegeneration. this model mimic quite well what is observed in early stages of sensory nerve fiber defect. studies have shown that sp and cgrp are involved in cutaneous inflammation regulation. in fact, these neuropeptides are released by sensory fibers and are pro-inflammatory mainly through recruitment of immune cells and vasodilation. thus, we studied gene expression of pro and anti-inflammatory cytokines by a rna array approach during pus formation in control and rtx mice. seven days after a single injection of rtx, epidermis, dermis and subcutaneous tissue layer were pinched with magnetic plates during hours. pressure induced a stage pus. gene expression was evaluated in each compressed area h after pressure. results showed mainly a down-regulation of gene expression in pus of rtx mice compared to control mice. a decrease of cgrp/sp in skin sensory small fiber increased pus formation associated with an increase of interleukins (il)- , il- , il- and il- expression and a decrease of il- expression in rtx mice. supplementary experiments with rt-qpcr for each cytokine will be necessary to confirm these preliminary results. these observations suggest a cgrp/sp role in regulation of cytokines expression during pus formation. the new inflammatory profile exhibited in this study might help in the design of new treatments improving the quality of life of neuropathic patients prone to developing bedsores. bhattacharyya bj , jayaraj nd , belmadani a , ren d , rathwell ca , hackelberg s , miller rj , menichella dm . department of neurology, northwestern university, chicago, il, usa; department of pharmacology, northwestern university, chicago, il, usa. painful diabetic neuropathy (pdn) is one of the most common and intractable symptoms of diabetes, affecting % of diabetic patients. the hallmarks of pdn are neuropathic pain and small fiber degeneration, manifested by the loss of dorsal root ganglion (drg) nociceptor axons. neuropathic pain is associated with nociceptor hyper-excitability in the absence of physiologically appropriate stimuli. in states of neuropathic pain, drg nociceptors become increasingly responsive to a variety of excitatory influences, including inflammatory cytokines. in particular, we have shown that stromal cell derived factor- (sdf- ) and its receptor cxcr are necessary for the generation of neuropathic pain in mouse models of pdn. however, the molecular mechanisms leading to the hyper-excitability of drg nociceptors in pdn are unknown, as are the mechanisms leading to small fiber degeneration. this fundamental gap in our knowledge represents a critical barrier to progress in developing novel therapeutic approaches for pdn. the objective of this study is to identify the molecular cascade linking cxcr /sdf- chemokine signaling to drg nociceptor hyper-excitability, neuropathic pain, and small fiber degeneration in pdn. drg nociceptors can be identified by a series of molecular markers, including expression of the sodium channel na v . . indeed, > % of na v . -expressing drg neurons are nociceptors. feeding mice a high fat diet (hfd) for several weeks induces glucose intolerance, obesity, and mechanical allodynia, a particular pain hypersensitivity associated with pdn. using the hfd model combined with dreadd receptor technology, we have shown that reducing excitability of na v . -expressing neurons prevents and reverse neuropathic pain, neuronal calcium overload, mitochondrial dysfunction, and small fiber degeneration. furthermore, we have shown that cxcr receptors are necessary for neuropathic pain and small fiber degeneration in pdn. taken together these data demonstrate that na v . nociceptor hyperexcitability in pdn is driven through the activation of cxcr receptors. inhibition of hyperexcitability can prevent and reverse the development of pdn. furthermore, these observations will advance our understanding as to how changes in excitability, calcium influx, and mitochondrial dysfunction in nociceptors contribute to neuropathic pain and small fiber degeneration in pdn, which is a critical barrier to progression for effective and disease modifying treatment for pdn. bianco m , terenghi f , gallia f , nozza a , scarale a , fayoumi mz , giannotta c , morenghi e , nobile-orazio e . poems (polyneuropathy, organomegaly, endocrinopathy, m protein and skin changes) syndrome is an unusual multisystem disease with neurological disability due to a severe disabling polyneuropathy, with high mortality by multiorgan failure. peripheral blood stem cell transplantation (pbsct) is considered the treatment of choice for poems while lenalidomide is the most promising therapy for patients not eligible for pbsct. the aim of the present study was to compare the long-term effects on clinical, biological and neurophysiologic parameters in patients with poems treated with lenalidomide or pbsct. the clinical, biological and neurophysiologic data were reviewed in poems patients treated with pbsct (n: ) or lenalidomide (n: ). the mrc sumscore on muscles, onls scale, vegf serum levels and nerve conduction studies were assessed before (t ) and after (t ) and years (t ) of treatment and the differences were compared using anova. combining the two groups of patients, there was a significant improvement after treatment in the mean mrc sumscore (t = ± ; t = ± ; t = ± ; p = . ), in the mean onls score (t = . + . ; t = . + . ; t = . + ; p = . ), in the ulnar mean distal motor latency (t = . ± . msec; t = . ± . msec; t = . ± . msec; p = . ), distal compound muscle action potentials amplitude (t = . ± . mv; t = . ± . mv; t = . ± . mv; p = . ), motor conduction velocity (t = . ± . m/sec; t = ± m/sec; t = . ± . m/sec; p= . ) and serum vegf levels (t vs t : p = . ; t vs t : p = . ). the difference was also significant when we separately analyzed patients treated with lenalidomide and pbsct and there was no difference between the two groups in any of the analyzed parameters. treatment with pbsct and lenalidomide significantly and similarly improved clinical, biological and neurophysiologic parameters in patients with poems syndrome up to two years. since pbsct may not be suitable for all patients, lenalidomide may represent an effective and a valuable alternative in these patients or in those relapsing after pbsct inducing a prolonged clinical, biological and neurophysiologic improvement. bis d , tao f , abreu l , sleiman p , hakonarson h , zuchner s and inherited neuropathy consortium. dr. j.t. macdonald department for human genetics, hussman institute for human genomics, university of miami, miami, fl, usa; center for applied genomics, the children's hospital of philadelphia, philadelphia, pa, usa. inherited peripheral neuropathies are clinically and genetically heterogeneous diseases that can cause distal muscular atrophy and sensory loss. alleles in over one hundred different genes have been shown to cause peripheral neuropathies; yet, greater than % of axonal neuropathy patients do not receive a genetic diagnosis. large scale exome studies are now beginning to be sufficiently powered to perform mutational burden analysis. this approach compares damaging allele frequencies of cmt cases with a control group to identify additional causes for neuropathies. this approach will also identify genes that require an oligogenic inheritance to cause a phenotype. in a deviation from the classic linkage-based and heuristic variant filtering approaches to gene identification, we are performing burden analyses in a large cohort of cmt families. in known neuropathy genes, we saw that neuropathy cases carried on average . rare, non-synonymous variants, while unrelated non-neuropathy controls harbored . variants (p= . e- , mann-whiney u-test). enrichment of rare, non-synonymous variants in cmt disease genes within inherited peripheral neuropathy cases suggests the presence of multiple weaker alleles in individual patients. we also performed an unbiased exome-wide gene-based burden analysis and ranked genes after multiple testing correction. several new candidate genes were identified that need further follow up conformational studies. a number of known cmt and related genes were observed in the list of top candidates. we are currently analyzing additional aspects of this sample and are actively seeking more cmt exomes to enlarge our study. in summary, statistical methods traditionally reserved for more 'common' phenotypes' increasingly are becoming available for rare disease genetics. c bl/ mice were treated with bortezomib alone or in combination with monastrol. neuropathic changes were assessed by nerve conduction studies and histological analysis. analysis of axonal morphology was performed with light and electron microscopy. anti-neoplastic properties of monastrol alone and in combination with bortezomib were assessed in different blood cancer cell lines. prolonged treatment with bortezomib induced a sensory neuropathy in mice. significant changes in axonal morphology correlated with reduced function of peripheral nerves. the administration of monastrol substantially ameliorated morphological features of axonal alterations and sensory neuropathy. cytotoxicity studies in blood cancer cell lines showed no interference of monastrol with the cytostatic effects of bortezomib. our data indicate that monastrol may alleviate bortezomib induced neuropathy. the favorable cytotoxic profile of monastrol makes it an interesting candidate as neuroprotective agent to prevent bortezomib-induced neuropathy. boczonadi v , meyer k , gonczarowska-jorge h , , bartsakoulia m , roos a , , bansagi b , zahedi rp , talim b , bruni f , kaspar b , , lochmüller h , boycott km , müller js , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; rinch, columbus, oh, usa; leibniz-institute für analytische wissenschaften-isas-e.v., dortmund, germany; capes foundation, brazil; department of pediatrics, hacettepe university children's hospital, ankara, turkey; dbbb,university of bari aldo moro, bari, italy; department of neuroscience, the ohio state university, columbus, oh, usa; department of genetics, cheo, university of ottawa, ottawa, canada. while autosomal dominant mutations in gars, encoding the glycyl-trna synthetase, have been identified in patients with charcot-marie-tooth peripheral neuropathy (cmt d) and distal spinal muscular atrophy type v (dsma-v), autosomal recessive mutations cause mitochondrial disease affecting skeletal muscle and heart. gars is a bi-functional enzyme and it is responsible for normal protein translation both in mitochondria and the cytoplasm. in this study we have focused on the mitochondrial function of the gars by investigating a mouse model (gars c r) , human fibroblasts and induced neuronal progenitor cell lines (inpcs). mild mitochondrial abnormalities were detected in skeletal muscle of the gars c r mice while no other tissues were affected. control and patient fibroblasts harboring gars mutation were directly converted into inpcs. we identified tissue specific impairment of mitochondrial function in neuronal cells carrying not only recessive but also dominant gars mutations, suggesting neuron-specific effects of mitochondrial alterations.comparative proteomic analysis of inpcs showed significant changes in mitochondrial proteins. furthermore, the reduction of the vesicle-associated membrane protein-associated protein b (vapb) and its downstream pathways in gars-deficient inpcs suggests that altered mitochondria-associated endoplasmic reticulum (er) membranes (mam) may also contribute to the motor neuropathy. boczonadi v , king ms , bansagi b , roos a , , eyassu f , borchers c , lane m , ramesh v , lochmüller h , pyle a , griffin h , smith ac , chinnery pf , , alan j robinson aj , edmund rs kunji ers , horvath r . jwmdrc, wtcmr, igm, newcastle university, newcastle upon tyne, uk; mitochondrial biology unit, medical research council, cambridge; leibniz institute of analytic sciences (isas), dortmund, germany; uvic-genome bc proteomics centre, vancouver, canada; paediatric neurology, royal victoria infirmary, newcastle upon tyne foundation hospitals nhs trust, newcastle upon tyne, uk. members of the mitochondrial carrier family (slc ) transport nucleotides, keto acids, amino acids, fatty acids, co-factors and inorganic ions across the mitochondrial inner membrane. several inherited diseases with very variable clinical presentations are associated with dysfunctional mitochondrial carriers. we report a patient with childhood-onset spinal muscular atrophy and mitochondrial myopathy caused by a homozygous mutation in slc a , encoding the mitochondrial oxodicarboxylate carrier (odc). the mutation renders the carrier dysfunctional and, consequently, -oxoadipate cannot be imported into the mitochondrial matrix. computer modelling of the metabolic defect caused by the mutation predicted that the impaired transport leads to accumulation of -oxoadipate, pipecolic acid and the known neurotoxin quinolinic acid, which were precisely confirmed by targeted metabolomics in serum and urine. exposure of -oxoadipate and quinolinic acid reduced the level of mitochondrial complexes in sh-sy y cells in-vitro suggesting a possible pathomechanism. here we demonstrate that -oxoadipate and quinolinic acid are toxic for spinal motor neurons and their increased levels may contribute to neuropathy. and families have been reported. interestingly, most mutations target the same amino acid residue (k e, k m, k n, k t) in the highly conserved -crystallin domain of the hspb protein. the spectrum of diseases caused by mutations in the hspb gene was recently expanded to distal myopathy. hspb is ubiquitously expressed, but is highly expressed in motor neurons and muscles. the hspb is a chaperone that participates in clearing misfolded poly-q containing proteins such as mutant huntingtin and ataxin- involved in respectively huntington's disease and spino-cerebellar ataxia. hspb directly interacts with the co-chaperone bag and their role in chaperone-assisted selective autophagy is well described. to delineate the molecular deficits and functional consequences of hspb mutations we generated a knock-in (ki) mouse model for the k n missense mutation mimicking the neuropathy phenotype. we observed that homozygous mutant mice (hspb k n/k n ) develop a progressive axonopathy, with decreased compound motor action potential amplitudes, and loss of large and medium myelinated axons. this results in locomotor deficits with an impaired performance at the rotarod and grip strength tests. at the ultrastructural level, the hspb -ki model displays severe signs of axon degeneration and a clear myofibrillar myopathy, as observed in some patients with hspb mutations. interestingly, hspb positive aggregates were found in the sciatic nerve and gastrocnemius muscle of our mutant mice. additionally, our model allowed us to generate hspb knock-out (ko) mice using the same targeting vector. strikingly, the homozygous hspb -ko animals do not show any sign of axonopathy and display a much milder myopathy than the hspb -ki animals. these data suggest that part of the pathomechanisms is due to toxic gain-of-function of the mutant protein. boutahar n , reynaud e , nasser y , camdessanché jp , antoine jc . university hospital, saint-etienne, france. dysimmune sensory neuronopathies (snn) depend on neuron cell death induced by an inflammatory reaction in dorsal root ganglia. we have recently identified the intracellular tyrosine kinase (trk) domain of the fibroblast growth factor receptor- (fgfr ) as the target of antibodies in a subset of patients with non paraneoplastic snn. fgfr is one of the four fgfrs and has been involved in sensory neurons maintenance during development and cell death induction after axotomy. fgfrs ligand fixation results in the activation of several intracellular pathways through adaptator protein interactions with the trk domain. in particular ras activation may lead to cell proliferation or apoptosis through erk / or p map kinase signaling. the p mapk pathway is also involved in neuronal cell death induced by nmda and ampa receptor activation. as fgfr is a cell surface protein, human antibodies may interfere with the receptor functioning as a growing number of evidence has showed with other cell surface antibodies in neurological diseases. to test this hypothesis we developed an in vitro model using fvbn mice cortical neurons culture exposed to a rabbit polyclonal antibody reacting with the trk domain of fgfr . comparatively to normal rabbit iggs, the fgfr antibody induced neuron cell death in a dose dependent manner. neuron cells were exposed to fgfr antibody concentration leading to - % cell death in absence or presence of the p mapk inhibitor sb and the expression of fgfr , glur subunit of ampa receptors and nr subunit of nmda receptor was measured by quantitative rt-qpcr. the fgfr antibody induced an upregulation of fgfr while the glur and nr subunits were modulated. these changes were prevented in presence of sb . these preliminary results indicate that anti-fgfr iggs may interfere with the functioning of the intracellular domain of the protein and the expression of nmda and ampa receptors through the p map kinase pathway. this model may be used to test the effect of human anti-fgfr iggs in vitro. braathen gj , tveten k , holla Øl , busk Øl , hilmarsen ht , svendsen m , høyer h . section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. next-generation sequencing (ngs) has during the last years entered the clinical diagnostics. ngs has proven to be very efficient in the diagnostics of disorders where multiple genes can be involved. our ngs-based targeted gene panel consists of hereditary neuropathy genes, i.e. mostly charcot-marie-tooth genes. this study is a retrospective study of clinic samples received between may and february . we describe the identified novel likely pathogenic sequence variants, according to international guidelines. in this period we identified novel, not previously described, likely pathogenic sequence variants in the following genes: aars, fgd , gan, hint , litaf, lrsam , mme, mpz, nefl, pmp , sbf , sh tc and yars. there is now a large range of genes causing hereditary peripheral neuropathies and many likely pathogenic sequence variants. likely pathogenic sequence variants are not only identified in old well established neuropathy genes but also in the newer genes like mme. modelling brown-vialetto-van laere syndrome in c. elegans mutations in slc a and slc a , which encode the riboflavin transporters rfvt and rfvt . patients with rfvt deficiency exhibit proximal and distal limb weakness, sensory ataxia, diaphragmatic paralysis, optic atrophy, sensorineural deafness and bulbar palsy. riboflavin is critical for the biosynthesis of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for carbohydrate, amino-acid and lipid metabolism. mutations in slc a reduce or abolish rfvt expression resulting in impaired riboflavin uptake into sensory and motor neurons. high-dose riboflavin treatment can improve or stabilise a patient's condition, however the optimum dose and long term effects of riboflavin treatment, and disease pathomechanisms remains poorly understood. to further understand the pathophysiological consequences of slc a mutations, we propose developing an animal model for bvvl. caenorhabditis elegans (c. elegans) are small round transparent nematodes extensively used for studying the genetics and molecular biology of neurodegenerative diseases. there are two c. elegans riboflavin transporter genes, rft- and rft- . based on protein sequence homologies and expression profiles for both genes, rft- is the ortholog of rfvt . the expression of rft- is regulated by riboflavin availability and knock-down of the rft- gene by sirna perturbs c. elegans development. our aim is to develop a knock-in c. elegans model of bvvl. human rfvt and c. elegans rft- protein sequences were aligned with clustal omega to identify conserved amino acid residues associated with bvvl mutations. the amino acid involving the l p rfvt mutation is conserved in rft- (residue l ). to create our model, we will introduce the l p rft- mutation into the rft- locus in c. elegans genomic dna using crispr/cas- technology. this bvvl c. elegans model will allow us to explore the pathogenic consequences of rfvt deficiency underlying motor nerve degeneration and to evaluate drug therapy regimes by determining the optimal riboflavin dose and treatment initiation, and trialing other compounds that may improve benefits seen with riboflavin supplementation. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune-mediated inflammatory disease of the peripheral nervous system. cidp can present chronic progressive or relapsing-remitting courses and can predominantly affect motor but also sensory nerve fibers causing weakness of proximal and distal muscles. it represents the most common chronic autoimmune neuropathy and is pathologically characterized by focal inflammatory-mediated demyelination followed by axonal degeneration. recently, we have developed a new animal model for cidp, the chronic-ean, induced in lewis rats by active immunization with s-palmitoylated p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) peptide. this model fulfills electrophysiological criteria of demyelination with axonal degeneration, a result confirmed by immunohistopathology. the late phase of the chronic disease is characterized by an accumulation of il- + cells and macrophages in sciatic nerves and as well as high serum il- levels. it is a reliable and reproducible animal model for cidp that can now be used for translational drug studies for chronic human autoimmune-mediated inflammatory diseases of the peripheral nervous system, particularly cidp, for which, there is a real need for new immunotherapies. the aim of this study was to test the therapeutic efficacy of ivig and a recombinant fc fragment (fcrec) in this new cidp animal model. treatments with ivig and fcrec proved effective in preventing further progression of cidp in rats. the therapeutic treatments not only decreased the maximal clinical scores of the cidp rats compared to albumin treatment but also abolished the disease chronicity. interestingly, a better efficacy of fcrec treatment compared to ivig was demonstrated at histological level, with the myelinated fibers well preserved and the greatly reduced accumulation of macrophages and il- + cells in sciatic nerves. ivig and fcrec therapeutic activities in this model can also be followed by measurement of antibodies in the serum and could therefore be used as biological markers. the current study provides for the first time direct evidence that ivig is effective in the treatment of cidp rats and suggests that a novel fcrec compound is more effective than ivig. it will contribute to the development of more effective and safer drugs for the treatment of autoimmune peripheral neuropathies like cidp. each time, one measurement with supramaximal stimulation according to the conventional ncs and another measurement with % enhanced stimulation intensity were conducted. we analyzed the results with special consideration of minimal and maximal fiber velocities and of the velocity with maximal fiber proportion. as a result, we detected statistically significant differences between cidp patients and controls in all these parameters. compared with controls, cidp patients had slower minimal and maximal fiber velocities and also the spectrum of motor conduction velocities was definitively shifted to slower velocities. slower minimal conduction velocities could be detected in some cidp patients by using the enhanced stimulation intensity. interestingly, in some cidp patients the conventional ulnar ncs were normal, but the collision technique showed fibers with a conduction velocity of less than m/s, indicating demyelination. to our knowledge, this is the first study using hopf's collision technique systematically in cidp patients. significant differences between the variation of ulnar motor nerve conduction velocity of cidp patients and controls could be detected. this method also showed signs of demyelination in some cidp patients with normal ulnar ncv. by enhancing stimulation intensity above the threshold of supramaximal stimulation in conventional ncs, the collision technique may be even more valuable. the clinical course of guillain-barré syndrome (gbs) is highly variable and some patients may develop treatment-related fluctuations (trfs) as an indication of ongoing disease activity and temporary treatment effect. other patients present as gbs, but subsequently develop repetitive relapses as an indication of acute-onset chronic inflammatory demyelinating polyneuropathy (a-cidp). this distinction is important because treatment may differ. we determined the frequency and clinical presentation of patients with gbs-trf and a-cidp in the first patients included in the international gbs outcome study (igos) with a follow-up of at least months. thirty-eight patients ( %) were excluded because of alternative diagnoses. of the remaining patients, ( %) had at least one trf, ( %) had a-cidp. preliminary analysis showed no significant differences between the groups (total-gbs, gbs-trf and a-cidp) for sex, age, sensory symptoms, cerebrospinal fluid results and mechanical ventilation. a-cidp patients had a median age of years (iqr - ), % was male, and all patients were treated in the acute phase of the disease (intravenous immunoglobulins (ivig) ( ), plasma-exchange (pe) ( ) or methylprednisolone (mp) ( )). gbs-trf patients has a median age of years (iqr - ), % was male and all patients received treatment (ivig ( ) and pe ( ). gbs-trf patients showed more antecedent events ( % versus %, p= . ), a higher gbs disability score (≥ ) at nadir ( % versus %, p= . ) and less frequently developed ataxia ( % versus %, p= . ) than patients with a-cidp. onset to nadir was longer in a-cidp than in gbs-trf ( days (iqr - ) versus days (iqr - ), p= . ) and the total gbs group ( days (iqr - ), p= . ). the time until the first clinical deterioration tends to be longer in the a-cidp patients (median days (iqr - ) versus median days in the gbs-trf group, not significant). the diagnosis a-cidp was made after a median of days . in conclusion, this first analysis identified distinctive characteristics of gbs-trf and a-cidp in support of a different pathogenesis that may help with early identification of these disorders in clinical practice. additional results will be presented at the conference. chronic inflammatory demyelinating polyneuropathy (cidp) is a disorder with a highly diverse clinical presentation, electrophysiological phenotype, response to treatment and outcome. this heterogeneity may indicate the presence of distinct subtypes of cidp, which may have a different pathogenesis and require more personalized treatment. the international cidp outcome study (icos) is a prospective, observational, international multi-center study that aims to describe this variation in clinical and electrophysiological subtypes and to define the clinical and biological determinants of these subtypes, disease activity, treatment response and outcome. in addition icos aims to provide an infrastructure for conducting new (therapeutic) studies in cidp, similar to the international gbs outcome study (igos). all patients fulfilling the efns/pns ( ) diagnostic criteria for cidp can be included in icos, independent of age, duration and severity of disease or treatment. we collect information on neurological deficits, diagnostic characteristics, various validated clinical outcome measures, previous and current treatment and we collect biomaterials (dna, cerebrospinal fluid, nerve biopsies and repeated serum samples). icos was started as a pilot study in dutch university centers. by february , patients were included in icos, patients recently diagnosed with cidp and previously diagnosed patients. included were ( %) males and ( %) females with a median age of years (iqr - ). the current cohort consists of classic (sensory-motor) cidp, madsam and pure motor variants. of the patients diagnosed in the past, patients ( %) were treated (intravenous immunoglobulins (ivig) ( ), prednisolone ( ), subcutaneous immunoglobulins (scig) ( ), dexamethasone ( ), plasma-exchange ( ) and ivig with methylprednisolone (mp) ( )). the recently diagnosed patients all received treatment (ivig ( ), dexamethasone ( ), plasma-exchange ( ) and patients were treated in a pilot study with ivig with methylprednisolone (mp)). the protocol has been evaluated and adjusted and will be shared with other researchers via the inflammatory neuropathy consortium and igos consortium. our aim is to include at least cidp patients worldwide with a minimum follow-up period of years. dominant mutations in glycyl trna synthetase (gars) cause inherited axonal neuropathy (charcot-marie-tooth type d). mutations in the mouse gars gene cause a similar phenotype, and represent valid disease models. we routinely use two mouse strains, one with a severe neuropathy, and one with a milder, later onset neuropathy. using these mouse models, we are exploring the mechanisms through which gars mutations cause peripheral axon degeneration. efforts include ribosome tagging to isolate ribosome-associated mrnas specifically from motor neurons, and non-canonical amino acid tagging to visualize and isolate newly synthesized proteins. taking advantage of the anatomy of motor neurons, we are able to analyze cell bodies separately from peripheral axons in both of these approaches. in addition, we are using these mouse models for preclinical studies testing gene therapy approaches to treat cmt d. consistent with our previous genetic studies in mice, knockdown of the mutant transcript, while preserving sufficient levels of the wild type, is a very successful approach when administered before the onset of neuropathy. we are now testing this approach in mice after the onset of symptoms, and in mice carrying a mutant gars allele associated with human disease. these studies are potentially translational, and also address mechanistic questions such as the timing and cell autonomy of the pathophysiology. burnor e , yang l , hao z , patterson k , quinn c , scherer ss , lancaster e . the university of pennsylvania, philadelphia, pa, usa; the second xiangya hospital, central south university, hunan, china. autoantibodies to two isoforms of neurofascin (nf and nf ) have been reported in patients with guillain-barre syndrome (gbs) or chronic inflammatory demyelinating neuropathy (cidp). it is not clear which of these responses reliably distinguish autoimmune neuropathies from other severe neuropathies, which responses are transient versus persistent, or which responses may target multiple isoforms of neurofascin. in addition, approximately % of neuropathy patients have no known cause, and it is unknown whether a subset of these patients may have autoantibodies to neurofascins. we have studied cohorts of patients with autoimmune neuropathy (n= ), genetic neuropathy (n= ), and idiopathic neuropathy (n= ) for igg and igm responses to neurofascins. neurofascin antibodies were found in ( %) of patients with autoimmune neuropathy, and ( . %) idiopathic neuropathy patients, but only % ( of ) in patients with genetic neuropathy. follow-up serum samples were available for positive cases. persistent responses were associated with chronic neuropathy while transient responses were seen in gbs or with remission of cidp. most patients had responses specific to either nf of nf . however, a particularly severe, treatment-resistant form of cidp, approaching a locked-in state, was seen in a patient with a unique response to all three isoforms of neurofascin (nf , nf , nf ). treatment of this patient with rituximab resulted in clinical improvement and resolution of the neurofascin antibody response. in conclusion, autoantibodies to neurofascins distinguish autoimmune neuropathies from severe genetic neuropathies, but the clinical phenotype may depend on the persistence and isoform specificity of the immune response. antibodies to the common domains shared by nf and nf may portend a severe but treatable neuropathy. a subset of idiopathic neuropathy patients may have an autoimmune mechanism. burns j , , sman ad , cornett kmd , wojciechowski e , , walker t , menezes mp , , mandarakas mr , rose kj , , bray p , , sampaio h , farrar m , , refshauge km , raymond j and the fast study group. university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia; university of new south wales, sydney, australia. exercise has undisputed benefits for human health and potentially as a treatment for neuromuscular disease. but there is also a risk of harm due to overwork weakness. we report the results of a -month randomised, double-blind, sham-controlled trial evaluating progressive resistance exercise of foot dorsiflexor muscles in pediatric cmt. sixty patients ( cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx ) aged - years were randomly assigned to undergo -weeks ( sessions) of moderate-intensity progressive resistance exercise or sham exercise. the primary endpoint was change in isometric dorsiflexion strength between groups assessed by hand-held dynamometry (expressed as a z-score based on age-and sex-matched normative reference values, positive values indicate an improvement in strength). the primary safety endpoint was change in muscle and fat volume of the muscles responsible for dorsiflexion by mri between groups. secondary outcomes were function (balance, long jump, -min walk test), walking ability ( d gait analysis), self-reported ankle instability, parent-reported quality of life (physical summary, psychosocial summary and global impression of change scores), and adverse events. fifty-five ( %) children completed the trial. adherence was comparable between exercise ( %) and sham ( %) groups. while patients experienced muscle soreness during training in the exercise group, adverse events did not differ between groups. the mean z-score for dorsiflexion strength increased in the exercise group by % at -months (from − . ± . to − . ± . ) and decreased in the sham group by %, mirroring the natural history of cmt (from − . ± . to − . ± . ). between-group ancova-adjusted difference at -months was . ( %ci, . to . ; p= . ). the mean scaled scores for mri muscle volume and fat volume were comparable between groups at months (p> . ). the global impression of change scores favoured the exercise group at -months (p= . ) and -months (p= . ). there was no other measurable effect of exercise. pre-specified subgroup analyses according to age ( - years vs. - years) showed a larger treatment effect with exercise in adolescents. targeted progressive resistance exercise was effective at halting progression of dorsiflexion weakness without detrimental effect on muscle morphology or other signs of overwork weakness in children with cmt. byung-nam y , yoon-ho h , suk-won a , seok-jin c , jung-joon s . inha university hospital, incheon, south korea; seoul metropolitan government boramae medical center, seoul national university, seoul, south korea; joong ang university hospital, seoul, south korea; seoul national university hospital, seoul, south korea. a years old female patient visited the hospital with weakness in both upper limbs from months under the suspicion of motor neuron disease with the finding of extensive denervation changes on electromyography. whole spine mri showed ventral nerve rootlet enhancement in the c and t bundles. the emg had confirmed active denervation changes in the muscles innervated by bilateral c ∼c roots. the cerebrospinal fluid culture test showed a protein (csf) elevation to mg / dl. she got a steroid pulse therapy. she had the symptoms of dry mouth and dry eyes during the recent years. a salivary gland scan test was performed for the possibility of sjögren's syndrome, and as a result, absorption of the contrast agent in both parotid and submandibular glands was decreased. the shammer test showed mm on the right side and mm on the left side. she was diagnosed of sjögren's syndrome. after weeks, overall strength improvement was observed, and bilateral shoulder abduction was improved to mrc grade or higher. the follow-up spine mri also showed that the initially seen ventral nerve root enhancement was disappeared. the case had visited the hospital with major symptoms of weakness and atrophy of the muscles, showing similar pattern to motor neuron disease, and was diagnosed as inflammatory polyradiculopathy and confirmed as primary ss during differential diagnosis. this case suggests that primary ss may induce inflammatory polyradiculopathy, which shows motor symptoms as major symptoms rather than sensory symptoms, and that a fast and accurate diagnosis is needed in terms that it can be treated with steroids and appropriate immune suppressive agents. traumatic injuries to peripheral nerves are frequent. however, effective pharmacological treatments are lacking. curcumin, a polyphenol found in rhizomes of curcuma longa, has been shown to develop antioxidant, anti-inflammatory, and neuroprotective properties. however, due to its poor hydrosolubility and its extensive metabolism, the use of large curcumin doses is required for therapeutic purpose. the aim of the present study was to investigate the effects of a local infusion of a low curcumin dose on nerve regeneration and functional recovery after sciatic nerve injury in rats. the experiments were conducted in g sd male rats submitted to unilateral sciatic nerve crush at d . curcumin was solubilized in polyethylene glycol and continuously administered using osmotic pumps ( . mg/day until d ) with a catheter delivering the drug near the lesion site. functional analyses using von frey, beam walking, static sciatic index (ssi) and grip strength tests were carried out at d (reference test) and every week after injury (d , d , d , d and d ). in addition, an evoked electromyogram was performed at d and d . after euthanasia (d ), nerve and muscle samples were collected and analyzed by light and electron microscopy. functionally, a significant improvement of the mechanical sensitivity (+ %) was observed at d in the curcumin-treated group (n= ) vs. vehicle group (n= ). in curcumin-treated group, skillful walking and finger spacing of the ipsilateral paw (ssi) were fully restored respectively at d and d contrary to vehicle group. furthermore, curcumin treatment improved the grip strength recovery (+ % at d ). the electrophysiological results indicated a full recovery of motor nerve conduction velocities (mncv) after days of curcumin treatment, while mncv remained altered in vehicle group ( % of the mncv at d ). morphometric analysis of nerve sections using g-ratio showed an improvement in the thickness of the myelin sheath in curcumin treated animals (+ % vs. vehicle group). histological investigation of gastrocnemius muscle indicated decreased neurogenic lesions in curcumin group. proteomic analysis is currently under investigation to understand the mechanisms involved in curcumin effects. our data could lead to the development of new therapeutic strategies in peripheral nerve regeneration using low doses of curcumin. previous studies suggest that the metabolic syndrome (mets) is associated with distal symmetrical polyneuropathy (dsp), and that diabetes, pre-diabetes, and obesity are the main metabolic drivers. the aim of this study is to investigate the association of mets components with retinal and cognitive function in a bariatric surgery cohort prior to surgery. patients were recruited from the adult bariatric surgery clinic at the university of michigan and lean controls from a research website (no mets components based on ncep/atpiii definition). participants underwent extensive metabolic phenotyping including a glucose tolerance test and fasting lipid profile. dsp was defined using the toronto consensus definition of probable clinical neuropathy. retinal function was measured with frequency doubling technology perimetry (average mean deviation), and cognitive function with the nih toolbox (composite score). univariate linear regression models were used to evaluate the association between mets components and retinal and cognitive function. to date, we have recruited bariatric surgery participants and lean controls. in the bariatric population, the mean (sd) age was ( . ) with % female compared with a mean age of . ( . ) with % female in the lean group. in the bariatric group, . % had diabetes, . % pre-diabetes, and . % normoglycemia. the dsp prevalence was % in lean controls, . % in normoglycemic, . % in pre-diabetic, and . % in diabetic bariatric participants (p< . for trend). retinal function was . ( . ), − . ( . ), − . ( . ), and − . ( . ) (p= . for trend), and cognitive function was . ( . ), . ( . ), . ( . ), . ( . ) (p= . for trend) in these same groups for lean controls, normoglycemics, pre-diabetics and diabetics, respectfully. pre-diabetes (− . , %ci: − . ,- . ) was the only mets component associated with retinal function, and waist circumference was the only one associated with cognitive function (− . , %ci − . ,- . ). dsp and retinal function, but not cognitive function decline with worsening glycemic status. similar to previous data for dsp, pre-diabetes and obesity are associated with retinal and cognitive function respectively. interestingly, while clinical dsp is common in this population, clinical retinopathy and dementia are not, indicating that dsp may be the first metabolic complication in the morbidly obese. and anti-cntn antibody-positive cases were confirmed by cba and were of igg subclass in half of them. by cba we identified additional / ( %) anti-caspr seropositive patients, whose isotype is currently being tested. sera of anti-nfasc and anti-cntn igg seropositive patients and patients with anti-caspr antibodies stained paranodes by indirect immunofluorescence on mouse teased nerve fibers. of note, seronegative patients for known antibodies showed reactivity against node and/or paranodes. compared to other seronegative cipd patients, seropositive patients had more frequently subacute onset of the neuropathy and a younger age at onset, particularly for nfasc or caspr antibodies. weakness was more severe and was often associated with proprioceptive loss, sensory ataxia and tremor. neuropathic pain was not a feature of caspr -seropositive patients. frequent findings were increased distal motor latencies and temporal dispersions on nerve conduction study and a higher protein level in csf. finally seropositive patients tended to have a higher disability and showed worst response to ivig. rituximab was effective in one patient with anti-nfasc antibodies and two patients with anti-cntn antibodies showed good and persistent recovery after cyclophosphamide. prevalence of antibodies was % in italian cidp patients and their presence was associated with distinctive clinical features. their determination, followed by characterization of igg subclass in positive cases, has clear clinical impact, by helping to guide therapeutic choices. the reactivity against nodal and paranodal components in sera from patients without known antibodies suggests that other targets could play a role in the autoimmune response in cidp and they still need to be identified. mutations in amynoacyl trna synthetases (arss), enzymes that catalyse the covalent attachment of amino acids to their cognate trna, are responsible for autosomal dominant cmt , intermediate cmt (cmt-i) and dhmn. we report the case of a male of italian ancestry who first presented with bilateral ankle clonus at three months, followed by toe walking and ankle instability. the ankle clonus subsided during adolescence. in the third decade he developed progressive walking difficulties followed by distal sensory loss. neurological examination at the age of revealed a steppage gait, distal lower limb weakness, decreased pinprick to the ankles, and reduced vibration sensation at the knees. reflexes were brisk in the upper limbs, reduced at the knees and absent at the ankles. muscle tone was increased in the lower limbs and plantar responses were extensor. nerve conduction studies revealed an axonal neuropathy. brain and spinal cord mri were normal. sanger sequencing of pmp , gjb , mpz , gdap and mfn were negative. sureselect focused exome sequencing (agilent technologies, santa clara ca, usa) demonstrated a c. g>a, p.arg his mutation in aars. the p.arg his mutation in aars has previously been reported in families with intermediate or axonal motor-sensory neuropathy, and in one case was associated with sensory-neuronal deafness. cns involvement has not previously been described with this mutation. mutations in aars have been associated with a range of phenotypes including cmti, cmt and dhmn with variable age on onset ranging from to years (mean years). of note, the aars p.gly arg mutation has been reported in a family with cmt and pyramidal signs. this study provides further evidence that pyramidal tract involvement can be an early feature of cmt n due to mutations in aars, further expanding the spectrum of arss-associated phenotypes. small fibres in the skin are vulnerable to damage in metabolic or toxic conditions such as diabetes mellitus or chemotherapy resulting in small fibre neuropathy and associated neuropathic pain (np). whether injury to the most distal portion of sensory small fibres due to a primary dermatological disorder can cause np is still unclear. recessive dystrophic epidermolysis bullosa, (rdeb) is a rare condition in which mutations of proteins of the dermo-epidermal junction lead to cycles of blistering followed by regeneration of the skin. damage is exclusive to the skin and mucous membranes, with no known direct compromise of the nervous system. it is increasingly recognised that most rdeb patients experience daily pain, the aetiology of which is unclear but may include inflammation (in the wounds), musculoskeletal (due to atrophy and retraction scars limiting movement) or np. in this study we investigated the incidence of np and examined the presence of nerve dysfunction in rdeb patients. around three quarters of patients presented with pain of neuropathic characteristics which had a length dependent distribution. quantitative sensory testing of the foot revealed striking impairments in thermal detection thresholds combined with an increased mechanical pain sensitivity and wind up ratio (temporal summation of noxious mechanical stimuli). nerve conduction studies showed normal large fibre sensory and motor nerve conduction however skin biopsy showed a significant decrease in intraepidermal nerve fibre density. autonomic nervous system testing revealed no abnormalities in heart rate and blood pressure variability however the sympathetic skin response of the foot was impaired and sweat gland innervation was reduced. we conclude that chronic cutaneous injury can lead to injury and dysfunction of the most distal part of small sensory fibres in a length dependant distribution resulting in disabling np. these neuropathic pain (np) following peripheral nerve injury is associated with hyperexcitability in damaged myelinated sensory axons which begins to normalise over time. we investigated the composition and distribution of shaker type potassium channels (kv channels) within the nodal complex of myelinated axons following injury. at the neuroma that forms after damage, expression of kv . and . (normally localised to the juxtaparanode) was markedly decreased. in contrast kv . and . , which were hardly detectable in the naïve state, showed increased expression within juxtaparanodes and paranodes following injury, both in the rat and in humans. within the dorsal root (a site remote from injury) we also noted a redistribution of kv channels towards the paranode. blockade of kv channels with dtx after injury reinstated hyperexcitability of a-fibre axons and enhanced mechanosensitivity. changes in the molecular composition and distribution of axonal kv channels, therefore represents a protective mechanism to suppress the hyperexcitability of myelinated sensory axons that follows nerve injury. members of the francophone anti-mag cohort group are listed in "appendix". appendix polyneuropathy with anti-mag igm antibodies is classically progressive, predominantly sensory, and distal with ataxia and sometimes postural-intention tremor. we assessed clinical, biological, electrophysiological, and histopathological features in patients with igm gammopathy and anti-mag antibody titres higher than btu. we focused on characteristics of patients according to the anti-mag antibody titres at diagnosis. we retrospectively and prospectively analysed standardized report forms of patients from fourteen french-speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset and last follow-up was . years ( . - . ). about . % of patients presented with a "variant" clinical phenotype independently of anti-mag titres (< or ≥ btu). this included acute or chronic sensorimotor polyradiculoneuropathies, paucisymptomatic sensory polyneuropathy and multifocal neuropathy. at the most severe disease stage, . % of patients were significantly disabled. anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in respectively , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. sixteen percent of patients did not meet the electrodiagnostic criteria of definite demyelinating neuropathy, independently of anti-mag titres (< or ≥ btu). nerve biopsy, performed in nineteen patients, provided support to the diagnosis of anti-mag neuropathy in some particular issues (low titres of anti-mag, unusual clinical or electrophysiological phenotype). we assessed the degree of probability (probable, possible or uncertain) that patient neuropathies are directly related to anti-mag antibodies, according to anti-mag titre, electrophysiological data and nerve biopsy characteristics if available. it appears uncertain in patients with low anti-mag titres ( . % of the whole population). the clinical phenotype didn't appear to be different according to anti-mag antibody titre. many of the patients with low anti-mag titres presented "genuine" anti-mag neuropathy as demonstrated by edx studies, clinical presentation and sometimes nerve biopsies. for a small proportion of these patients, a direct relation between neuropathy and anti-mag antibodies is uncertain due to atypical clinical presentation, axonal neuropathy pattern or nerve biopsies, and positivity of antigangliosides antibodies. we assessed therapeutic management, response to immunotherapies and adverse events in a cohort of patients presenting with igm gammopathy and anti-mag antibody titres higher than btu. we retrospectively and prospectively analysed standardized report forms of patients from french speaking neuromuscular centres. mean age at onset was . years (range - . ). mean time between symptoms onset to diagnosis and last follow-up were respectively . ( - ) and . years ( . - . ). anti-mag antibody titres at diagnosis were low (< btu), medium ( - ) or high (≥ ) in , and % of patients. patients presented with mgus or waldenström's macroglobulinemia in respectively % and % of cases. seventy eight percent (n = ) of patients received immunotherapies. transient response to ivig or plasma exchanges at six month was observed in less than and % of patients respectively. chemoimmunotherapies and rituximab were more frequently administered in the group of patients with malignant hemopathies (n = ) compared to mgus (n = ) (mean lines of therapy = . , range - , sd . versus . , range - , sd . , p = . ). more than % of patients (n = ) received rituximab monotherapy. clinical worsening, mostly transient and reversible, was observed in / patients after rituximab. clinical response to rituximab at months and/or during - months follow-up period was observed in . % of patients, and correlated with anti-mag titre ≥ btu ( / responder versus / non-responder patients, p = . ). at - months follow-up, responder patients presented shorter symptom duration compared to non-responders, though not significant after logistic regression ( . years, range . - . , sd . in responder patients versus . years, range . - . , sd . , in non responder patients, p = . ). in some cases, electrodiagnostic studies were recorded during rituximab treatment follow-up and showed in responder patients a clear improvement of motor conduction velocities. these data may support another clinical trial to study benefice of rituximab in anti-mag neuropathies early in the disease. it raises issues about the value of incorporating electrodiagnostic parameters as end-points. schwann cells (scs) are essential for axon integrity and myelination in the uninjured pns. after pns injury, scs function as "first responders" , undergoing phenotypic re-programming and orchestrating many processes that lead to functional nerve repair. receptors in scs that contribute to sc repair programs remain incompletely understood. we identified a member of the ionotropic glutamate receptor (igr) family, n-methyl-d-aspartate receptor (nmda-r), in scs that is upregulated after nerve injury and acts as a co-receptor with ldl-receptor related protein (lrp ). lrp is a well-known regulator of the sc response to pns injury. herein, we used pcr to profile igr expression in cultured rat scs. obligate receptor subunits required for assembly of nmda-rs, ampa-rs, and kainate receptors were identified. treatment of rat scs with - microm glutamate or . - . microm nmda robustly activated akt and erk / . the response was transient and bimodal; glutamate concentrations greater than microm failed to activate cell-signaling. phosphoprotein profiling demonstrated other cell-signaling and transcription factors regulated by glutamate in rat and human scs, including p s -kinase, glycogen synthase kinase- , ribosomal s kinase, c-jun, and creb. activation of cell-signaling by glutamate in scs was blocked by eliminating calcium from the medium, by the selective nmda-r antagonist, mk , and by genetic silencing expression of the obligate nmda-r nr subunit. phosphoinositide -kinase/pi k functioned as an essential upstream activator of both akt and erk / in glutamate-treated scs. by activating pi k and erk / , glutamate promoted sc migration. glutamate ( microm) or nmda ( microm) injected into crush-injured sciatic nerves robustly activated p-erk / in both myelinating and non-myelinating scs in vivo. these results identify igrs as potentially important cell-signaling receptors in scs that may promote axon-glial interactions. understanding the function of sc nmda-r is important given current efforts to develop nmda-r-targeting drugs for patients with pain, depression, and alzheimer's disease. while frequently overlooked in a therapeutic context, scs are extremely important in the pathogenesis of chronic neuropathic pain. if these drugs modulate the activity of sc nmda-r and sc physiology, the response to pns injury may be altered and the possibility that neuropathic pain develops increased. candayan a , atkinson d , durmus tekce h , parman y , jordanova a , battaloglu e . department of molecular biology and genetics, bogazici university, istanbul, turkey; center for molecular neurology, antwerp university, antwerp, belgium; istanbul medical school, istanbul university, istanbul, turkey. charcot-marie-tooth (cmt) disease is a group of inherited peripheral neuropathies affecting one in individuals worldwide. the disease presents both clinical and genetic heterogeneity. so far, mutations in genes and loci are associated with cmt with autosomal dominant, autosomal recessive, x-linked and mitochondrial inheritance. despite the advances in genetic testing, approximately % of all cmt patients worldwide remain without a molecular diagnosis. we have investigated unrelated cmt patients of turkish descent, in all of which pmp duplication has been excluded previously. we used multiplex amplification of specific targets for resequencing (mastr) assay to sequence exonic regions of common cmt genes. recurrent mutations were identified in cases in mfn , gjb , mpz and hint genes. we have also identified novel variants in cases in mfn , pmp , gars, aars, ighmbp and gdap genes, all of which are very rare or not present in the variation databases and are predicted to be pathogenic by in silico tools. familial segregation analyses are ongoing for novel variations. mfn and gjb genes were the most commonly mutated causative genes in this cohort. cases without molecular diagnosis after the mastr testing are candidates for further analyses such as whole exome sequencing or whole genome sequencing. outcomes of the current study and our previous experience with turkish cmt patients suggest a high genetic heterogeneity. our insight is that different genetic strategies or larger panels are essential to determine the causes underlying cmt especially in regions where rare recessive types of the disease can be observed due to high frequency of consanguineous marriages. capoccitti g , giannini f , ginanneschi f , casali s , insana l , rossi a . university of siena, siena, italy. several efforts have been made to elaborate new electrophysiological criteria for early diagnosis of guillain-barré syndrome (gbs) and to differentiate demyelinating (aidp) from axonal (aman/amsan) forms. the aims were to verify the diagnostic power of total cmap (tcmap) duration, firstly applied in gbs field. this parameter was compared with commonly used neurophysiological measures, including negative phase cmap duration (ncmap), and was added to modified rajabally criteria. we reviewed the clinical and electrophysiological data of patients with gbs (level or of brighton clinical criteria). each patient underwent at least two neurophysiological studies, the first within weeks, the second between and weeks from symptom onset. at least four motor and three sensory nerve conduction studies were recorded for each test. regarding early diagnosis, the binary logistic regression model with multiple variables, including ncmap duration, showed that the features of predictive model presenting greater significance (p < . ) were tcmap duration, sural sparing and a-waves. among these, tcmap duration showed greater significance (p = . ). the tcmap was diffusely prolonged in aidp compared to aman/amsan, already in first examination and confirmed in the second one. roc analysis for tcmap duration in aidp vs. aman/amsan showed: cut-off . ms, auc . , ppv . %. we propose the tcmap duration as a new useful electrophysiological measure for early diagnosis of "generic" gbs and for early differentiation between aidp and aman/amsan. moreover, the prolongation of tcmap, the presence of a-waves and sural sparing represent a strongly diagnostic predictive triad of aidp. aifm (apoptosis-inducing factor, mitochondrionassociated- ) has captured great attention from biomedical researchers due its critical role in the regulation of cell apoptosis. this flavoprotein is typically located in the mitochondrial intermembrane space where it is associated with respiratory chain complex-i. upon a cell-death insult, aifm is cleaved into a kd protein that is released into the cytosol. the kd peptide may enter the nucleus to trigger chromosome condensation and fragmentation, initiating a caspase-independent pathway of apoptosis. however, this nuclear translocation may be blocked by cytosolic heat-shock protein- (hsp ) that binds with the fad domain (aa - ) of aifm . mutations in aifm gene have resulted in several clinical phenotypes, including a family with cmtx (glu val). clinical deficits in these patients usually involve multiple organs. in this study however, we identified a family with a novel missense mutation (phe leu) in aifm that developed a late-onset sensory motor axonal polyneuropathy by nerve conduction criteria. the proband and affected siblings exhibited distal muscle weakness and atrophy with normal cognitive and cranial nerve functions. there was no obvious phenotype from other organs. interestingly, this phe leu mutation affects a highly conserved amino acid at the center of the fad domain. we hypothesize that this mutation impairs the binding between aifm and hsp , leading to an enhancement of cell-death signaling. this family therefore provides a unique opportunity to explore how altered apoptotic signaling affects peripheral nerve system. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr chronic inflammatory demyelinating polyneuropathy (cidp) can be the key symptom leading to diagnosis of associated lymphoma. patients with diagnosis of cidp according to efns/pns criteria associated with b cell lymphoproliferative disorders (bld), in one center, between november and november were included. demographical, clinical, nerve conduction, immunological, histological data and response to treatment were recorded retrospectively. eight patients ( men), median age yo were included. onset of polyradiculoneuropathy was either chronic (n= ) or acute (n= ). neurological condition led to diagnosis of bld in all but one case, because of onset (n= ) or worsening of neuropathy (n= ). clinical presentation was that of cidp in patients or pseudo-canomad in and plexopathy in one. lymphoma type was: lymphoplasmacytic (n= ), diffuse large b cell (n= ), small lymphocytic (n= ), marginal zone (n= ), unclassified small b cell (n= ). only patients presented with lymphadenopathies. bld was diagnosed in all patients on myelogram or bone marrow biopsy, performed because of cytopenia (n= ), atypical (n= ) or severe (n= ) neuropathy. monoclonal gammapathy was identified in / patients (igm n= , igg n= ). neuromuscular biopsy was performed in patients and disclosed endoneural infiltration in . anti-neuronal or antigangliosides antibodies were positive in patients. none of the patients presented a systemic autoimmune disease, hemolytic anemia associated with bld (n= ). immunomodulating treatment was administered in all patients (ivig n= , plasma exchange n= , steroids n= ) and immunosuppressants (n= ). immunochemotherapy for lymphoma was initiated because of lymphoma type or severity in cases, in cases because of the associated neuropathy. median follow-up was of months after treatment initiation. four out of patients treated within months of neurological onset improved as well as one out of patients whose preexisting neurological condition had worsened. two patients presented neurological relapse during progression of lymphoma. two patients died. unusual presentation of cidp -i.e., rapid progression or treatment failure -should lead to further testing for associated lymphoma. because general symptoms and lymphadenopathy often lack, diagnosis requires analysis of bone marrow with lymphocyte phenotyping. early treatment with immunochemotherapy was associated with better prognosis in our series. cardiac scintigraphy is a useful tool for the diagnosis, prognosis and pre-symptomatic early detection of familial amyloidosis-associated neuropathies three main types of familiar amyloidosis: transthyretin (ttr), apolipoprotein a and gelsolin. cardiac involvement is a leading cause of morbidity and mortality; one new described mutation strongly related with isolated cardiac amyloidosis is the ttr val ile. the discovery of tests that allow early diagnosis of cardiac involvement in amyloidosis and to infer about the etiology of the disease is of major importance. in a cohort of patients with different types of familiar ttr amyloidosis, we aimed to assess the role of mtc- , -diphosphono- , -propanodicarboxylic acid ( mtc-dpd) in detecting myocardial amyloid infiltration. we enrolled four patients diagnosed with late familiar amyloidosis, which mutations were documented at deoxyribonucleic acid analysis: three patients with ttr val met mutation and one patient with ttr val ile mutation. three patients were asymptomatic for cardiac involvement and one patient (val ile mutation) had a previous diagnosis of heart failure. myocardial uptake of mtc-dpd scintigraphy was semiquantitatively and visually assessed at five minutes and three hours.the uptake of mtc-dpd highly demonstrated amyloid in cardiac area in two out the three cases of ttr val met and in ttr val ile. ttr val ile case presented the highest uptake due to the exclusive deposition of amyloid in cardiac area resulting in severe heart failure. in hereditary transthyretin-related amyloidosis, including the mutations ttr val met and val ile, mtc-dpd cardiac scintigraphy can identify infiltration even in asymptomatic individuals, allowing an early diagnosis of cardiac compromise in this group of diseases. we can consider that this non-invasive test would be a tool with potential importance in the diagnosis, prognosis and pre-symptomatic early detection of cardiac amyloidosis, giving emphasis on its applicability in familial forms of amyloidosis. cervellini i , galino j , zhu n , birchmeier c , bennett dl . ndcn university of oxford, oxford, uk; max-delbrück-center for molecular medicine, berlin, germany. erk/mapk pathway has a critical role in pns development since its involvement in many physiological processes. sustained erk / mapk activation in schwann cells enhances myelin growth during development and overcomes signals ending myelination leading to a continuous myelin production. however, strong activation of erk has also been shown to cause schwann cells dedifferentiation and demyelination in vivo. our aim was to investigate whether a mild activation of this signalling pathway in adult schwann cells (scs), by expression of gain of function mek dd allele, could have a beneficial role in remyelination and regeneration after injury. erk/mapk activation in adult scs in plpcreer t ;mek dd mutant mice, did not affect myelination during development. following sciatic nerve injury, wallerian degeneration was enhanced in mutants pushing towards a dedifferentiation stage of scs as previously described. however, mapk activation was detrimental during regeneration with a delay in functional recovery and a negative impact in both myelinated and non-myelinated fibres compared to controls. one month after injury the total number of axons in mutant sciatic nerves was half of the controls. although no differences in g-ratio have been found in the two groups, mutants presented a higher number of myelinated axons showing myelin disruption with start of myelin decompaction, lack of cajal bands, abundant sc processes surrounding axons and a shorter sc elongation, as seen by decreased internodal distance. in addition, we found a negative effect of mapk activation also in small diameter axons with the presence of abnormal remak bundle structures, reduced number of c-fibres/remak bundle and a significant decrease in intra epidermal nerve fibres density in the skin. we concluded that mild mapk activation has a different role in development and remyelination where negatively affects axon survival, myelin stability, remak bundle formation and small fibres regeneration. cervellini i , galino j , zhu n , bao lu , bennett dl . ndcn, university of oxford, oxford, uk; harvard medical school, boston, ma, usa. neprilysin (nep) is an endopeptidase which has been of interest due to its potential role in neurodegeneration and pain as a consequence of its ability to degrade amyloid and substance-p respectively. nep expression is not limited to cns and it has been reported to be expressed in schwann cells, nodes and schmidt-lanterman incisures. our interest in this gene was related to recent findings that have associated homozygous and heterozygous nep mutations with charcot-marie-tooth type- . in old mice lacking nep subtle morphological changes have been reported. our aim was to determine whether nep expression was modulated by nerve injury and to investigate its role in axon regeneration and re-myelination. we find that nep gene expression was decreased after nerve crush and furthermore was dependent on the growth factor (and pro-myelin signal) neuregulin- . in control mice nep expression was transiently reduced and returned to baseline at day after injury, in neuregulin- knock-out (ko) mice, in which re-myelination was impaired, the expression was still decreased at day . in assessing behavioural measures of locomotor and sensory function one month after sciatic nerve crush, nep ko mice showed a functional regeneration comparable to wt, as seen by sciatic functional index measurement, beam and toe spreading tests. the only significant difference we observed between wt and ko was in the sensorial test, showing ko mice recovering faster in the pinch test by days after crush. the results for all the tests at baseline did not differ between the two groups. detailed histological analysis of nerve repair was undertaken using electron microscopy. there was no difference between wt and ko in total axon number, g-ratio, axon diameters and other myelin features one month post crush. in summary, although nep expression is regulated by nerve injury in a neuregulin- dependent fashion this endopeptidase is dispensable for axon regeneration and re-myelination after nerve injury in the rodent. diabetes neuropathy is a common complication of diabetes, and neuropathic pain has a detrimental impact on quality of life. this study investigated sensory nerve excitability properties to elucidate the axonal changes of diabetic neuropathy. a total of diabetes patients ( type ii, and type i) were enrolled in this study. clinical assessment, nerve conduction studies, and nerve excitability testing data were analyzed to determine axonal dysfunction in diabetic neuropathy. among those patients, seventeen subjects had complained of spontaneous painful sensation over feet or hands (painful cohort), and seventy-eight patients had no sensory symptoms or decreased the sensation over foot (non-painful cohort). sensory nerve excitability of the painful cohort showed reduced late subexcitability (p= . ), increased superexcitability (p= . ) in compared to the non-painful cohort. there is no difference in disease duration, blood glucose levels (hba c) between these two cohorts. these findings suggested the possible pathogenesis of painful sensory axons might be hyperpolarized or slow potassium channels dysfunction. these insights our further understanding of painful diabetic neuropathy, and may provide a basis for neuroprotective or therapeutic approaches for painful polyneuropathy. the main purpose of this study was to assess the clinical feasibility of diffusion tensor imaging (dti) for the diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp). between march and december , we prospectively enrolled patients with definite cidp according to the efns criteria and two control groups: healthy volunteers matched on age and sex and patients with cmt- a. using a -t magnetic resonance imaging scanner, we obtained dti scans of brachial plexus of these groups and prepared fractional anisotropy (fa) maps, and compared these values between groups. adc values and cervical nerve roots diameters on stir sequences were evaluated too. two neuroradiologists, blinded to clinical informations, reviewed mri studies independently. in all patients with cidp, we also performed clinical evaluation and electroneuromyography. significantly decreased fa values (p< . ) and increased adc values were observed in cidp patients compared with healthy subjects. there is no significant difference between cidp and cmt group. inter-observer concordance was excellent for fa values ( c= . ; p< . ) and moderate for adc values ( c= . ; p< . ) and cervical nerve root diameters ( c= . ; p< . ). there is a significant correlation between fa and disease duration (r = − . , p < . ), inclusion mrc score (r = . , p < . ) and between fa mesured on c c and incat score at inclusion (r = − . , p < . ). no significant correlation is observed between fa and electrophysiological indices. compared with healthy subjects, cervical nerve root diameters were significantly increased (p< . ) in patients with cmt and cidp. contrary to fa values, moderate level of concordance was found between inter-observers measurements of diameters (cclin = . ). our preliminary data prove the clinical feasibility and reproductibility of dti for the evaluation of plexus and cervical nerve roots in patients with cidp. cheng yj , teng a , goh ejh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. the sural-sparing pattern of the sensory nerve action potentials (snap) of guillain-barré syndrome (gbs) has been attributed to greater immunological injury of the blood-nerve barrier at its most vulnerable regions. we asked if entrapment sites, such as median nerve at the wrist, are more predisposed than the distal nerve endings to such injury. we compared the median snap with radial snap measured antidromically at digit in gbs patients whose nerve conduction study showed the sural-sparing pattern. the terminal nerves at digit are of similar length, but those of median nerve are prone to compression, often subclinically, at the carpal tunnel while those of radial nerve are not. we defined the sural-sparing pattern as a greater decrease in median and or ulnar snap than that of the sural, compared to age and height-matched normal controls. a total of gbs and miller fisher patients from our institution's database were studied. patients had the sural-sparing pattern, of whom had pre-existing carpal tunnel syndrome. of the remaining patients with sural-sparing, had abnormal median snap at digit , while had both abnormal median and radial snaps at digit . none had isolated abnormality of the radial digit snap. among the cases that had abnormal median and radial snaps at digit , the mean percentage decrease when compared to age and height matched norms was greater in median nerve compared to radial nerve ( % and % respectively). of the patients without sural-sparing pattern, had normal snaps; patient had inexcitable sensory nerves while the other had a length-dependent decrease in snap. in the latter patient, unlike those with sural-sparing, there was no differential decrease of median snap over radial snap at digit . our findings suggest that the disruption of blood nerve barrier at entrapment sites rather than the distal nerve endings may underlie the pathophysiology of the sural-sparing pattern seen in gbs. chiba a , uchibori a , gyohda a . kyorin university, tokyo, japan. serum igg anti-gq b antibody is the most specific biomarker for fisher syndrome and its related disorders (fs-rd), but approximately - % of the patients are seronegative for it in conventional assays (gq b-seronegative). some molecules need ca + cation to interact with their ligands, and antibodies with such a property (ca + -dependent antibodies) are reported. we have found that such a ca + -dependency is also present in igg anti-gq b antibody, and majority of gq b-seronegative patients with fs-rd have this type of antibodies. in patients with final clinical diagnoses as fs-rd (fisher syndrome, guillain-barré syndrome with ophthalmoplegia, bickerstaff brainstem encephalitis, and acute ophthalmoplegia), were seropositive for igg antibodies against gq b-related antigens (isolated gq b in , and gq b-conatinig complexes in two) in conventional elisa using phosphate-buffered saline. in the remaining patients, eight ( %) turned positive for igg antibody against gq b-related antigens (isolated gq b in seven and gq b-conatinig complexes in one) in elisa using ca + -added tris-buffered saline. the reaction strengths increased depending on ca + concentration, and reached to nearly maximum level in the physiological concentration. all the patients with the ca + -dependent antibodies were also positive for igg antibody against gt a-related antigens, suggesting that the terminal disialo residue common to both the gangliosides would be important as an epitope also for the ca + -dependent antibodies. in the patients with ca + -non-dependent antibodies, only two showed increased titers of igg anti-gq b antibody by adding ca + , and showed significantly decreased titers. this difference in the effect of ca + -addition between ca + -dependent and ca + -non-dependent antibodies suggests that ca + would not be just an enhancer of the antigen-antibody reaction. there are four single bonds between the two pyranose rings in the terminal disialo, and those rotatable bonds make it possible for the disialo structure to take various conformations. a molecular model shows that the distance between two minus-charged carboxy groups in the disialo could vary from nearly zero to approximately , pm and that the disialo would take specific conformations, if divalent ca + cation, which size is approximately pm in diameter, interacts with these two minus-charged groups. the ca + -dependent antibodies might recognize such particular conformations of gq b. charcot-marie-tooth disease (cmt) is a genetically and clinically heterogeneous disorder with variable inheritance modes. it is characterized by loss of muscle tissue and touch sensation, predominantly in the feet and legs but also in the hands and arms in the advanced stages of disease. as several molecules have been reported to have therapeutic effects on cmt, depending on the underlying genetic causes, exact genetic diagnostics have become important for executing personalized therapy. aminoacyl-trna synthetase (arss) genes encode enzymes responsible for charging trna with corresponding amino acids. arss are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. specifically, arss attach amino acids to their cognate trna molecules in the cytoplasm and mitochondria. recent studies have demonstrated that mutations in genes encoding arss can result in neurodegeneration, raising many questions about the role of these enzymes in neuronal function. mutations in six cytoplasmic ars genes have been reported as the cause of cmt. this study was performed the whole exome sequencing to identify genetic defects in korean cmt patients from unrelated families. variants were sorted with cmt gene list that includes almost genes were related cmt neuropathy, and additionally sorted wes data as ars genes. capillary sequencing for family members and more than controls revealed five novel mutations, c. g>a (p.d n), c. c>t (p.s f), and c. c>a (p.p h) in gars; c. c>a (p.p t) in mars; _ ga>at (p.d i) in yars gene in each family. the mutation sites were well conserved between different species and each mutation were located in the well-conserved catalytic domain or between two catalytic domains or anticodon-binding domain. in silico analysis predicted all mutations may affect protein function. clinical features were similar to those reported in other countries, but differed in terms of age at onset and degree of disability. we believe that those novel ars mutations are the underlying causes of the each family. a -year-old man presented with a -year-history of weakness in biltearal upper limbs. he was complaining of intermittent fasciculation of upper and lower limbs with gradually worsening of paresthesia for years. dysphagia and dysarthria were also presented years ago. there was no patient affected muscle weakness and bulbar symptoms in his family members. in neurological examination, the patient had weakness in biltereal upper and lower limbs (mrc grade ) and prominent distal sensory loss were combined in length dependant pattern. deep tendon reflexes were absent on bilataral biceps and knee joints. in nerve conduction study, there was consistent with demyelinating sensorimotor polyneuropathy. molecular diagnostic analyses those spinobulbar muscular atrophy (sbma) and mutation related to peripheral myelin protein (pmp ) gene were performed and confirmed expansion of expansion of a polymorphic cag in androgen-receptor (ar) gene and deletion of pmp gene. smba, also known as kennedy disease, is an adult-onset, x-linked recessive trinucleotide, polyglutamine (poly-g) disorder caused by expansion of a polymorphic cag tandem-repeat in exon of ar gene on chromosome xq - . charcot-marie-tooth disease (cmt) is the most common hereditary neuropathies and cmt cases with motor conduction velocities(mcvs) of upper limb below m/s are defined as demyelinating (cmt ) and those with mcvs above m/s are defined as axonal (cmt ). most families with cmt linked to duplication of pmp gene on the short arm of chromosome ( p . ), called cmt a. the reciprocal deletion of pmp gene is a responsible genetic defect in % of hereditary neuropathy with liability to pressure palsy (hnpp). these "classical" phenotypes of cmt a and hnpp have been considered which are determined by different mutation mechanism of the same gene. however, an overlap of cmt a and hnpp due to pmp gene deletion was reported that suggestion the phenotype of hereditary neuropathies may differ variably. herein, we report a patient who simultaneously presented clinical and electrophysiologic features of smba and cmt a with genetical confirmation of cag expasion and deletion of pmp gene. charcot-marie-tooth type a disease (cmt a) is an inherited peripheral neuropathy stemming from overexpression of pmp protein in schwann cells due to the duplication of the pmp gene. this leads to abnormal schwann cell differentiation and dysmyelination, eventually leading to axonal loss and muscle wasting. no approved treatment is currently available for cmt a. we conducted a systems biology level analysis of the signaling network putatively underlying the processes driving cmt a pathology. based upon this, we identified and tested three repurposed drugs -baclofen, naltrexone and sorbitol -alone and in combination to determine their ability to rescue aberrant myelination in cultures derived from cmt a transgenic rats overexpressing pmp gene. to this end, we studied a validated in vitro co-culture model of sensory neurons and schwann cells adapted to -well culture plates. this model allows measurement of the appearance of myelin proteins as an index of the physiological process of in vivo myelination. total myelin length was quantified with an automatic image analyzer following pmp immunostaining. we first determined the full dose-response curves of single drugs, emphasizing their promyelinating activities. we then tested binary combinations of very low and inactive doses of each drug and compared these to the activity of the combination of the three, namely pxt . whereas combination of any two drugs was not significantly active at the doses tested, combination of all three produced a synergistic improvement in myelination. these findings clearly demonstrate the necessity of using pxt over its single components and highlight the value of pleiotropic combinational repurposing of drugs at low doses as a novel approach for rapid drug development in cmt a and other disorders. autonomic dysfunction is frequently observed in guillain-barré syndrome (gbs) and affects approximately % of the patients. it has been shown that the sweating function can be impaired in gbs. the aim of the present investigation is to summarize the current knowledge on sweating disturbances in gbs patients. we have used appropriate terms to systematically search for references published until and indexed in the following databases: medline, embase, lilacs and cochrane. the inclusion criteria were a diagnosis of gbs and a description of the methods used to test the sudomotor function. the search was limited to the english language. relevant information about study design, methods of assessing the deficit of sweating, patient's characteristics and main results were collected. we selected original references for the final analyses. the majority of the studies were cross-sectional in nature and there were two longitudinal studies. the severity of sweating impairment varied according to the applied method, ranging from normal to almost sympathetic nervous system failure. in seven research papers, the sympathetic skin response was used to evaluate the sudomotor function in patients, and approximately % demonstrated abnormal results. however, researchers used different stimulation protocols and parameters to interpret their results. regarding whole-body sweating test, four research papers applied the thermoregulatory sweating test in patients and they showed areas of anhidrosis on the lower limbs in all of them. eight patients presented sweating impairment on the upper limbs and abdominal wall. results on the sudomotor axon reflex test suggested a length-dependent pattern of sweating loss according to one case report. in another study, eight gbs patients were tested only on the distal leg and foot dorsum and the authors proposed an association between post-ganglionic sudomotor function and antiganglioside antibodies. the present literature review showed that the studies of sweating disturbances in gbs patients included only small cohorts. future studies with larger patients sample sizes are necessary to investigate the patterns of sweating loss in gbs and their changes along the follow up. funding: grants # / - and # / - , são paulo research foundation (fapesp). sensory neuronopathy (sn) represents a rare subgroup of peripheral neuropathies characterized by degeneration of primary sensory neurons at the dorsal root ganglia on the spinal cord. depending on the neuronal population affected, its clinical presentation may manifest as gait ataxia, proprioceptive sensory loss and positive and negative sensory symptoms. although a few reports have mentioned areas of anhidrosis in sn, we were not been able to find previous case series studies on the sweating function in sensory neuronopathies. the aim of the present investigation was to study the whole-body distribution of sweating on both anterior and posterior surfaces in patients diagnosed with sn. quantitative sensory testing for cold and warm sensation threshold (method of levels) was performed on the dorsum of the hands and feet in a randomized order. we tested the thermoregulatory sweating using a sweat chamber ( to ∘ c air temperature and % relative humidity). the oral and skin temperature was monitored and the test time did not exceed min. in order to study the sudomotor axon reflex we employed the q-sweat device on standardized body sites. the test was performed on both sides, simultaneously. we included seven patients (three male; mean age . years) with a mean disease duration of . months (range - ) and a confirmed diagnose of sn. patients presented an asymmetrical loss of cold and warm threshold on hands or feet compared with healthy control (p< , ). regarding the tst results, we found a striking variation of sweating disturbances, ranging from small areas of anhidrosis on the trunk to complete failure of the sympathetic nervous system. two patients underwent the axon reflex test and there was an asymmetrical and mostly distal pattern of sweating loss in one of them and a distal-symmetrical on the second one. our findings indicate a great variability of sweating losses in sn, not overlapped to the sensory loss areas. currently, we are testing more patients in order to confirm our results. funding: grants # / - , # / - and # / - , são paulo research foundation (fapesp). diet, exercise, and inflammation are established modulators of peripheral nervous system function, including pain. prior work examining exercise consistently demonstrates a benefit on heightened pain from a number of acute and chronic pain models. in the present work, we investigated several parameters of peripheral nerve function relevant to pain in rats bred for high (high capacity runners, hcr) or low running capacity (low capacity runners, lcr). the longtime selective breeding of these rat substrains has created divergent intrinsic aerobic capacities and predisposition of metabolic conditions between lcr and hcr rats. examination of the role of sex in the development of chronic pain has established key differences in males and females. to understand gender specific differences, this study focused on female rats to understand the role of metabolic status and peripheral nerve function in females. our analysis identified numerous parameters of peripheral nerve function relevant to pain and neuropathy that are different among lcr and hcr female rats. lcr female rats display reduced hind paw mechanical sensitivity, increased hind paw intraepidermal nerve fiber density and trka-positive epidermal axons, increased numbers of langerhans and mast cells in the hind paw dermis, and increased overall fat mass relative to body weight compared to female hcr rats. examination of sensory and motor nerve conduction velocities, thermal sensitivity, and mrna expression of selected genes relevant to peripheral sensation found no differences between hcr and lcr females. together these results suggest that a genetic component of aerobic capacity and metabolic status can influence sensory sensitivity and specific aspects of inflammation and immune responses in peripheral tissues, which may lead modify the animal's responses to tissue damage and painful stimuli. the lcr and hcr rat model will provide a useful model in the future to assess the involvement of metabolic status in the development of pain. ivig is often considered treatment of first choice in chronic inflammatory demyelinating polyradiculoneuropathy (cidp) because of its rapid onset of action and its relatively safe long-term adverse event profile. clinical trials published so far focused on a loading dose of . g/kg ivig and/or a standard maintenance dosage of . g/kg ivig once every weeks, but have not investigated different dosing options. this study is a prospective, double-blind, randomized, parallel group, multi-center phase iii efficacy study and will be conducted in centres in canada, eu, russia, ukraine and australia. adult patients with definite or probable cidp according to the efns/pns criteria will be enrolled and randomized : : to receive either . g/kg or . g/kg or . g/kg ivig (panzyga ® ) for seven maintenance infusions at -week intervals during the dose-evaluation phase. the starting loading dose will be . g/kg ivig (panzyga ® ) for all patients. primary objective: efficacy measured as percentage of responders (decrease in adjusted incat score by at least point) in the . g/kg ivig (panzyga ® ) arm (given every weeks) at week as this should corroborate the existing and published evidence on efficacy of ivig in cidp. secondary outcome: percentage of responders at week in the . g/kg and . g/kg ivig (panzyga ® ) arms relative to baseline and compared to the . g/kg arm. the procid study aims to confirm published clinical results obtained with the . g/kg standard dose and will in addition evaluate one higher and one lower maintenance dose, with the aim to offer cidp patients a more adequately dosed and effective treatment policy. * este medicamento no se encuentra comercializado en españa. understanding the rate of disease progression in patients with charcot-marie-tooth disease (cmt), both within and between subtypes is important for clinical prognosis and is crucial for clinical trial design. due to the progressive nature of cmt, intervening at the earliest stages of the disease is a priority. measuring progression of a disease with both motor and sensory deficits requires a multi-item composite scale. the cmt pediatric scale (cmtpeds) is a well-tolerated psychometrically robust -item scale measuring fine and gross motor function, strength, sensation and balance for children and adolescents aged - years with cmt. the aim of this study was to determine the rate of disease progression of children and adolescents within and between genetic subtypes of cmt. ( female) participants aged - years enrolled in the inherited neuropathies consortium were included in this study. demographic, anthropometric and diagnostic information were collected at baseline and -year follow-up. disease progression was measured with the cmtpeds. on average cmtpeds scores progressed at a rate of . points over -years ( % change from baseline, p< . ). there was no difference in rate of disease progression between males and females. of the most common genetic subtypes, participants with cmt a/pmp duplication progressed by . points ( % change from baseline, p< . ), nine participants with cmt b/mpz mutation progressed by . points ( % change), six participants with cmt a/mfn mutation progressed by . points ( % change) and seven participants with cmt c/sh tc mutation progressed by . points ( % change). participants with cmt a progressed faster than those with cmt a (p= . ). children with cmt a progressed consistently during childhood and adolescence while children with cmt b and cmt a progressed faster during childhood than adolescence. overall, children with cmt progress at a significant rate over -years according to the cmtpeds. understanding the rate at which affected children deteriorate is essential for adequately powering clinical trials of disease-modifying interventions. queen square, london, uk; department of neurology, university of iowa carver college of medicine, iowa city, ia, usa; department of neurogenetics, the national hospital for neurology and neurosurgery, ucl institute of neurology, london, uk; department of molecular neuroscience, ucl institute of neurology, london, uk and national hospital for neurology and neurosurgery, queen square, london, uk. in recent years, targeted ngs panels have changed the diagnostic work-up in patients with inherited neuropathies. however, there is limited data on the impact of targeted ngs panels on the diagnosis of cmt patients in everyday practice. the aim of this study was to investigate the impact of targeted ngs panels on the diagnosis of cmt across two tertiary referral centres in the united kingdom (london) and united states (iowa). in london, patients with a diagnosis of cmt (previous pmp duplication and common cmt genes excluded in appropriate cases) underwent targeted ngs panel sequencing covering genes associated with cmt and additional genes associated with hsp or als. a variable number of genes, ranging from to , were analysed depending on the clinical phenotype of the patients. a definite molecular diagnosis was achieved in cases ( %) including pathogenic and likely pathogenic mutations in sh tc ( cases), gjb ( cases, including cases with mutations in the promoter and 'utr regions), gdap ( cases), fgd ( cases), aars ( cases), ighmbp ( cases), mpz ( cases), nefl ( cases). vus were further identified in patients. the diagnostic rate was higher in demyelinating cmt cases ( / , %), compared to cases with axonal cmt ( / %), dhmn ( / , %) and hsn ( / , %). in iowa, patients were investigated by ngs panels covering to genes associated with cmt. a molecular diagnosis was reached in / ( %), and in particular / ( %) demyelinating and / ( %) axonal cmt cases. the most frequent genes identified were gjb ( cases), mfn ( cases), sh tc ( cases) and ighmbp ( cases). vus were identified in patients, including cases with novel variants in aars, warranting additional testing such as segregation of the variant in the family or functional validation studies. in clinical practise, targeted ngs panels represent an effective approach for the diagnosis of cmt. the lower diagnostic rate in london is likely to be due to prior sanger sequencing and exclusion of mutations in common cmt genes in this patient population. more than genes are known to cause cmt and an even larger number are known to cause peripheral neuropathy as part of a more complex neurological disorder. despite the use of custom panels, a significant proportion of patients with inherited neuropathy have no molecular diagnosis. the aim of this study was to investigate the diagnostic yield of a disease-associated gene exome (sureselect focused exome, agilent technologies, santa clara ca, usa) in the diagnosis of cmt and in cases with complex neurological syndromes associated with neuropathy. thirty-one patients with molecularly undiagnosed inherited neuropathy were analysed with sureselect focused exome sequencing. six patients had a more complex phenotype including learning difficulties, cerebral white matter changes, ataxia and pyramidal tract involvement. a genetic diagnosis was achieved in / ( %) of cases by detecting a mutation in cmt-associated genes mpz ( cases), aars, nefl, bscl , bicd and trpv . of note, six cases had mutations in genes which are not covered by currently available diagnostic targeted ngs panels, including kif a, polg, mme ( cases), dnajb , and a novel candidate gene. the average coverage was higher compared to the usual coverage of whole-exome sequencing; % of the targets were covered at x or more, and % of the targets were covered at x or more. this study provides evidence that the sureselect focused exome is a useful tool for the diagnosis of cmt and complex neurological disorders and provides further insight into the phenotypic spectrum of genes associated with inherited neuropathy. changes in the cis-regulatory sequences of a gene's untranslated regions (utr) are increasingly recognised as a significant cause of inherited disease in humans. for example, variants in the non-coding region of gjb account for % of all mutations in our cohort of cmtx patients. one of the biggest challenges in analysing the large number of non-coding variants in a gene is identifying those that are disease-causing and those which are polymorphisms. the aim of this study was to implement a reliable method for the in-vitro functional validation of non-coding variants in the promoter and 'utr regions of gjb . in our cohort of cmtx patients we have previously identified seven mutations (c.- _ inst, c.- g>a, c.- t>g, c.- t>c, c. t>c, c.- g>c, c.- c>t) in the promoter region and one novel mutation in the 'utr (c. c>t), which were considered likely to be pathogenic based on the clinical phenotype, segregation in affected family members and absence in control databases. we have now generated a luciferase-based reporter system and optimised it in a hela cell line. mutations in the promoter region were generated by site-directed mutagenesis using a commercially available gjb promoter clone (genecopoeia). our preliminary results show a reduction of luciferase activity for the c.- t>c and c.- _ inst mutations compared to the wild-type promoter. this difference was increased when transcription factors sox or egr were co-transfected with the t>c and c.- _ inst mutations respectively. validation of other variants is currently ongoing. if successful, our study will provide a useful tool for the validation of mutations in non-coding regions of gjb . moreover, it will constitute a proof-of-principle approach to the functional validation of non-coding variants in other cmt genes known to cause disease by a loss of function. recessive and dominant mutations in leucin-rich repeat and sterile alpha-motif-containing (lrsam ) have been associated with cmt p. lrsam is a ubiquitin e ligase containing a ring domain in its c-terminal, which is crucial for correct protein folding and ubiquitination activity. to date, the majority of dominant mutations reported have resulted in a frame shift disrupting a major portion of the ring-domain, although point mutations in this domain have also been described. the aim of this study was to report the prevalence, clinical features and genetic findings of patients with cmt p in our centre. we performed targeted next-generation sequencing in genetically undiagnosed cmt patients and identified cases with heterozygous mutations in lrsam ( . %) from unrelated families. the mutations identified included frameshift insertions and deletions, a non-frameshift deletion and non-sense and missense point mutations. all of the mutations were novel and were located in or flanking the ring domain. the average age of disease onset was in the rd decade but an earlier onset was reported in two cases. four had a positive family history in keeping with autosomal dominant inheritance. symptoms at presentation were heterogeneous and encompassed distal numbness, unsteadiness, distal weakness of upper or lower limbs and foot deformities. positive sensory symptoms, including tingling and shooting pains, and cramps were also frequently reported. neurological examination showed mild to moderate distal atrophy and weakness, with early ankle plantar flexion involvement in three patients. loss of vibration and reduced joint position sense were often prominent in the lower limbs and appeared to be disproportionate to the degree of weakness and impairment of pinprick sensation. ankle jerks were absent but knee and upper limb reflexes could be normal or brisk. after an average disease duration of years, all but one patient was able to walk independently. nerve conduction studies showed a sensory and motor axonal neuropathy with normal conduction velocities. our study highlights that mutations in lrsam are a relevant cause of cmt and are associated with prominent large fibre sensory loss. in recent years, the implementation of ngs panels for the molecular diagnosis of cmt has increased the number of patients with a genetic diagnosis. nevertheless the interpretation of a particular variant as disease causing can be challenging especially when multiple variants are identified in a single patient. we report two illustrative cases of such challenges. the first index case was born of non-consanguineous healthy parents. he presented with falls in early childhood. over the years he developed foot deformities and progressive length dependent weakness. he had multiple orthopaedic operations to his feet. the past history was also notable for kyphoscoliosis, sensorineural deafness from the age of and bilateral cataracts. nerve conduction studies at the age of revealed a demyelinating neuropathy consistent with the clinical phenotype of cmt . his younger brother had a similar, although more severe phenotype. a litaf (c. c>t,p.pro ser) mutation was identified by sanger sequencing and was present in both affected brothers but also in the unaffected sister. ngs for cmt -associated genes was therefore performed and identified two compound heterozygous pathogenic mutations detected in sh tc (c. c>t, p.arg *; delg, p.arg serfs* ), which segregated with the disease in the family. the second case describes a brother and sister with early onset demyelinating cmt associated with scoliosis and cranial nerve involvement. the male proband underwent ngs and a single previously reported pathogenic intronic splice-site mutation in sh tc (c. - a>c) was found. relative read-depth analysis of ngs was performed to look for possible copy number variants in sh tc , thus identifying a deletion of exon , which was confirmed by long pcr. cruz-velásquez gj , miramar-gallart md , alarcia-alejos r , roche-bueno jc , rodríguez-valle a , capablo-liesa jl . neurology service, university hospital miguel servet, zaragoza, spain; genetics unit, clinical biochemistry service, university hospital miguel servet, zaragoza, spain. charcot-marie-tooth disease (cmtd) defines a clinical and genetically heterogeneous group of inherited peripheral neuropathies characterized by chronic motor and sensory impairment. the type cmtd- also known as cowchock syndrome, is the product of the mutation in the apoptosis inducing factor mitochondria associated gene (aifm ). it is a slowly progressive, recessive, x-linked disease characterized by axonal neuropathy, deafness, and cognitive impairment. our purpose is to describe new cases, brothers, children of non-consanguineous parents, with a characteristic phenotype and a new mutation in the aifm gene. both siblings present from childhood, progressive weakness in lower limbs with diffuse amyotrophies. needing tenotomy before the year due to equinovarus foot. likewise they develops sensory deafness and one of them requires unilateral support at and the other wheelchair at , this one need a pacemaker for an atrioventricular block at . with brain functions and normal language, sensorial deafness, proximal and distal weakness in the four limbs with intense amyotrophies, predominantly distal. tactile and painful sensitivity decreased in glove and sock pattern. an extensive metabolic and biochemical study was normal. the electroneurography demonstrates an axonal neuropathy without response in most of the nerves explored. the electromyography shown a myogenic pattern with distal predominance. brain mri was normal in both cases. through a genetic study by exoma targeting genes associated with cmt and inherited related neuropathies was identified the homicigosis mutation in the aifm gene (p.glu lys), located in the chromosomal region xq . (cmtx ). cowchock syndrome is a rare entity, with few cases described in the literature. the in silico analysis indicates in of the predictors used (provean, sift, polyphen , lrt, mutationtaster, mutationassessor and condel) , that it is a deleterious variant. we audited all patients with peripheral neuropathy caused by a paraprotein, who received treatment and attended king's college hospital peripheral nerve service between - . patients were identified retrospectively from our database of patients attending the peripheral nerve outpatient clinic. clinical information was obtained retrospectively from hospital electronic patient records. we excluded patients with poems syndrome or in whom the neuropathy was not felt to be caused by the paraprotein. we identified patients with a diagnosis of paraproteinaemic neuropathy. we excluded four who did not fulfill the diagnostic criteria and eleven who had received no treatment or were under diagnostic study. we included patients in the final audit. ( %) had igm paraprotein. the haematological diagnosis was monoclonal gammopathy of undetermined significance (mgus) in %, waldenstroḿs macroglobulinaemia %, and lymphoma or plasmacytoma %. after treatment, overall ( %) patients improved neurologically, ( %) stabilised, and ( %) worsened. in the patients who received more than one type of treatment, we analysed outcomes according to the most powerful treatment received. patients received rituximab alone of which ( %) improved, ( %) stabilised and ( %) worsened. nine patients received rituximab combined with cyclophosphamide or bendamustine, of which ( %) improved, ( %) stabilized and ( %) worsened. eight patients received intravenous immunoglobulin, of which ( %) improved, ( %) stabilized and ( %) worsened. four patients received other chemotherapy, of which improved, stabilised, worsened. two patients received corticosteroids and both worsened. there was improvement in / ( %) with mag antibodies and / ( %) without. there was improvement in / ( %) with mgus and / ( %) with haematological malignancy. there was improvement in / ( %) with kappa light chains and / ( %) with lambda. factors associated with better outcome (by univariate analysis) were negative mag antibodies, kappa light chain, and haematological malignancy. there was no significant difference between treatments in the proportions who improved. cumberbatch m , cox a . addenbrooke's hospital, cambridge, uk. treatment of patients with autoimmune neuropathies such as chronic inflammatory demyelinating polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) has centred on the use of intravenous immunoglobulin (ivig). however, ivig therapy is associated with systemic side-effects, treatment wear-off effects and regular hospital attendance. subcutaneous immunoglobulin (scig) is an efficacious alternative that can be flexibly dosed, self-administered at home and avoids the 'peaks' and 'troughs' observed with ivig. these factors may combine to improve patient satisfaction and alleviate hospital capacity issues. here, we report on clinical and patient experience of switching from hospital-based ivig to home-based manual push scig for the treatment of cidp and mmn. this was a clinical case series of patients ( cidp, mmn; mean age . years) who were clinically stable on ivig and wished to switch to manual push scig. starting scig dose was equivalent to the final ivig dose for each patient (mean . g/kg week). clinical efficacy (medical research council sum score, -m walk, modified inflammatory neuropathy cause and treatment score, overall neuropathy limitations scale, romberg test) and patient-reported outcomes ( -item short form health survey , life quality index [lqi] ) were assessed at baseline and at regular intervals until the final visit ( - months after switching). at baseline, patients cited 'convenience' as their primary reason for switching to scig. eight patients completed the full assessment period and successfully undertook administrations at home (via hospital-at-home service in cases). dose adjustments, based on clinical need, were required in patients. treatment efficacy and patient quality of life, measured by sf- , were maintained after switching to scig; overall patient satisfaction, measured by lqi, increased from % to %. in the lqi, 'convenience', 'travel time/cost' and 'interference-work' were significantly improved (p< . ) after switching to scig therapy. adverse events included mild erythema and localised swelling, as expected for a ml subcutaneous injection. these findings suggest that manual push scig therapy is a viable alternative to ivig for patients with cidp and mmn, as it maintains disease stability, is more convenient for patients and may help ease hospital capacity concerns. cumberbatch m , soares regua d , cox a . addenbrooke's hospital, cambridge, uk. intravenous immunoglobulin (ivig) is used to treat a number of chronic autoimmune neurological diseases. in most centres, infusions are given at slow rates as there is a perception that this reduces the risk of adverse events (aes). this results in longer in-patient admissions, or frequent day case attendances, impacting on both the patients' quality of life and hospital capacity. however, there is little evidence to suggest that slow infusion rates are required. we used the manufacturer recommendations to optimise infusion rates and reduce the time patients spend in hospital. we report a retrospective audit which describes the impact of different ivig infusion rates on patients' clinical condition. the audit comprised three -month assessment periods: january-june (cohort ; infusion rates of . ± . ml/kg/hr, n = ); january-june (cohort ; . ± . ml/kg/hr, n = ) and july-december (cohort ; . ± . ml/kg/hr, n = ). clinical data were reviewed to determine: patient demographics, duration of infusion; time spent in hospital; and incidence of aes. the three cohorts were well matched in terms of patient demographics ( patients were treated in all treatment periods). cohorts and had significantly shorter treatment episodes than cohort ( . and . vs. . hours, p< . ), spent less time on the unit over the month period ( . and . vs. . hours, p< . ) and had fewer admissions/patient ( . and . vs. . , p< . ). the overall incidence of confirmed aes (mainly headaches) was similar across the cohorts (cohort : %; cohort : %; cohort : %). these findings indicate that increases in ivig infusion rate are well tolerated and significantly reduce treatment time, which benefits patients and offers potential cost savings and reduced pressures on hospital capacity for healthcare providers. cunningham me , yao d , meehan gr , barrie ja , willison hj . university of glasgow, glasgow, uk. one mechanism of injury in the acute motor axonal neuropathy (aman) form of guillain-barré syndrome (gbs) is the attack of peripheral nerve axons by anti-ganglioside antibodies (agabs). rodent models have demonstrated that that binding of these antibodies activates the complement cascade, resulting in the insertion of the terminal component, membrane attack complex (mac) into the axonal membrane. complement activation also results in the release of anaphylatoxins, which are known to recruit phagocytic immune cells to the site of injury. our current in vivo mouse model of agab and complement-mediated injury are acute and severe, resulting in respiratory distress over several hours of such magnitude to warrant termination of experimental procedures. to observe and potentially target immune cell infiltration following agab and complement-mediated injury, a subacute model extending over days is required. here, we demonstrate the development of such a model. to compare differences in immune cell infiltration subacutely under control and injury conditions, mice with endogenous expression of egfp in monocytes and macrophages underwent a modified agab and complement-mediated injury, resulting in a less severe phenotype than previously published models. six days following injury, immune cells in the diaphragm were compared by immunofluorescence and flow cytometry. flow cytometry found overall presence of neutrophils was significantly increased in the diaphragm. macrophages were also increased in injured mice, although did not achieve statistical significance at this timepoint. these results were reflected in immunofluorescent staining of the diaphragm where egfp+ macrophages were quantified surrounding the neuromuscular junction (the primary injury target in this model). the development of an extended mouse model of agab and complement-mediated injury is important, since acute models do not take into consideration either the late-term effects of complement-mediated activation at the nerve membrane, or the recovery phase. future studies will look at the effect of inhibiting complement activation on the presence of immune cells in distal motor nerves. family- is a large australian family with an autosomal dominant form of dhmn (dhmn : omim % ) -a group of length-dependent neurodegenerative disorders affecting the lower motor neurons leading to chronic disability. we recently reported a novel . mb chromosomal insertion within the dhmn locus which we hypothesise is likely to cause disease by dysregulating the expression of one or more nearby genes. studying gene dysregulation in peripheral nerve disease is challenging as the relevant tissues (spinal cord and peripheral nerve) are not easily accessible in patients. therefore, alternative strategies are needed to elucidate the disease mechanisms and pathways involved in peripheral nerve degeneration. to address this problem, we have devised a two-tiered strategy to assess dysregulation of candidate genes using patient lymphoblast and fibroblast cell lines. these cell lines can be easily established, are minimally invasive to obtain, and will harbour the natural mutation and genetic background of patients. our strategy firstly uses lymphoblast gene expression profiles as an initial screening tool to prioritise candidate genes for assessing altered expression. differentially expressed genes will then be modelled in c.elegans where behavioural and nerve morphology can be assessed. using rt-pcr, we have screened dhmn candidate genes in patient and control lymphoblast cell lines. eighteen candidate genes were expressed in lymphoblasts. twelve of the eighteen genes were prioritized for further analysis based on expression in both lymphoblast and neural tissues. quantitative analysis using qrt-pcr taqman assays revealed that ube c, was differentially expressed between patients and controls. it is important that patterns of differential expression can be recapitulated in neural cell-specific models. as part of our second strategy, we have generated patient and control induced pluripotent stem cell derived motor neurons (ipsc-mns) from reprogrammed fibroblasts. using this model, we will perform rna-seq and qpcr experiments to examine disease-relevant alterations in gene expression in neural tissue. we predict that utilization of these two strategies will shed light on the pathogenic mechanisms underlying the dhmn insertion and provide useful insights of pathways leading to peripheral nerve degeneration. the outcome of guillain-barré syndrome remains unchanged since plasma exchange and intravenous immunoglobulin were introduced over years ago. pathogenesis studies on gbs have identified the terminal component of complement cascade, the membrane attack complex, as a key disease mediator and thus a therapeutic target. the inhibition of complement in guillain-barré syndrome (ica-gbs) trial looked at the first use of c pathway inhibition with eculizumab in humans with gbs in a randomised, double-blind, placebo-controlled trial. its primary outcome was to look at safety and tolerability of administration concomitantly with ivig and in the context of severe (gbs disability score or greater) disease. participants were recruited for a month period, with regular follow up. subjects were screened, with ( %) being randomised. the two main causes for failure to proceed were participant concerns around eculizumab side effect profile, specifically the meningitis risk, and also intercurrent infection precluding treatment. five received eculizumab for four weeks, alongside standard intravenous immunoglobulin treatment, with receiving placebo. the safety outcomes, monitored via adverse events capture at each trial visit, showed eculizumab to be well tolerated and safe when administered in conjunction with ivig. the most common adverse events were mild derangement in transaminases or infection. there were no infusion reactions. primary and secondary efficacy outcomes were captured via gbs disability scores, mrc sum scores, rasch overall disability scores and overall neuropathy limitation scores. for the primary efficacy outcome at weeks after recruitment, of placebo and of eculizumab-treated subjects had improved by or more grades on the gbs disability score. all patients had improvements in other measured parameters. this trial highlights the challenges in recruiting acutely unwell patients, due to time constraints and intercurrent infection. although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in gbs are warranted. charcot-marie-tooth disease (cmt) affects about one in . people. currently more than genes have been identified, with the most different phenotypes. the majority of cases in western countries are autosomal dominant and classified as demyelinating and axonal according to electroneuromyography (enmg). the clinical condition is characterized by weakness and predominant sensory changes in the feet and hands. sometimes there are different phenotypes. recently, variants in heterozygotes in the hars gene (histidil-trna synthetase) have been described associated with cmt called type w.to report a case of cmt-sensitive phenotype with a probable new mutation in hars gene (p.leu arg).a male patient, adopted son, caucasian, drug addict, for three years suffered pain in lower limbs, of great intensity, refractory to drug treatment. the examination showed retrognathism, abolition of patellar and achilleas reflexes, painful and thermal anaesthesia and apalesthesia in the feet.the enmg showed reduced sensory action potentials in sural and superficial fibular nerves. laboratory investigations for painful polyneuropathy of thick and fine fibres was normal. sural nerve biopsy revealed axonal predominance neuropathy. exome sequencing revealed a mutation in the hars gene with a pathogenic variant in heterozygosity, with replacement of the amino acid leucine at position by arginine. our patient, although we did not know the antecedents, presented painful polyneuropathy, whose genetic research, although not unequivocal, indicated a variant called cmt w. few cases of this variant were described, with several mutations. our case revealed mutation hitherto unknown (p.leu arg). we conclude by the importance of a thorough genetic evaluation, in cases of sensory polyneuropathy of unknown cause. small fiber neuropathy (sfn) is a condition that affects the small a -and c-fibers, leading to severe neuropathic pain and autonomic dysfunction. several sodium channel gene mutations have been found in patients with sfn, with scn a-gene mutations being the most frequent. because current available sodium channel blockers are not selective for na v . , these treatments often result in numerous side effects. lacosamide is an anticonvulsant that targets specific sodium channels with a slow-inactivation state, while sparing those with normal activity. several mutations of the scn a-gene with an impaired slow-inactivation of na v . have been found in patients with sfn. therefore, a positive effect of lacosamide on pain reduction in these patients is expected. the primary objective of this study was to determine the effect of lacosamide versus placebo on pain in subjects with scn a-associated sfn. secondary objectives were to determine the effect of lacosamide on autonomic symptoms, sleep interference, and quality of life, and to examine the safety and tolerability. the lacosamide-efficacy-'n'-safety in sfn (lenss) study was a randomized, placebo-controlled, double-blind, crossover-design study. subjects were randomized to start with lacosamide and end with placebo or vice versa. during both of the two phases of the study, the subjects were treated for a period of eight weeks of mg bid, preceded by a titration period, and ended by a tapering period. patients filled in a pain diary twice daily and scored a set of validated questionnaires on autonomic symptoms, sleep interference, and quality of life at multiple study visits. in total patients with scn a-associated sfn were included between november and february . the subjects had a median age of years, ranging from to years. sixty percent of the patients were female. the final results of the study, including the primary and secondary outcomes, will be presented. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces must be in intimate contact with nerve fibres to have a proper function, but it has been shown that this is compromised due to the foreign body reaction (fbr). this fbr is the first response of the nonspecific immune system against an implanted device and is characterized by a first inflammatory phase followed by a second antiinflammatory and fibrotic phase. this process results in the formation of a tissue capsule around the interface causing function loss due to the physical separation between the active sites of the electrode and nerve axons. taking this into account, here we have tested several anti-inflammatory drugs such as dexamethasone, ibuprophen and maraviroc to reduce macrophage activation as well as clodronate liposomes to reduce monocyte/macrophage infiltration. moreover, sildenafil have been administered as an antifibrotic drug to reduce collagen deposition in a fbr model with longitudinal parylene c-based intraneural devices implanted in rat sciatic nerve. briefly, animals were systemically treated with dexamethasone, ibuprophen, sildenafil, maraviroc or clodronate liposomes for two weeks, and nerve damage, inflammatory reaction and matrix thickness around the implant were assessed. treatment with dexamethasone, ibuprophen or clodronate liposomes significantly reduced the inflammatory response in the nerve in comparison to saline group while sildenafil or maraviroc had no effect on iba positive cells infiltration in the nerve. however, only dexamethasone was able to significantly reduce the matrix deposition around the implant after two weeks of treatment. these results support the idea that inflammation triggers the foreign body response in peripheral nerves and a potent anti-inflammatory treatment with dexamethasone could have a beneficial effect on lengthening intraneural interfaces lifespan. de la oliva n , del valle j , navarro x . department of cell biology, physiology and immunology, institute of neurosciences, universitat autònoma de barcelona and centro de investigación biomédica en red sobre enfermedades neurodegenerativas (ciberned), bellaterra, spain. intraneural interfaces functionality decreases over time, among other factors, due to the foreign body response (fbr), which encapsulates the implanted devices and physically separates the active sites from the nervous tissue. here we have studied the fbr to parylene c or polyimide thin devices implanted in rat sciatic nerves, assessing thickness of the tissue capsule, signs of inflammation and nerve damage. we have characterized the responsible cells of this response and several molecular mediators over months of implant to find differences between the fbr to both materials. after weeks of implant, the inflammatory response due to the surgery was already decreased, whereas in the implanted nerves it reached its highest levels to then decrease at chronic time points. besides, the amount of foreign body giant cells (fbgc), as a result of macrophage fusion, found in the tissue capsule around the implant also increases progressively to reach a maximum after weeks. on the other hand, molecular analysis of the environment revealed a peak of inflammatory cytokines during the first day of implant to return to standard levels thereafter. however, an increase on ccls molecules was found at later time-points for both materials. with regard to the capsule thickness, all the devices were surrounded by a tissue deposition which appeared soon after the implantation. however, in the case of polyimide devices, the tissue capsule showed a peak weeks after the implant and signs of remodeling thereafter, while the parylene c devices showed a second increase from to weeks in comparison to polyimide devices. immunohistochemical and electron microscopy analysis revealed two different cell types implicated in the fbr in nerve to both materials: macrophages, in close contact with the interface, and fibroblasts which appear after weeks surrounding the tissue capsule. although further analyses are needed to elucidate the differences in the fbr to parylene c and polyimide polymers, these results can help to determine therapeutic targets in order to reduce this response and to improve the intraneural interfaces lifespan. delmont e , antoine jc , paul s , boucraut j , attarian s . referral centre for als and neuromuscular diseases, marseille, france; referral centre for neuromuscular diseases, saint etienne, france; immunology laboratory, saint etienne, france; immunology laboratory, marseille, france. peripheral neuropathies with antibodies against myelin associated glycoprotein (mag) are chronic sensory neuropathies characterized by the presence of an igm monoclonal gammopathy and high levels of anti-mag antibodies. these antibodies recognize a specific epitope called human natural killer (hnk ) shared by nk lymphocytes and several components of the peripheral nerve (mag, p , pmp , sgpg, phosphocan). recently an elisa test has been developed to detect antibodies against hnk epitope. our objectives were to determine the sensitivity and the specificity of anti-hnk antibodies in the diagnosis of anti-mag neuropathy and to know if these antibodies were correlated with the severity of the disease. anti-hnk antibodies were assessed in anti-mag neuropathies and in negative controls: chronic inflammatory demyelinating polyradiculoneuropathies (cidp), miller fisher syndromes, sensory neuronopathies, length-dependant axonal sensory polyneuropathies, healthy controls. in anti-mag neuropathies, were recorded age, disease duration, incat sensory sum score (iss), overall neuropathy limitation scale (onls), rasch-built overall disability scale (rods), mrc sum score, anti-mag antibodies titer, peak dosage of the igm monoclonal gammopathy. anti-hnk antibodies were measured with gangliocombi™ mag elisa test and anti-mag antibodies with anti-mag autoantibodies elisa test both from buhlmann company. anti-hnk antibodies were positive in / anti-mag neuropathies, and in / controls (sensitivity %, specificity %). in anti-mag neuropathies, anti-hnk titer was correlated with sensory deficiency evaluated with the iss score (r= . , p= . ) and with disability evaluated with the rods (r= − . , p= . ) and onls scales (r= . , p= . ). anti-hnk titers were not related to age, disease duration, mrc sum score, anti-mag antibodies titer, peak dosage of the paraproteinemia. anti-mag antibodies titers were associated with none of the characteristics of the patients with anti-mag neuropathy. anti-hnk antibodies have good sensitivity and specificity in the diagnosis of anti-mag neuropathy. compared to anti-mag antibodies, their value is that their titers are related to the disease severity. these results need to be confirmed in a larger prospective cohort. chronic inflammatory demyelinating polyradiculoneuropathy (cidp)is a heterogeneous and treatable immune-mediated disorder that critically lacks biomarkers to support diagnosis. recent evidences indicate that paranodal proteins (contactin- , contactin-associated protein- , and neurofascin- ) are the targets of autoantibodies in a subset of patients with cidp showing distinct clinical presentations. particularly, these biomarkers appear to have clinical relevance and help to orientate therapeutic choice. here, we examined five patients presenting an igg reactivity against the nodes of ranvier and the axon initial segment. using a proteomic approach, cell-based assays and elisa, we identified neurofascin- (nfasc ) and neurofascin- (nfasc ) as the main targets of autoantibodies at the nodes of ranvier. four patients displayed predominantly antibodies of the igg subclass, whereas one patient presented igg antibodies that activated the complement pathway in vitro. these antibodies recognized different epitopes than the previously described anti-neurofascin- igg suggesting different pathogenic functions. accordingly, patients with anti-nfasc / igg showed a distinctive clinical presentation. most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. tremors or neuropathic pain were not observed. four patients presented with a subacute-onset and sensory ataxia. of interest, the neuropathy occurred concomitantly with nephrotic syndromes in two patients and with an igg -related retroperitoneal fibrosis in one patient. this suggested that autoantibodies could be responsible for the occurrence of both disorders. intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient improved following cyclophosphamide and rituximab treatment. clinical remission was found to correlate with the depletion of anti-nfasc / antibodies and the loss of igg reactivity toward the nodes of ranvier. in addition, recovery of conduction block and of distal motor amplitude were observed following remission and suggested a nodo-paranodopathy. our data demonstrate that nodal antigens are the target of autoantibodies in a subgroup of patients with cidp. this emphasizes that the pathogenic mechanisms involved in chronic immune-mediated demyelinating neuropathies are broad and may include dysfunctions of the nodes of ranvier. mutations in the neurofilament heavy (nefh) gene have been recently identified as a rare cause of autosomal dominant, axonal charcot-marie-tooth disease (cmt ). the clinical spectrum of this condition remains to be delineated. we report two french families with an axonal, predominantly motor, dominantly inherited form of cmt caused by two previously unreported mutations in the nefh gene. twelve patients belonging to two different families were included in the study. they displayed an axonal motor and sensory neuropathy, with no mutations in known axonal cmt genes. a remarkable feature in all patients was the early involvement of proximal muscles of the lower limbs, occurring approximately to years after the onset of motor deficit. proximal weakness affected predominantly the iliopsoas muscle, whereas quadriceps and hamstring muscles were relatively preserved. muscle weakness and muscle wasting progressed rapidly, with most of the patients requiring walking assistance after years of disease evolution. three patients in family had brisk reflexes. nerve-conduction velocity studies displayed evidence of a motor and sensory axonal neuropathy predominantly affecting the lower limbs. original deletions of nucleotides near the end of the coding sequence of nefh were identified: in family , c. _ del (p.lys argfs* ), and in family c. _ del (p.lys glyfs* ) causing a frameshift. interestingly, this frameshift leads to the loss of the terminating codon and to the translation of additional amino acids encoding a cryptic amyloidogenic element, suggesting that this type of mutations could induce protein aggregation. consistently, we showed that overexpression of the mutated forms of nefh in a human neuroblastoma cells induced the formation of protein aggregates. we also observed that it triggered caspase activation and apoptosis. using electroporation of chick embryo spinal cord, we confirmed in vivo that mutated nefh formed aggregates and triggered apoptosis of spinal cord neurons. altogether, this suggests that these mutations in nefh cause protein aggregation and neurotoxicity in neurons expressing nefh. progressive loss of such neurons would explain the early motor involvement and the pyramidal signs observed in some patients. our results provide a physiological explanation to the presence of cmt and als clinical features in affected patients. del valle j , , delgado-martínez i , righi m , santos d , , cutrone a , bossi s , d'amico s , micera s , , navarro x , . neuroprosthetic devices that are aimed to restore sensorimotor limb function of amputee patients require highly selective electrodes designed to establish a tight relationship with the nerve, allowing the bidirectional transduction of signals between nerve fibres and the interface and enabling close-loop control from the user. differently from extra-or intraneural interfaces, regenerative nerve electrodes are designed to enable electrical interface with regrowing axonal bundles of injured nerves, aiming to achieve high selectivity for recording and stimulation. however, most of the developed designs pose an obstacle to the regrowth mechanisms due to low transparency and cause an impairment of the nerve regeneration. in this work, we present a novel double-aisle planar regenerative electrode, a new type of highly transparent, non-obstructive regenerative electrode, which allows the selective stimulation and recording of separated nerve fascicles. the design consists of a thin and flexible double-sided electrode longitudinally inserted across a conduit thus creating two separated aisles in which regenerating fascicles can independently regrow after nerve transection. electrodes implanted in acutely transected nerves of rats showed the capability of selectively stimulating and recording different fascicles inserted in the aisles. moreover, chronic implantation of the electrode in a nerve gap of mm after sciatic nerve section allowed for fascicle regeneration and reinnervation of distal muscles as confirmed by the high number of myelinated axons inside each aisle, good biocompatibility, and adequate nerve conduction. in addition, three and six months after implantation, independent stimulation and recording of each separately regenerated fascicle were possible. our results demonstrate the potential contribution of the doubled-aisle regenerative electrode to selectively interface different fascicles of an injured nerve with no deleterious effects on nerve regeneration. therefore, this multi-aisle regenerative electrode may be suitable for neuroprosthetic applications, such as prostheses for the restoration of hand function after amputation or severe nerve injuries. demichelis c , garnero m , franciotta d , cortese a , callegari i , mancardi gl , schenone a , leonardi a , benedetti l . department of neuroscience, rehabilitation, ophthalmology, genetics, maternal and child health, university of genoa and irccs aou san martino-ist, genoa, italy; laboratory of neuroimmunology, irccs, "c. mondino" national neurological institute, university of pavia, pavia, italy; u.o. neurology, asl imperiese, imperia, italy. querol et al. showed that neurofascin (nf ) antibodies identify a chronic inflammatory demyelinating polyradiculoneuropathy (cidp) phenotype characterized by severe polyradiculoneuropathy, poor response to intravenous immunoglobulins (ivig), and disabling tremor. neurological improvement after therapy with rituximab has been previously reported in three patients with cidp with igg anti-nf antibodies. herein we describe the acute-onset of a case of cidp positive for nf igg antibodies resistant to conventional therapies and responsive to rituximab. the patient is a year-old woman who presented acute onset ataxia and gait disturbances; her symptoms progressed over two weeks and distal weakness, numbness and paresthesias appeared too. the nerve conduction study was suggestive for a motor-sensory polyradiculoneuropathy mainly demyelinating. the cerebrospinal fluid analysis showed elevated protein level and normal cellular count. the patient was initially diagnosed with guillain-barré syndrome (gbs) and treated with plasma exchange without improvement. an ivig cycle was started with a partial relief but at the time of admission to the rehabilitation center the patient still had a marked weakness in all four limbs. after six months she presented a further clinical deterioration and she was restricted to wheelchair. there was no response to additional treatment with ivig, while pulse corticosteroid treatment determined a significant clinical improvement. during the next months, despite the maintenance of steroid therapy, the patient presented a progressive deterioration and she was again restricted to wheelchair. postural and intention tremor appeared at upper limbs and became progressively more disabling. anti-nf ab dosage resulted positive. rituximab was administered at a dosage of mg/m /weekly for weeks. after three months the tremor improved, allowing her to eat independently and the patient was able to walk with bilateral support. antibodies anti-nf were negative. after six months she walked without support and she was able to stitch crochet. as previously reported, in this case a cidp positive for igg nf developed severe polyradiculoneuropathy with predominant distal weakness, ataxia, disabling tremor and resistance to conventional therapies. interestingly the onset was gbs-like. the correct identification of these cidp subtypes has diagnostic, prognostic and therapeutic implications. rituximab con be useful in these patients. demir Ö , yazıcı t . department of neurosurgery, university of gaziosmanpaşa school of medicine, tokat, turkey; department of neurosurgery, kent hospital, giresun, turkey. it is still challenging problem to maintain motor and sensory functions of peripheral nerve after nerve transection. after the nerve injury, calcium concentration in the damaged area increases. then the calcium ions act like cytotoxic agents in the damaged area. nifedipine is calcium channel blocker. we aimed to investigate the effects of nifedipine on nerve regeneration by modulating calcium in the damaged area. twenty-four swiss albino male rats were divided into two groups. left sciatic nerve transection surgery was performed to the all rats in both groups. then the all transected nerves were sutured primarily with epineural interfascicular method. in the experimental group, the anastomosis sites were wrapped with a piece of gel foam soaked into diluted nifedipine solution. in the control group, the anastomosis sites were wrapped with a piece of gel foam soaked into saline solution. we evaluated the effect of nifedipine by using functional, electro-physiological and histopathological studies after the surgeries. in the postoperative second week, walking test was performed and sciatic function index was calculated. in the postoperative third week electroneuronography (enog) was performed. there are significant differences between two groups. nifedipine improved nerve recovery functionally (p< . ) and electro-physiologically (p< . ). in the postoperative fourth week, we performed histopathological examination. in the experimental group with nifedipine there were more organized axons that reached the aim. we conclude from these results that nifedipine is an effective nerve protective agent when used locally at the anastomosis site after the transection of the nerve. the lack of effective, disease-modifying therapies for cmt highlights the need for novel preclinical models suitable for drug discovery. studies in rodent models of cmt tend to be time-consuming, and findings so far have translated poorly into clinical trials. primary and induced pluripotent stem cell (ipsc)-derived neuronal cultures are an established model of neurological diseases. however, due to the random distribution of neuronal bodies and neurites that happen when plating these cells, this system is not ideal to investigate axonal, length-dependent processes like peripheral neuropathies and particularly cmt. to optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on motor neuron neurospheres. we differentiated motor neurons from human induced pluripotent stem cells, purified them by magnetic sorting and cultured them in suspension until they formed neurospheres. floating neurospheres can be maintained in agitation for months as a reliable source of motor neurons. after neurospheres are platted, axons rapidly grow out of them in a radial fashion, resembling dorsal root ganglia cultures. this configuration allows for a better visualization of axons in imaging studies and for continued axonal growth over at least a -day period. axons grew at an average rate of micrometers/day and reached up to cm in length. neurospheres can be fixed and stained allowing for morphological analysis and investigation of protein distribution in axons. this system is also ideal for time-lapse imaging to study axonal transport of organelles and neurofilament kinetics. lastly, our motor neuron neurosphere system lends itself well for high content screening platform. neurospheres can be plated in -well plates where multiple compounds can be tested and the axons easily imaged by a high content screening microscope. in summary, we developed a new platform to investigate motor axons in vitro, which are particularly useful to study length-dependent processes such as inherited peripheral neuropathies and may facilitate the identification of new therapeutic compounds using high content screening systems. mutations in the neurofilament light chain (nfl) gene cause autosomal dominant axonal charcot-marie-tooth neuropathy (cmt e). nfl is a major component of the neuronal cytoskeleton, and is believed to function in conjunction with nfm and nfh to provide structural support for the axon and regulate axon diameter. despite the significant advances in understanding its biological basis, there is still no effective, disease-modifying therapy for cmt e, in part due to the paucity of preclinical models suitable for drug discovery. the development of novel preclinical platforms that can faithfully mimic mechanisms of axonal degeneration in vitro would be an essential and valuable resource to better understand the biology of cmt e and identify potential targets for therapy development. to address this, we generated control and cmt e patient-derived motor neurons and cultured them in suspension until they formed neurospheres. immunostaining of cmt e neurospheres with nfl and tubb antibodies revealed numerous areas of nfl accumulation in n s cmt e axons, resembling the accumulations of mutant nfl protein seen in the processes of catecholaminergic neuronal cell line cad overexpressing several nfl mutants. further analysis demonstrated that areas of nfl accumulation were also immunopositive for nfh, pnfh and nfm and that at least nfl and nfm co-localized in the same areas of deposits. taken together, these results demonstrate that abnormal axonal neurofilament distribution is a feature of cmt e ipsc-derived motor neurons and involve all three neurofilament subunits. we also developed an image analysis routine to allow for automatized quantification of neurofilament distribution. preliminary quantification of nfl signal intensity revealed that axons from patients have a weaker nfl signal compared to control axons, but present several signal peaks above the range observed in controls, which related to the areas of nfl accumulation. these results suggest that nfl accumulates in certain regions of cmt e axons but is reduced in the areas with no accumulation. these findings can be readily adapted into a high content screening platform and will be used to identify compounds able to reverse this axonal phenotype. in summary, we identified a strong axonal phenotype in human cmt e motor neurons with potential as a screening platform for drug discovery. nodal and paranodal proteins have been identified as antigens in peripheral inflammatory neuropathies, however the frequency and clinical relevance of antibody responses against these targets remain poorly investigated in gbs. patients with acute onset inflammatory neuropathies were identified by exploration of the local databases of the departments of neurology and the institute of neurology of the medical universities in innsbruck and vienna. patient data, electrophysiological classification and presence of anti-gangliosid antibodies were retrospectively retrieved by review of patient records. only patients with typical clinical presentation and electrophysiological results consistent with one of the subtypes of gbs were included in the study. among forty-nine patients, thirty-five were classified as aidp, six aman, three amsan, three mfs, and two pharyngo-cervico-brachial gbs. of the included patients had anti-ganglioside-antibodies. ten patients with the initial suspicion of aidp had a disease duration of more than months and were reclassified as cidp. all patient and twenty sera of control patients with non inflammatory polyneuropathy were screened by an optimized tissue based assay using rat brains for immune responses against surface antigens, and by cell-based assays with transfected hek cells for antibodies against contactin (cntn ), contactin (cntn ), contactin-associated-protein (caspr ) and neurofascin- (nf ). in the tissue based assay some of the patients showed a light neuropil staining. none of gbs patient's sera had antibody reaction to cntn , cntn , caspr or nf in cell-based assays. among the cidp patients, two patients demonstrated reactivity against cntn with similar clinical presentation as previously described. none of the control patients had any antibody reaction to the performed tests. our results suggest that antibody responses to cntn , cntn , caspr or nf are absent in austrian gbs patients, although more patients will be screened to substantiate these preliminary results. furthermore, it remains to be established whether antibodies against cntn may predict a chronic course in acute onset inflammatory neuropathies. a few variants of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described with a frequency of - %. their relation and possible evolution into typical-cidp remain unclear, as is their treatment response possibly because of differences in diagnostic criteria. we used the data from a web-based database on italian patients with cidp to determine the frequency and characteristic of these variants, the possible evolution into typical-cidp, and their treatment response. all the patients were assessed at study entry and the disease course before inclusion was analyzed. by february- , we included patients ( men, women), aged - years (median years) with a mean disease duration of . years (range . - years) and complete data available from . based on the clinical data and our revised diagnostic criteria, patients ( %) were classified to have atypical cidp at onset and for the following two years including with dads ( %), with motor cidp ( %), with sensory cidp and cisp ( . %), with lewis-sumner syndrome ( . %), and with recurrent cranial neuropathy. at study entry, patients ( %) had progressed into typical cidp after - years (median years) while ( %, % of total) still had atypical cidp after . - years (median years) with a similar proportion of progression ( - %) within each group. the diagnosis of atypical cidp at entry fulfilled efns/pns criteria in ( %). csf studies were diagnostic in / ( %) patients, nerve biopsy in / ( %), and nerve imaging in / ( %) tested patients. similarly to typical cidp, % of treated patients with atypical cidp improved after treatment with a proportion of response varying from % to % in the different forms. most patients with sensory or motor cidp had however an unsatisfactory response to steroids. this study shows that he proportion of patients with atypical cidp varies during the course of the disease with almost % of the patients evolving into typical cidp within years from onset. in addition, response to treatment is frequent in atypical cidp even if not all the forms respond to the same therapies. only few studies investigated the frequency of antecedent events and comorbidities in patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and little is known on the role of possible predisposing factors, dietary, and lifestyle habits, on the onset and progression of the disease. we used the data from a web-based database on italian patients with cidp to determine the frequency of antecedent events and comorbidities and the possible role of predisposing factors including lifestyle and dietary habits and exposure to toxic agents, using a structured questionnaire. partners of patients served as controls. impairment was evaluated using the mrc sumscore and disability with incat and r-ods scales. logistic regression was used to calculate odds ratio (or) with % confidence interval (ci) for the risk of cidp. sex and disease-duration were included as covariates. by february- , patients were enrolled, with complete data on patients for antecedent events and comorbidities and patients and controls for lifestyle habits. ninety-two patients ( %) reported an antecedent event, mostly infection or vaccination ( %). one or more comorbidity were present in % of the patients including hypertension ( . %), thyroid disorders ( %) and diabetes ( . %) and in % influenced the choice of initial therapy. exposure to toxic environmental agents (odds ratio [or] = . ; % ci, . - . ), cigarette smoke (or = . ; % ci, . - . ), and dietary supplements (or = . ; % ci, . - . ) were associated with a higher risk of cidp while rice consumption was associated with a reduced risk (or = . ; % ci, . - . ). concerning disease severity, more severely affected patients more frequently consumed raw-meat (or = . ; % ci, . - . ) and white meat (or = . ; % ci, . - . ), while rice (or = . ; % ci, . - . ) and soft drink consumption (or = . ; % ci, . - . ) and physical activity were associated with lower disability (or = . ; % ci, . - . ). this study confirms that comorbidities are frequent in patients with cidp and often influence the choice of initial therapy. in addition preliminary data show that toxic exposure and some lifestyle and dietary habits may influence the onset and progression of cidp. doppler k , schuster y , weishaupt a , sommer c . department of neurology, university hospital würzburg, würzburg, germany. autoantibodies against the paranodal protein contactin- have recently been described in patients with cidp. in most patients, autoantibodies of the igg subclass are predominant and are supposed to be pathogenic. the role of igg anti-contactin- is so far unclear. in the present study, igg of three different patients, one with igg anti-contactin- , one with a low titer of igg anti-contactin- and one with a high titer of igg anti-contactin- , and of controls were injected into the sciatic nerves of lewis rats. nerve conduction studies of the injected nerve and motor and sensory testing were performed before and after injection. conduction blocks and motor deficits were detectable in the two patients with high titers of igg and igg , not in the patient with low titers. the percentage of conduction blocks was . % in rats injected with igg of the igg patient and % in those injected with igg . motor deficits were detectable in both patients with conduction blocks but were most apparent in the patient injected with igg of the igg patient. no differences in sensory testing were observed. conduction blocks and motor deficits improved after five days and were normal after seven to eight days. our data give the first evidence of pathogenicity of igg anti-contactin- autoantibodies, not only igg . igg of the igg patient induced a more severe clinical and electrophysiological phenotype compared to the igg patient. remarkably, this reflected the clinical phenotype of the patients, as the igg patients showed an acute-onset of sensorimotor symptoms at the time of blood withdrawal whereas the igg patient presented with a more chronic course of disease. doppler k , frank f , koschker a-c , reiners k , sommer c . department of neurology, university hospital würzburg, würzburg, germany; endocrinology and diabetes unit, department of medicine i, university hospital würzburg, würzburg, germany. axoglial dysjunction and paranodal demyelination have been discussed as potential mechanisms of nerve fiber damage in diabetic neuropathy. studies on human tissue are limited, as nerve biopsies are invasive and only rarely performed in patients with confirmed diabetic neuropathy. skin biopsy has recently been suggested as a good tool to analyze paranodal and nodal changes of myelinated fibers. in the present study, we analyzed the paranodal and nodal region in myelinated fibers of skin biopsies of patients with diabetic neuropathy, patients with diabetes mellitus without neuropathy, and normal controls. immunofluorescence of skin sections with antibodies against caspr, neurofascin, sodium channels and myelin basic protein was performed to assess paranodal/nodal architecture, segmental demyelination and myelinated nerve fibers. staining with antibodies against protein gene product . was used to quantify unmyelinated nerve fibers. we found an increase of elongated ranvier nodes and a dispersion of neurofascin at the distal leg in patients with diabetes mellitus with and without neuropathy and at the finger in patients with diabetic neuropathy. an increased dispersion of caspr was only found in biopsies of the finger in patients with diabetic neuropathy. our data show that skin biopsy is an appropriate tool to analyze nodes of ranvier in patients with diabetes mellitus. structural nodal changes are detectable in diabetic neuropathy, and even in diabetic patients without neuropathy. dourado me , fernandes u , vital al , ramos e , urbano jc , sena a , queiroz jw , jeronimo smb . federal university of rio grande do norte, natal, brazil. the erasmus gbs outcome score (egos) is a validated prognostic model that uses acute phase and easy-to-obtain clinical characteristics to determine outcome at months in patients with gbs. this study aims to assess the validity of egos in rio grande do norte, brazil, and to compare with another european study. data collected prospectively from a cohort of patients with gbs of rio grande do norte, brazil, between june and august , was assessed. ninety patients were excluded for missing data or diagnoses of miller fisher syndrome and atypical forms of gbs. to calculate the egos, the gbs disability score was assessed in the second week of disease and at months. to compare this study with the european one in independent group proportions, we used the student's t-test, being considered statistically significant p< , . the patients included were divided in four groups based on egos. thus, patients had egos between and ; had egos between . and . ; had egos and had egos between . and . in the first, second, third and fourth group, ( %), ( . %), ( . %) and ( . %) of the patients were unable to walk independently after six months of the disease, respectively. overall, of the patients analyzed, ( . %) had poor outcomes in this study. in the european paper, based on the same group division, of ( . %), of ( %), of ( %) and of ( %) were unable to walk independently. comparing both studies, the patients of this study were younger, more seriously ill in the first weeks and with more sensitive deficits. there were no difference relative to sex, cranial nerves deficits and presence of anti-gangliosides antibodies. using the student's t-test for ability to walk after months according to egos stratification, we achieve in the first group p= . ; in the second p= . ; in the third p= . ; and in the fourth p< . .the egos did not have a good capacity to predict the ability to walk after months of gbs in rio grande do norte, brazil. historically, guillain-barré syndrome (gbs) epidemics are rarely seen. between and , we treated and followed cases of gbs in the state of rio grande do norte with a yearly incidence of . / , . no seasonality was observed. the mean age of the patients was years (range, - ), with % of the cases younger than years. demyelinating variant was the most frequent subtype of gbs. in march , the first report of autochthonous transmission of zika virus (zikv) was determined in natal, brazil. later that month, we documented an increase in incidence of gbs in natal, brazil. the incidence in was of . / . . of the cases of gbs diagnosed in , were diagnosed from march through may, which coincided with the outbreak of zikv in natal, brazil. eighteen patients ( % of the cases) had a history of rash and fever prior to onset of gbs symptoms, with the median age of years (range, - ) . the electroneuromyography studies of these patients indicated that ( . %) had acute inflammatory demyelinating polyneuropathy, ( . %) had acute motor axonal neuropathy, and ( . %) was inconclusive. the mean time from onset of zikv infection symptoms to onset of the gbs were days (range, - ). the mean time of nadir was days (ranged, - ) . cranial neuropathies were present in patients ( . %). nine patients were bedridden ( %) and ( . %) required mechanical ventilation. the mean protein content of the central spinal fluid was . g/l, with the white blood cell count below /mm in all patients. they were all treated with intravenous igev. they all improved quickly. anti-gm was negative in all patients. rt-pcr was negative for dengue, chikungunya and zika. serum mac-elisa igm for zika and dengue was made in patients and it had % of positivity. prnt for zika and dengue had % positivity. in summary, we report a geographically and temporally defined cluster of gbs associated with an outbreak of acute rash in the state of rio grande do norte, brazil. as the prevalence of diabetes mellitus continues to increase worldwide, diabetic complications represent a growing burden to patients and society. distal symmetrical polyneuropathy (dsp) is a common complication that affects up to % of diabetic patients. dsp reduces patient quality of life due to chronic pain, ulcerations, and may lead to lower extremity amputations. despite its high prevalence, the mechanisms underlying diabetic dsp are poorly understood and several mechanisms are believed to play a role. we hypothesize that diabetic dsp arises from microvascular complications characteristic to diabetes. specifically, capillary dysfunction -disturbances in capillary flow patterns -is a likely candidate to explain development of dsp, as it can limit oxygen and nutrient delivery to nervous tissue, causing nerve dysfunction and damage, and thus development of dsp. we will study this hypothesis utilizing the state-of-the-art blood flow imaging techniques to visualize and quantify endoneureal blood flow and then link these findings with measures of dsp (e.g. nerve conduction velocity; intra-epidermal nerve fibre density) in animal models. we will include several animal models of diabetic dsp caused by either type or type diabetes. two photon microscopy and optical coherence tomography allow visualisation and quantification of capillary transit times and blood flow within peripheral nerves at high resolution. we hypothesise that changes in blood flow patterns and subsequent impairment of nutrient and oxygen delivery to nervous tissue precede the onset of diabetic dsp. if our experiments support this prediction, we will attempt to develop interventions that improve capillary blood flow to prevent or delay the development of dsp. duman o , saracoglu m , haspolat s , bozkurt o . department of child neurology, akdeniz university hospital, antalya, turkey. axonal polyneuropathies are very heterogeneous group of diseases which are very rarely seen during infantile age. some of them may be accompanied by developmental retardation, severe muscle weakness and progressive course. we aimed to present two cases of axonal sensorymotor neuropathy with infantile onset and atypical course. our -year-old boy patient was admitted to our clinic for progressive gait loss since one month. he was the first offspring of consanguineous parents with normal prenatal and natal history. electromyography revealed axonal sensorymotor polyneuropathy. metabolic and cerebrospinal fluid (csf) examinations for etiology were all normal. brain and spinal magnetic resonance imaging (mri) were normal. he had partial benefit from oral steroid treatment. in the course of disease along with four neuropathy attacks he had significant benefit from serial intravenous immunoglobulin treatments in two years clinical course. a month-old girl who is the first offspring of nonconsanguineous parents was admitted to the clinic for acute tetraparesis. axial sensorymotor polyneuropathy was detected in the electromyography. metabolic and cerebrospinal fluid (csf) examinations were normal. she had three more acute polyneuropathy attacks during ivig cessation period. both patients revealed with serial immunoglobulin treatments but unresponsive to riboflavine treatment. we aimed to discuss our rarely seen and the pathogenesis is not completely understood cases' course. serial ivig treatment may be helpfull for such patients' treatment. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominantly inherited disorder caused by mutations of the transthyretin (ttr) gene. the mutant amyloidogenic transthyretin protein causes the systemic accumulation of amyloid fibrils that result in organ dysfunction. ttr-associated fap is a progressive and fatal disease, if left untreated, and should be considered in the differential diagnosis of any person presenting with a progressive polyneuropathy, particularly with accompanying autonomic involvement. the clinical, electrophysiological, histopathological, and genetic characteristics of patients from turkey ( female, male) from eleven families with polyneuropathy and mutations in ttr were evaluated. two patients had no family history of ttr-fap and were considered as sporadic cases, and the remainders were familial cases displaying an autosomal dominant inheritance pattern. sequence analysis of the ttr gene revealed five mutations (p.val met, p.glu gln, p.gly glu, p.glu gly and p.gly glu). most common mutation was p.val met (in unrelated families). mean age at disease onset was . ± . years (range - years). the most commonly reported initial complaint was paresthesia in the feet (asymmetric in three patients). four patients ( male) with the p.glu gln mutation presented with carpal tunnel syndrome. two patients with the p.gly glu mutation showed episodes of dysarthria and hemiparesis, consistent with this genotype. seven patients died during the follow-up period as a result of systemic involvement. this study suggests that our cohort of ttr-fap patients from turkey exhibits clinical and genetic heterogeneity. ebenezer gj , liu y , judge dp , cunningham k , truelove s , carter nd , sebastian b , byrnes k , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; division of cardiology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. effect of amyloid deposition on cutaneous nerves was assessed in subjects with pathogenic ttr variants and control subjects. three groups of subjects each including ttr-fap patients, age/gender-matched healthy subjects and disease controls as well as ttr mutation carriers without neuropathy (ttr-nopn) and with al-amyloid underwent neurological examination and mm skin biopsies. micron sections were stained with anti-pgp . , anti-ttr and congo red. amyloid burden with imagej, intraepidermal (ienfd) sweat gland (sgnfd) and pilomotor densities (pmnfd) measured and correlations between amyloid burden, fiber subtype, neuropathy impairment score-ll (nis-ll) and nis sensory subscore were evaluated. ienfd, sgnfd, and pmnfd were all significantly reduced in ttr-fap patients vs. healthy controls while mutation carriers had intermediate reductions. lower nerve fiber densities were associated with nis-ll (p< . ). congo red staining revealed brilliant red amyloid deposits with apple-green birefringence within dermal collagen, sweat glands, and arrector pili muscles. amyloid infiltration was observed in the endoneurium and perineurium of small fiber sensory and autonomic nerves that innervate sweat glands and arrector pili muscles. cutaneous amyloid deposition was detected in % of ttr-fap and not in healthy or disease controls subjects. both al and / ttr-nopn subjects were congo red positive. amyloid burden was inversely correlated with ienf (p< . , r=− . ) sgnf (p< . , r=− . ), pmnf (p= . , r=− . ) distal leg densities, and correlated with nis-ll (p= . , r= . ) and nis sensory subscore (p= . , r= . ). wild-type ttr staining was less prominent in pathogenic ttr carriers. the diagnostic sensitivity and specificity to detect amyloid in skin were % and % in ttr-fap. the repeat measurement of the amyloid burden from the same section with imagej was r = . , p< . and different sections from the same biopsy was r = . and p< . . we conclude that endoneurial amyloid contributes to sensory and autonomic nerve injury. amyloid burden correlated strongly with sensory/autonomic axon densities and nis-ll. skin punches offer a convenient alternative to establishing a tissue diagnosis. amyloid burden is an attractive biomarker marker for ttr-fap and treatment effect. the study was supported through a grant from pfizer. ebenezer gj , truelove s , polydefkis m . department of neurology, johns hopkins university, baltimore, md, usa; department of epidemiology, johns hopkins bloomberg school of public health, baltimore, md, usa. we investigated differences of unmyelinated sensory nerve fibers in the distal limb among healthy-weight subjects (bmi < kg/m ) and overweight/obese subjects (bmi ≥ kg/m ), aged - years. subjects underwent neurological examination and mm skin punches from distal leg (dl), thigh (dt), and proximal thigh (pt) sites, from which micron sections were stained with anti-pgp . antibody; intraepidermal nerve fiber density (ienfd; fibers/mm) and epidermal thickness were assessed. a second dl biopsy was processed for electron microscopic examination and both thick and thin sections were examined for ultrastructural changes. multivariable linear regression models were used to assess the effect of age, gender, height and weight. after controlling for height, age, and obesity, females were found to have lower distal leg ienfd (− . ; p= . ). increasing age and height were significantly associated with decreasing dl ienfd, with decreases of − . fibers/mm per years (p<. ) and − . fibers/mm per cm (p< . ), respectively. even after controlling for height, being overweight/obese was associated with reduced dl ienfd, with . fibers/mm lower dl enfd than healthy-weight individuals (p=. ). these findings remained consistent across distal thigh and proximal thigh enfd, though not all associations remained significant. the epidermis was thicker in obese subjects across the lower limb, most pronounced at the distal leg (μm, mean± sd, healthy-dl: ± . , dt: . ± . , pt: . ± . , obese-dl: . ± . , dt: ± . , pt: . ± . ). under em very few intact dermal nerve bundles were identified at the proximal thigh sites. the atrophic and degenerating axons were seen with perineurial infiltration by dense collagen in obsess/overweight subjects but not age/gender matched controls. obesity further accelerates attenuation of epidermal nerve fibers across the lower limb even after controlling for other associated factors. echaniz-laguna a , geuens t , petiot p , péréon y , adriaenssens e , haidar m , capponi s , maisonobe t , fournier e , dubourg o , degos b , salachas f , lenglet t , eymard b , delmont e , pouget j , juntas morales r , goizet c , latour p , timmerman v , stojkovic t . strasbourg university hospital, strasbourg, france; peripheral neuropathy group, vib department of molecular genetics and institute born bunge, university of antwerp, antwerpen, belgium; lyon university hospital, lyon, france; nantes university hospital, nantes, france; hôpital de la pitié-salpétrière, paris, france; nice university hospital, nice, france; marseille university hospital, marseille, france; montpellier university hospital, montpellier, france; bordeaux university hospital, bordeaux, france. in this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins hspb and hspb and investigate the functional consequences of newly discovered variants. among unrelated patients with distal motor neuropathy, we identified mutations in hspb ( index patients/ ; . %) and hspb ( index patients/ ; . %) genes. patients have slowly progressive distal ( %) and proximal ( %) weakness in lower limbs, mild lower limbs sensory involvement ( %), foot deformities ( %), progressive distal upper limb weakness ( %), mildly raised serum creatine kinase levels ( %) and central nervous system involvement ( %). we found a broad range of disease onset with some patients presenting with foot drop at the age of years, and others presenting symptoms only after years. disease progression was slow in all patients, and even with a disease duration of more than years patients were still able to walk. none of our patients were wheelchair dependent. muscle pathology, nerve pathology and electrophysiology showed in all cases a slowly progressive, mostly symmetrical and predominantly distal motor axonal neuropathy. mild sensory involvement was observed upon nerve conduction studies, mostly the lower limbs, in % of cases. we identified hspb and hspb mutations, including respectively and not previously reported. transmission was either dominant ( %), recessive ( %) or de novo ( %). three missense mutations in hspb (pro ser, gly asp, gln arg) cause hyperphosphorylation of neurofilaments, while the c-terminal mutant ser leu triggers protein aggregation. two frameshift mutations (leu fs, ala fs) create a premature stop codon leading to proteasomal degradation. two mutations in hspb (lys met/asn) exhibited increased binding to bag . we demonstrate that hspb and hspb mutations are a major cause of inherited motor axonal neuropathy. mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins. eftimov f , querol l , rajabally ya and on behalf of all participants of the st enmc workshop. academic medical center, amsterdam, the netherlands; hospital de la santa creu i sant pau, universitat autònoma de barcelona, spain; aston university, birmingham, uk. although chronic inflammatory demyelinating polyneuropathy (cidp) is a treatable neuropathy further research is urgently needed to define the diagnostic clinical and electrophysiological boundaries of cidp and its subtypes, and to define the role of biomarkers in supporting the diagnosis, monitoring disease activity and predicting response to treatment and outcome. in recent years, several national registries and biobanks have been developed to enable systematic data collection in cidp. an international registry with large number of patients is needed to allow answering many important questions and develop validated prognostic models to predict outcome in individual patients with cidp. at the inflammatory neuropathy consortium (inc) meeting in , the inc members agreed that a european neuromuscular center (enmc) workshop would be the ideal setting to reach a consensus on the infrastructure of database and biobanks. the st enmc workshop will take place on may - , with participants representing different countries. primary objective of the workshop is to reach a consensus on inclusion and exclusion criteria, core sets and recommended sets of clinical data, diagnostic data and follow-up points and a manual of operations for collection of biomaterials. a secondary objective is to construct an infrastructure to allow sharing data between different databases and biomaterials. conclusions of the consensus meeting and outline of further perspectives will be presented at the peripheral nerve society meeting in . eichinger kj , burns j , cornett k , bacon c , shepherd m , mountain j , sowden j , shy r , shy me , herrmann dn . clinical outcome assessments that measure functional ability are important endpoints for clinical trials. dr. burns has led the development/validation of a functional outcome assessment (cmtpeds) for individuals with charcot marie tooth disease (cmt) ages - years in the inc rdcrn. the cmtpeds is reliable and sensitive to change. however a validated functional outcome measure (fom) for adults with cmt is needed. our data in - year-olds indicated that the cmtpeds could be modified for adult use. however, some items of the cmtpeds (e.g. balance beam and jumping) have floor effects in adults with cmt. we have developed an adult cmt-fom modeled on the cmtpeds, and refined based on literature review, patient interviews, a large-scale cmt patient survey and expert opinion. the cmt-fom is a performance-based scale comprising items that are combined to form a composite score to quantify functional ability of adults with cmt. the cmt-fom shares items with the cmtpeds. four items were added to measure functional abilities relevant to adults (sit to stand, meter walk/run, stair climb, and timed up and go test). the cmt-fom scoring mirrors the cmtpeds. to generate a score ranging from - , raw item scores are converted to z scores, based on age-and sex-matched normative reference values form the norms project and categorized to a - likert along a continuum of impairment levels. we have conducted a pilot study of the cmt-fom in adults with cmt a ( male, female, age . ± . yrs) of differing severity (cmt exam score (cmtes) range - ). the cmt-fom is feasible, individuals were able to complete all items, and takes minutes to perform. the mean cmt-fom score was . ± . (range - ). concurrent validity of the cmt-fom is supported by an association with the cmtes (r = . ). the overall score did not demonstrate floor or ceiling effects. in summary the adult cmt-fom is well-tolerated and captures upper and lower limb strength, dexterity, balance, speed, ambulation and endurance. the cmt-fom requires validation in a large longitudinal cohort, prior to application in clinical trials. estilow t , glanzman am , burns j , cornett kmd , menezes mp , shy r , moroni i , foscan m , pagliano e , pareyson d , laurà m , bhandari t , muntoni f , reilly mm , finkel rs , sowden j , eichinger k , herrmann dn , shy me , yum sw , ramchandren s and on behalf of the inherited neuropathies consortium. cmt is associated with progressive impairment of the hands. reducing this impairment by treating children who are in the early stages of the disease is crucial. studies assessing measures of cmt hand function in children and their associations with patient-reported outcomes are lacking. we analyzed the upper extremity items from the cmt pediatric scale (cmtpeds) and pediatric cmt quality of life scale (pcmt-qol) in children ages - years enrolled in the inherited neuropathies consortium, to explore the relationships between measures of hand function (impairment, activity, and activities of daily living), and patient-reported outcomes. weak grasp ( %), hand pain ( %), and tremor ( %) were prevalent impairments. performance on activity level tasks, the nine hole peg test ( hpt) and functional dexterity test (fdt), were impaired in % and % of the cases, respectively. patients reported difficulty "sometimes" to "always" in opening a jar/lid ( %), zipping/buttoning ( %), writing ( %), carrying a plate without spilling food ( %) and putting on shoes ( %). patients reporting tremor showed significant differences on the hpt (p=. ). grip strength was shown to have a moderately significant correlation with performance on the fdt (r=. ; p<. ). stepwise multiple linear regression showed that grip strength (beta=−. ; p<. ), hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to open a jar (adjusted r =. ; p<. . grip strength (beta=−. ; p<. ), and fdt (beta . ; p<. ) were predictive of ability to carry plate without spillage (adjusted r =. ; p<. ). grip strength (beta=−. ; p<. ), hpt (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to put on shoes (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and fdt (beta . ; p<. ) were predictive of ability to zip/button (adjusted r =. ; p<. ). hand pain (beta=. ; p<. ) and hpt (beta . ; p<. ) were predictive of ability to use a pen/pencil (adjusted r =. ; p<. ). children with cmt present with frequent limitations in adl performance impacting qol. the upper limb measures of the cmtpeds are associated with hand performance and interventions to improve grip strength and reduce pain should be investigated further with respect to their impact on improving function, and ultimately qol. evans me , morrow jm , wastling s , sinclair cdj , fischmann a , shah s , emira ak , hanna mg , yousry ta , thornton js , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; neuroradiological academic unit, ucl institute of neurology, london, uk; university of basel hospital, basel, switzerland. responsive outcome measures are needed in charcot-marie-tooth disease (cmt) to allow adequately powered clinical trials to test novel therapeutics. we have shown high responsiveness of mri quantified intramuscular fat accumulation in calf muscles of cmt a patients over months. the aim of the present study was to assess the responsiveness and longitudinal validity of quantitative mri over a year follow-up period. we undertook two further sets of quantitative mri, myometric and clinical assessments in the original mrc centre cmt a quantitative mri cohort. mri sequences included fat quantification using the point dixon fat-water separation method, t quantification and magnetisation transfer imaging. of the patients with genetically confirmed cmt a were assessed at baseline ( male, mean age . ± . years), underwent repeat assessments a median on months (data already published), underwent repeat assessments at a median of months, and underwent a final assessment at a median of months. the primary outcome measure currently being analysed is mean calf muscle fat fraction at a single axial slice a fixed distance distal to the knee joint. results of this analysis and correlation with clinical measures will be presented at the peripheral nerve society meeting. fainmesser y , dori a , , drory ve , . department of neurology and neuromuscular service, tel-aviv medical center, tel-aviv, israel; department of neurology, sheba medical center, ramat gan, israel; sackler faculty of medicine, tel aviv university, israel. the use of anabolic drugs by those who wish to increase lean body mass is widespread and not well supervised medically. a years old man was referred for evaluation of a slowly progressive sensory and motor disturbance in the distribution of the left ulnar nerve. his symptoms began months after repeated self-injections of anabolic steroids and vitamin e into the biceps and triceps brachii muscles bilaterally. his examination showed increased muscle mass of the injected muscles with a hard-rubbery consistency, atrophy and weakness of left interossei, mild weakness of the bilateral biceps brachii and sensory loss in an ulnar nerve distribution. nerve conduction studies showed a left ulnar neuropathy with reduced motor and sensory response amplitudes and denervation in the left first dorsal interosseus, as well as mild myopathic changes with significantly reduced insertional activity in both biceps muscles. mri of the soft tissues of the arms showed massive fibrosis and infiltration of fat in the arm muscles compressing the ulnar and median nerves on the left. neurolysis of the left ulnar nerve was performed, and the ulnar nerve was found enclosed in a fibrotic mass throughout the entire length of the upper arm. the brachial artery and the median nerve were similarly enclosed in fibrotic tissue and were released. a biopsy of the affected muscles showed muscle necrosis and fibrosis. the patient was treated with physiotherapy and losartan with only mild improvement in the consistency of the muscles, but without clinical improvement in ulnar nerve function, and worsening of nerve conduction. this case illustrates severe peripheral nerve damage due to entrapment in massive muscle fibrosis following improper intramuscular injection of anabolic steroids for cosmetic purposes. fabrizi gm , testi s , høyer h , braathen gj , squintani g , bertolasi l , ferrarini m , taioli f , cabrini i , pancheri e , cavallaro t , tonin p . department of neuroscience, biomedicine and movement, university of verona and department of neuroscience, aoui verona, italy; section of medical genetics, department of laboratory medicine, telemark hospital, skien, norway. inherited diseases of nerve and muscle may overlap phenotypically or coexists as facets of the same disorder. two families whose probands were initially diagnosed with a lower motor-neuron (lmn) syndrome and a hereditary distal motor neuropathy (dhmn) turned out to represent a vcp (valosin-containing protein)-related syndrome and a gne (udp-n-acetylglucosamine -epimerase/n-acetylmannosamine kinase)-related "distal myopathy" . both conditions shared the presence of rimmed vacuoles (rv) in muscle biopsies. in family , the year-old male proband had a lmn syndrome manifesting at age years (wasting/weakness, cramps, fasciculations of thigh muscles, later involving the distal upper limbs). his year-old brother had lower limb weakness and diffuse pain in bones/joints since age . the father was diagnosed with a "muscular dystrophy" thirteen years before dying a years; by age years he had developed a behavioural frontotemporal dementia (ftd). electrodiagnosis (edx) disclosed myopathic changes with ongoing denervation together with a mild sensory-motor axonal polyneuropathy in the proband, and a chronic sensory-motor axonal polyneuropathy in the brother. mri showed fatty replacement of weakened muscles in both siblings and diffuse changes of bones consistent with paget disease (pd) in the elder sibling. in family , a year-old african woman was affected by weakness and wasting starting at the distal lower-limb muscles at age years and soon progressed proximally; parents were not consanguineous and two sisters out of six siblings, deceased in their thirties for post-partum complications, had a similar disease. edx mainly disclosed a neurogenic process with ongoing denervation. muscle biopsies from the three family- patients and from the family- proband showed a myopathy with rv. next-generation sequencing demonstrated a heterozygous c. c>t change of vcp leading to a known pathogenic p.arg cys substitution in all family- patients, and two compound heterozygous c. g>a and c. g>a changes of gne in the family- proband leading to known p.ala thr and p.ala thr substitutions. vcp is known to cause autosomal dominant amyotrophic lateral sclerosis- , charcot-marie-tooth disease type y or inclusion body myopathy (ibm) with pd and ftd (ibmpfd); gne causes the autosomal recessive nonaka myopathy (alias ibm ). both cases emphasize the clinical and neurophysiological heterogeneity of those disorders. fargeot g , vandendries c , labeyrie c , viala k , theaudin m , adams d . neurologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neuroradiologie, crmr nnerf, aphp, filnemus, chu bicêtre, le kremlin-bicêtre, france; neurologie, groupe hospitalier universitaire pitié salpêtrière, paris, france. the diagnosis of cidp is often challenging, especially when electrophysiological signs of demyelination are lacking. plexus mri has documented nerve abnormalities in small series of typical cidp but its contribution in patients with no electrophysiological signs of demyelination remains to be proved. we report the results of plexus mri in a serie of patients suspect of having cidp without efns pns definite electrophysiological criteria. we did a retrospective study of patients consulting in kremlin bicetre and pitié salpétrière hospital. lumbar or brachial plexus mri (or both) were performed and we assessed nerve trophicity, t -stir signal intensity and gadolinium enhancement as well as the topography of abnormalities. a consensus diagnosis was made by a group of experts (based on clinical data and other supportive criteria) and allowed us to classified patients in "cidp" or "other diagnosis". the practical contribution of plexus mri to the diagnostic algorithm has been studied. diagnosis of cidp was made in patients. mri was abnormal in % of cidp patients and showed nerve roots hypersignal/hypertrophy/enhancement in respectively . / . / . % of patients. the pattern of abnormalities was often asymetrical ( . %), diffuse ( . %) or multifocal ( . %). after unblinding, the mri confirmed the diagnosis of experts in . % of patients and changed the diagnosis in % of patients. plexus mri has shown to be useful in our serie to confirm the diagnosis of experts or to modify it in patients ( %) . the "classical" pattern described in definite cidp (diffuse nerve root hypertrophy and hypersignal) was documented in % of our cidp patients whereas less typical pattern (focal or multifocal abnormalities, hypersignal without hypertrophy) was found in %. plexus mri seems usefull when facing patients suspected of having cidp when electrophysiological criteria are not met: both symetrical diffuse or asymetrical multifocal patterns can be found and should be always correlated to the clinical examination and other supportive criteria. the specificity of such abnormalities remains to be studied. feely sme , rebelo a , abreu l , tao f , bacon c , zuchner s , shy me . university of iowa, iowa city, ia, usa; dr. john t. macdonald department of human genetics and hussman institute for human genetics, miller school of medicine, university of miami, miami, fl, usa. mutations in bcle-associated athanogene (bag ) have been shown to cause a distal myofibrillar myopathy and cardiomyopathy that can severely affect children or only affect adults depending upon the particular mutation. children with severe cardiomyopathy and myopathy have also developed axonal peripheral neuropathy, consistent with the known localization of bag in neurons as well as in muscle. we have identified two large autosomal dominant families with adult onset charcot-marie-tooth disease (cmt ) with the identical novel missense mutation pro ser, a codon previously shown to cause severe or mild myopathy depending on the amino acid substitution. these families expand the phenotypes caused by mutations in bag to include cmt and provide an additional example of adult onset cmt that may previously have been diagnosed as chronic idiopathic axonal neuropathy (ciap). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. myelin protein zero (mpz), expressed only by myelinating schwann cells, has sequence alterations that have previously been reported to cause demyelinating, intermediate, and axonal forms of charcot marie tooth (cmt) disease. we describe a rare duplication in mpz which is causing an early onset, demyelinating form of cmt in our patient. patient was a product of a normal pregnancy and delivery. early milestones were delayed. she began walking at months of age. she was not able to keep up with her peers and was the slowest runner. she started wearing smos at years of age and afos by years of age. she was diagnosed with scoliosis at years of age and started wearing a brace at years. her examination at years of age showed that her fdi, apb, and adm were all / bilaterally. weakness in her lower extremities included / foot eversion and / great toe dorsi flexion. she had tight heel cords bilaterally. pinprick sensation was reduced throughout in her upper and lower extremities and absent at her toes bilaterally. vibration sensation with a rydel tuning fork was absent at her toes and ankles and reduced at her knees and fingers. nerve conduction studies were performed and revealed no responses in all sensory nerves tested with the exception of the radial nerve which had normal latency and mildly slowed conduction velocity ( m/s). prolonged latencies, demyelinating range slowing (between - m/s), and low cmap amplitudes in almost all segments of the median and ulnar motor nerves were also observed. these findings were consistent with a hereditary sensorimotor demyelinating polyneuropathy. she was diffusely areflexic and her total cmt pediatric score (cmtpeds) was / which is in the severe range. her cmt neuropathy score (cmtns) was / in the moderate range. the duplication of mpz has previously been identified as a rare cause of cmt b. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). feely sme , saade d , shy me . carver college of medicine, university of iowa, iowa city, ia, usa. mutations in egr cause a severe, demyelinating form of cmt, cmt d. we describe a novel mutation in egr which led to extreme variability in severity in a family. proband was a year old girl who was the product of a normal pregnancy and delivery. early milestones were on time. problems with walking started at years of age. at . she was unable to use stairs, run, or jump. at she was wearing bilateral afos. by she was using a wheelchair and started breathing assist at night. she lost arm lift but could still hold a pen. by she could not ambulate independently and breathing assist was required day/night. she lost the ability to write and developed a head drop. by years she could not sit up. on exam her head and neck muscles were / . upper limbs, deltoids, biceps, wrist ext/flex, finger ext, fdi, and adm were / bilaterally. triceps were / ; finger flexors and apb were / bilaterally. lower limbs were / and she had contractures on the right. sensory examination was normal. she was diffusely areflexic. ncvs were absent. both the cmtns and cmt pediatric score were severe at / and / . exome sequencing revealed a r l variant in egr which was likely pathogenic. the probands' mother also had this mutation. she had no reported symptoms at the age of with strength at / throughout with the exception of left foot eversion which was +/ and great toe dorsi flexion which was -/ bilaterally. sensory exam showed a decrease of vibration and pinprick sensation at left toe. she had a normal gait, tandem gait and could toe walk, but not heel walk. she was diffusely areflexic. ncvs showed absent sensory responses. motor ncvs showed her latencies were prolonged and velocities reduced ( - m/s). cmtns was mild ( / ). additional family members who had the r l mutation were evaluated including maternal grandmother who was moderately impaired ( / ) and maternal aunt who was severe ( / ). no other mutations that could cause another known neuropathy or myopathy were identified and mitochondrial sequencing was normal. inherited neuropathy consortium is part of the nih rare diseases clinical research network (grant# u ns - ). fisgun a , luan x , hoke a . johns hopkins university, baltimore, md, usa. molecular mechanisms that underlie slow distal axonal degeneration seen in chemotherapy induced peripheral neuropathy (cipn) are unclear. however, several identified molecular targets suggest shared mechanisms with wallerian degeneration. since spontaneous mutation in wlds mice and genetic deletion of sarm gene lead to slow wallerian degeneration, we asked if wlds or sarm knockout (ko) mice are resistant to distal axonal degeneration induced by several chemotherapy agents. we chose chemotherapeutic drugs from different classes of agents, paclitaxel (taxane), cisplatinum (platin-based drugs) and bertozomib (proteasome inhibitor), to model cipn in mice. primary outcome measure was evaluation of epidermal nerve fibers in the hind paw plantar footpads. secondary outcome measures included thermal sensation and nerve conduction studies. sarm ko mice were almost % protected against development of sensory neuropathy but the protection in wlds mice was partial. this study confirms the pivotal role sarm plays in mediating axonal degeneration and identifies inhibition of sarm activity as a potential therapeutic target for prevention of cipn. florio f , scapin c , ferri c , feltri ml , wrabetz l , d'antonio m . myelin biology unit, san raffaele scientific institute, milan, italy; hjkri-university of buffalo, ny, usa. myelin protein zero is the most abundant structural protein in myelin of the pns. in humans, more than mutations in p are associated with hereditary neuropathies. deletion of serine causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in mice (wrabetz et al., ) . p s del protein is misfolded and is retained in the er where it gives rise to a dose-dependent unfolded protein response (upr) (pennuto et al., ) . the upr results in the activation of transcriptional and translation control programs that reduce protein synthesis and increase the folding and degradative capacity of the cell. usually, when this first response is not sufficient the cells may activate apoptosis resulting in cell death. however, in p s del schwann cells there is no cell death suggesting that these cells may respond differently to chronic stress. transcriptomic analysis performed on p s del nerves showed increased expression of transcription factors normally present only in the early phases of differentiation such as c-jun, sox and id (d' antonio et al. ) . in order to understand the role of the expression of these factors in the peripheral nerve myelination we used ex vivo and in vivo approaches and we showed that sox and id act as negative regulators of myelination. these results suggest that the expression of sox and id may contribute to the hypomyelination observed in p s del mice. as such, we reasoned that their ablation could ameliorate the phenotype. surprisingly, the ablation of these factors in the p s del mouse severely worsens the neuropathy bursting schwann cell differentiation and increasing the expression of both p wild type and mutant allele with concomitant exacerbation of the upr. this suggests that the overexpression of early differentiation factors in schwann cell under chronic er-stress is an adaptive mechanism that limits differentiation and reduces the expression of toxic proteins. intravenous immunoglobulin (ivig) is the treatment of choice for the guillain-barré syndrome (gbs). the working mechanism of ivig in gbs is undefined, but most likely all potential effects are dose dependent. the pharmacokinetics (pk) and pharmacodynamics (pd) of ivig in gbs are highly variable between patients and a rapid consumption or clearance of ivig is associated with poor recovery. in the current study we developed a model to predict the pk of a standard dosage of ivig ( . g/kg for consecutive days) in individual patients with gbs. non-linear mixed-effects modelling (nonmem) was used to construct a model based on a cohort of gbs patients, with a total of sequential serum igg levels. the final model accurately describes the day to day increment in igg levels during the -day course and the initial rapid fall and graduate decline to steady-state levels thereafter. we explored several potential covariates that improved the predictive capabilities and decreased the between-subject variation in the model. the model including these covariates were evaluated successfully (bootstrap analysis) and through numerous simulation studies each based on (simulated) gbs patients. in conclusion, a first accurate and robust nonmem model for the pk/pd of standard ivig treatment in gbs was developed. the model can be used to predict the pk in individual patients applying a few simple baseline characteristics. in addition, the effect of different treatment regimens of ivig in gbs on a population pk/pd level can be simulated. this modeling technique is a new tool to optimize the pk in individual patients and the study design for new trials with ivig in gbs. forese mg , pellegatta m , rivellini c , podini p , quattrini a , previtali sc , taveggia c . axonal neuregulin (nrg ) type iii is an essential instructive signal for peripheral nervous system (pns) myelination, as its expression determines whether axons are myelinated as well as the thickness of the myelin sheath. we recently demonstrated that gamma-secretase cleavage of nrg type iii generates an axonal intracellular fragment, which translocates in the nucleus to upregulate the expression of the prostaglandin d synthase (l-pgds) gene in neurons. l-pgds catalyzes the conversion of prostaglandin h into prostaglandin d (pgd ). we also showed that specific inhibition of l-pgds activity impairs in vitro myelination. accordingly, myelin in l-pgds null mice is noticeably thinner, thus indicating that l-pgds is a new modulator of developmental pns myelination. previous studies have shown that prostaglandins are involved in the process of wallerian degeneration (wd) and axonal regeneration after injury. thus, to determine whether l-pgds and pgd could be important in pns regeneration and remyelination, we performed sciatic nerve crush injury in months old l-pgds null and wild type control mice and we analysed nerves by morphologic, biochemical, histological and molecular approaches at different time points (t) after crush. we focused on three phases: degeneration (t -t ), axon regeneration (t -t ) and remyelination (t ). our results indicate that in l-pgds null mice the amount of myelin proteins synthesized after crush as well as the number of remyelinated fibers do not change, suggesting that l-pgds might be dispensable for remyelination. however, we observed an increased number of macrophages in null nerves during regeneration (t ), possibly as a consequence of an increase in the blood-nerve barrier (bnb) permeability, indicating potential alteration in the regeneration process in l-pgds null mice. these results suggest that l-pgds could have a different role in developmental pns myelination and after injury. whether other prostaglandins and synthases might compensate for l-pgds activity is currently under investigation. fornasari be , , raimondo s , , ronchi g , , crosio a , budau ca , el soury m , muratori l , , tos p , battiston b , geuna s , , gambarotta g . department of clinical and biological sciences, university of turin, italy; neuroscience institute cavalieri ottolenghi, turin, italy; microsurgery unit, health and science city, cto, turin, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. to repair nerve gaps following severe peripheral nerve injuries, chitosan tubes were proved to give good results, comparable with those obtained with nerve autografts, the gold standard technique. to further improve peripheral nerve regeneration using chitosan tubes, a conduit enrichment strategy was developed using longitudinal skeletal muscle fibres, which have been previously shown to be good fillers in the "muscle in vein" experimental paradigm, where they played a trophic and a structural role. to this aim, rat median nerve gaps were repaired using two different conduits: mm chitosan tubes filled with a longitudinal piece of pectoralis major muscle ("muscle in tube") and hollow chitosan tubes. samples were harvested at early time points ( , , , days) for biomolecular and morphological analysis, and later ( months) for stereological analysis. autologous nerve grafts were used as gold standard positive control in the early time points. biomolecular analysis carried out on in vitro degenerating muscle and on "muscle in tube" at early time points show that the muscle produces high levels of soluble isoforms of neuregulin , a key factor for schwann cell survival and activity, usually released by schwann cells after nerve injury. functional assay and stereological analysis carried out on the distal part of regenerated nerve months after nerve repair, show no significant differences in the regeneration outcome between hollow chitosan tube and "muscle in tube" groups. therefore, we conclude that for short gaps (≤ mm), both hollow chitosan tube and "muscle in tube" are good techniques to repair nerve defects and we suggest that the "muscle in tube", which spontaneously releases neureg-ulin , might be a promising strategy to promote regeneration when the gap is longer or the repair is delayed in time. foucquier j , bertrand v , jouve e , truillet r , mandel j , laffaire j , blin o , magy l , lehert p , , hajj r , guedj m , cohen d , attarian s . pharnext, issy-les-moulineaux, france; aix marseille université, aphm, marseille, france; hôpital dupuytren, limoges, france; university of melbourne, melbourne, vic , australia; faculty of economics, louvain, belgium. charcot-marie-tooth disease type a (cmt a) is a rare disease belonging to a group of inherited, progressive, chronic motor and sensory peripheral neuropathies. the charcot-marie-tooth neuropathy score (cmtns) (shy et al., ) and the overall neuropathy limitations scale (onls) (graham & hughes, ) are considered as the main clinical scales for evaluating progression of disability associated with cmt. as cmt a is a slowly progressive neurodegenerative disease, the choice of endpoints and their ability to monitor small changes over time remain a major concern for clinical drug development. with this in mind, we studied a cohort of french cmt a patients with a follow-up ranging from months to . years resulting from the merge of two multicentre clinical trials (micallef et al., ; attarian et al., ) and a non-interventional study (unpublished) . the sensitivity to change of both cmtns and onls were assessed using a mixed effect model estimating annual progression with time in years, cmtns or onls baseline value as covariates, study centre as a fixed factor and patients as a random effect to account for the repeated measures. disease progression was estimated to be + . points per year on the cmtns (p = . ) and + . points per year on the onls (p = . x − ), both corresponding to a deterioration of impairment and disability. while both endpoints have similar and favourable properties, our set of observations led us to conclude that the onls could be more promising to monitor disease progression in cmt a. frasquet m , , lupo v , mas f , , vílchez r , chumillas mj , , espinós c , sevilla t , , . hospital universitari i politècnic la fe, valencia, spain; instituto de investigación sanitaria la fe, valencia, spain; centro de investigación príncipe felipe, valencia, spain; eresa, valencia, spain; centro de investigación biomédica en enfermedades raras (ciberer), valencia, spain; departamento medicina, universitat de valencia, valencia, spain. mutations in the bicd gene are a cause of dominant spinal muscular atrophy, lower extremity predominant (smaled). we report six patients belonging to three spanish families who carry three different novel mutations in the bicd gene. we describe clinical, electrophysiological and magnetic resonance imaging (mri) data. we provide results of muscle biopsy of one patient and skin biopsy for the study of epidermal nerve fiber density (enfd) of other two patients. genetic diagnosed was reached using a gene panel for genetic testing of cmt and dhmn. three novel mutations in the bicd gene that segregated with the disease were detected: p.val gly; p.tyr his and p.s l. the most frequent clinical phenotype consisted of mild weakness in proximal muscles of lower limbs combined with foot deformities. one patient had prominent sensory symptoms and abnormalities on sensory examination. other two patients had minor sensory abnormalities on examination. in one patient sensory and motor nerve action potentials were reduced, in the rest of patients electrophysiological studies showed normal motor and sensory nerve responses, with chronic denervation predominantly in muscles of lower limbs. mri studies at the level of tight and calf were performed in all patients. the most affected muscles were rectus femoris, vastus lateralis and medial gastrocnemius. mri studies at the level of feet were obtained from five patients and showed that there was not fatty infiltration in intrinsic foot muscles. mri at the level of pelvis muscles performed in four patients showed marked fatty infiltration of gluteus medius muscle in two of them. muscle biopsy performed in one patient showed myopathic features. skin biopsy was performed in two patients of the same family. in the older patient, who had minor sensory abnormalities on examination, there was a marked reduction of enfd that followed a length-dependent pattern. in conclusion, we report three new pathogenic mutations in the bicd gene. in our study we include mri findings at the level of pelvis and feet, which allow us to better define the pattern of muscle involvement related with this gene. our results also raise the subject of a possible sensory involvement in the disease. hereditary neuropathy with liability to pressure palsies (hnpp) is an autosomal dominant disorder that usually results from deletions in the pmp- gene. the neuropathy is unique in that it manifests with recurrent mono-neuropathies at common sites of compression. while spontaneous recovery from episodes of nerve injury usually occurs, it is often incomplete, and over time patients may develop a length dependent polyneuropathy. given the relapsing/remitting nature of hnpp symptoms, standard clinical scores, such as the cmt neuropathy score, are not effective at capturing the severity or progression of the disease. a specific tool is therefore needed for measuring clinical severity of hnpp in preparation for emerging clinical trials. in the current study, we evaluate a new pilot measure, the hnpp severity score (hnpps). the score is composed of patient reported questions addressing current and prior sensory and motor symptoms, and the impact of symptoms on quality of life, followed by items based on a motor examination. total scores vary from - , with higher scores indicating increased disease severity. in this study, the hnpps was administered to patients with genetically confirmed hnpp at the ucl institute of neurology. subjects included males and females with a mean age of years (+/− , range - ). the mean hnpps was . points (+/− . , range - ) and the data did not demonstrate major skew. the cronbach alpha for the hnpps was . , and items based on the physical examination showed the least variability. a modest correlation was observed between the hnpps and the cmt examination scores (pearson correlation . , ci . - . ). we conclude that the hnpps may be a useful measure of clinical severity in hnpp, and should be refined in larger patient cohorts. fridman v , sillau s and on behalf of the inherited neuropathies consortium (inc) . university of colorado hospital, aurora, co, usa; university of iowa hospitals and clinics, iowa city, ia, usa. the most common of the hereditary neuropathies (hn) is cmt a, an autosomal dominant demyelinating neuropathy that results from duplications in the pmp- gene. recent advances in defining the pathomechanisms of the disease have led to an increasing number of potential therapies; however, the absence of reliable natural history data and the paucity of sensitive clinical outcome measures have been barriers to effective clinical trials. the charcot marie tooth neuropathy score (cmtns) was developed to quantify impairment and measure progression in hn. it was observed that while the score discriminates well between mildly and severely impaired patients, it tends to cluster together patients in the middle range of severity. to improve the score's sensitivity, rasch analysis-based weighted category responses were developed. we report a longitudinal study of weighted cmtns and cmt examination scores (cmtes) over a three-year time frame in patients with cmt a. baseline, one year, two year and three year wcmtnsv /wcmtesv scores were available for / , / , / and / patients respectively. mean wcmtns (sd)/wcmtes (sd) scores were as follows: . ( . )/ . ( . ) at baseline, . ( . )/ . ( . ) at one year, . ( . )/ . ( . ) at two years and . ( . )/ . ( . ) at three years. a mixed regression model showed significant change in wcmtns and wcmtes at years (mean change from baseline at years was . points (p=. ) for wcmtns and . points (p=. ) for wcmtes. significant change as compared to baseline was also seen at years (mean change from baseline . points (p= . ) for wcmtns and . points (p= . ) for wcmtes. we conclude that weighted cmtnsv scores show change over the first three years of the inherited neuropathies consortium natural history study and are a helpful measure of progression in cmt a. fridman v , novak p , david w , macklin ea , mckenna-yasek d , walsh k, oaklander al , brown r , hornemann t , eichler f . massachusetts general hospital, boston, ma, usa; university of massachusetts medical school, worcester, usa; university hospital zurich, zurich, switzerland. hsan is an autosomal dominant, severe sensory motor polyneuropathy caused by mutations to serine palmitoyl-coa transferase. mutations shift the substrate preference from serine to alanine leading to formation of neurotoxic -deoxysphingolipids ( -deoxysl). treatment with high-dose l-serine has been shown to reduce -deoxysl accumulation and improve neuropathy in transgenic hsan mice. we report a two-year, delayed-start, placebo-controlled clinical trial evaluating the safety and efficacy of oral l-serine in hsan . eighteen hsan subjects were equally randomized to l-serine ( mg/kg/d) or placebo for year. at -weeks, the placebo group crossed-over and all participants took open-label l-serine for one additional year. sixteen subjects completed their -week visit, and no serious adverse events related to l-serine were reported. participants randomized to l-serine experienced a decline in charcot marie tooth neuropathy scores (cmtns) over year relative to placebo (− . units, % ci − . to − . , p = . ). both groups improved in the second year of the study, with a diminished difference in cmtns at weeks (− . units, % ci − . to . , p= . ). skin biopsies from the distal leg site were largely devoid of intra-epidermal nerve fibers (ienf), but at year, a greater increase in ienf density was seen in participants on l-serine versus those on placebo (median change of vs. fibers/μm , p= . ). -deoxysl levels declined among participants on l-serine versus those on placebo after one year of treatment ( % decrease in deoxysphinganine vs. % increase on placebo, p < . ), and placebo participants experienced similar declines in -deoxysl levels after crossing over to l-serine. we conclude that l-serine is a safe and potentially efficacious treatment for hsan . fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the relationship between guillain-barré syndrome (gbs) and malignancy is uncertain. under the diagnostic criteria of paraneoplastic neurological syndrome (pns) by euronetwork, , neuropathy with no definite onconeural antibodies identified due to gbs has been classified as "non-classical" paraneoplastic disorder. we retrospectively analyzed data of consecutive patients admitted with gbs from birmingham, u.k. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to calculate the relative cancer risk using different definitions and determined characteristics of malignancy-associated gbs. patients were classified according to definitions: ) all cases of malignancies excluding preceding diagnoses > year before gbs and with no evidence of malignant disease activity at the time of gbs diagnosis, and ) malignancies diagnosed > years post-gbs onset as per pns criteria. total number of gbs patients with malignancy was ( . %). a total of patients ( . %) fulfilled requirements for inclusion as malignancy-associated gbs as per existing criteria for pns. associated malignancy consisted of angioimmunoblastic t-cell lymphoma ( ), poorly differentiated squamous cell carcinoma of nasal septum ( ), gastric adenocarcinoma ( ), hepatocellular carcinoma due to hepatitis b ( ), rectal carcinoma with liver metastasis ( ) and myelodysplastic syndrome ( ). malignancy was globally commoner in our gbs cohort compared to the general population (odds ratio: . ; ci: . - . ; p = . ). however, this was unconfirmed if paraneoplastic criteria were applied. gbs patients with cancer were significantly more likely to be older (p = . ), hyponatremic (p = . ) and demonstrate more electrophysiological axonal loss (p < . ). cerebrospinal fluid (csf) protein levels were lower in the malignancy group (p = . ) and neurological improvement was less likely (p = . ). in-patient mortality was significantly higher in patients with malignancy (p < . ). none of the patients in the malignancy group had positive anti-ganglioside antibodies or anti-neuronal antibodies (anti-hu, yo, ri, crmp ). we conclude global cancer risk is higher in gbs than in the general population, although definition-dependent. application of the strict published criteria for paraneoplastic syndrome reduced the number of cases and suggested absence of a link. malignancy requires consideration in elderly, hyponatremic subjects with normal csf protein, severe electrophysiological axonal loss who fail to improve post-treatment. fu liong h , aude-marie g , anne-catherine an , emilien d , julien c , shahram a , yusuf r . anti-myelin associated glycoprotein antibody (anti-mag) neuropathy is a slowly progressive neuropathy resulting in disability through distal sensory more than motor deficits and tremor. there are currently no evidence-based treatments for anti-mag neuropathy and the effect of the disease on quality of life (qol) in this patient population is unknown. our objective was to assess determinants of qol in patients with anti-mag neuropathy. the sf- questionnaire was assessed in patients, from marseille, angers (france) and birmingham (united kingdom). routine clinical evaluations included mrc sum score, incat sensory score, inflammatory rasch built overall disability score (i-rods), ataxia score, jamar grip dynamometry, timed meter-walk, neuropathic pain symptom inventory (npsi) score, and fatigue severity score (fss). there were males and females. mean age was . years (sd: . years). mean disease duration was . years (s.d.: . years). there were no significant differences between the french and u.k. cohorts in terms of gender distribution, age, disease duration, anti-mag antibody titre. all physical assessments, including mrc sum score (p= . ), jamar grip dynamometry (p= . ), onls (p= . ), irods (p= . ), incat sensory score (p= . ), -meter timed walk (p= . ), ataxia score (p= . ), tremor score (p= . ), were comparable. prevalence and/or severity of pain (p= . ), fatigue (p= . ), restless legs syndrome (p= ) and cramps (p= . ) were also similar. physical component summary (pcs) and mental component summary (mcs) of the sf questionnaire were significantly lower than in reported normal subjects of both countries (p< . ). all sf- qol domains correlated with i-rods, except mcs for which significance was however approached (p= . ). pcs correlated with mrc sum score, ataxia score, timed m-walk, tremor, jamar grip dynamometry, npsi pain score, fss and level of social support. mcs correlated exclusively with fss and level of social support. in multivariate regression, pcs was associated independently with i-rods (p< . ) and npsi pain score (p= . ), whereas mcs was associated independently with fss (p= . ). qol is accurately predicted in anti-mag neuropathy by the i-rods and fss, lending support to their use in clinical and research settings. effective measures to improve qol should include tremor and neuropathic pain treatment, fatigue management and improved social support. fu liong h , yusuf r . regional neuromuscular clinic, queen elizabeth hospital, university hospitals of birmingham, birmingham, uk; school of life and health sciences, aston brain centre, aston university, birmingham, uk. the electrophysiological diagnosis of guillain-barré syndrome (gbs) is dependent on a number of abnormalities affecting different parameters in peripheral nerves on nerve conduction studies (ncs). diagnostic sensitivity varies with different electrodiagnostic criteria, practices as well as extensiveness of nerve study. however, the most efficient method of performing electrophysiology for guillain-barré syndrome (gbs) is unknown. we retrospectively analyzed electrophysiological data of consecutive gbs patients from birmingham, uk ( - ), studied ≤ weeks post-onset. we first identify abnormal nerves from various regions which produced gbs electrodiagnosis using the recently described criteria by rajabally et al. we subsequently used pre-established hypothetical nerve conduction study protocols to determine the potential optimal method of achieving electrodiagnosis. we found the sensitivity of electrophysiology for each gbs subtype was dependent on the upper and lower limb nerves tested. in acute inflammatory demyelinating polyneuropathy (aidp), abnormalities were predominant in the arms, whereas leg abnormalities predominated in axonal gbs. in aidp, the most common abnormal parameters were distal motor latency ( . %) and conduction block ( . %), and the most frequently affected nerve was the median ( . %). prolonged f-waves were present in % and f-waves were absent in %. mcv was the least frequently abnormal ( . %) with demyelinating range slowing, significantly lower than cb or dml prolongation (p< . in both cases). in axonal gbs, reduced motor amplitudes ( . %) and conduction block ( . %) were the most common parameters, and the most frequently abnormal nerve was the tibial ( . %). f-waves were absent in . %. . % of all motor nerves were unexcitable, significantly more common in lower limb nerves compared to upper limb nerves (p= . ). on comparison of different hypothetical ncs protocols ( unilateral protocols with nerves, with nerves as well as the protocols with -nerves and with exclusive bilateral upper limb and lower limb testing), unilateral -nerve (median, ulnar, common peroneal and tibial) testing produced the highest diagnostic sensitivity for both aidp ( . %) and axonal ( . %) gbs. electrodiagnostic sensitivity in gbs is thus dependent on nerves tested and parameters considered. each subtype preferentially involves specific nerves and parameters. these findings may help per-procedure interpretation, improve electrodiagnostic sensitivity, and reduce patient discomfort. funakoshi k , nagashima t , kokubun n , hirata k , yuki n . department of neurology, dokkyo medical university, tochigi, japan; department of neurology, mishima hospital, nagaoka, japan. recently, igg anti-ganglioside complex (gscs) antibodies have been reported in patients with guillain-barré and miller fisher syndrome. some researchers (nobile-orazio et al., ) reported igm anti-gscs antibodies in multifocal motor neuropathy (mmn) and other chronic immune-mediated neuropathies. in ten of eleven mmn patients with anti-gm antibody, there was a decreased reactivity to gm /gd a compared to single antigen gm . similarly, in one of two chronic inflammatory degenerative polyradiculoneuropathy (cidp) patient with anti-gm antibody, there was a decreased reactivity to gm /gm , gm /gd a, gm /gd b, gm /gt b compared to gm . these relationships were defined negative interaction. in one cidp with anti-gd b antibody, anti-gm /gt b and -gm /gt b reactivity increased although gm and gm were negative (positive interaction). in japan, on the other hand, this correlation remains unclear. sera were investigated from one mmn patient ( y/o male) with anti-gm , and anti-gd a antibody, one cidp patient ( y/o female) with anti-gm , and one subacute sensory motor polyradiculoneuropathy ( y/o male) with anti-gd b, and anti-gq b. igm antibodies to gscs gm /gd a were tested with a mixture of gm and gd a (each pmol/well) as antigen. anti-gm /gd a antibodies were judged to have positive interactions when the optical density was . greater than the sum of the antibodies against individual gm and gd a, and negative interactions when the optical density was % or less compared to the antibodies against individual gm or gd a. antibodies to at least one combination of two of the six gangliosides (gm , gm , gd a, gd b, gt b, and gq b) were similarly tested and judged for positive or negative interactions. in one mmn, anti-gm /gd a had negative interactions. in one cidp, anti-gm /gd a, and anti-gm /gq b had negative interactions. in one subacute sensory motor polyradiculoneuropathy, various anti-gscs antibodies including gd b or gq b had negative interactions. in the cidp patient mentioned above, anti-gm /gt b or anti-gm /gt b had no positive interactions. the relationship between this attenuated reactivity presumably driven by adjacent gangliosides and the mechanism of chronic immune-mediated neuropathies needs to be clarified. galino j , cervellini i , zhu n , stöberl n , fricker fr , lee g , hütte m , lalli g , bennett dl . the nuffield department of clinical neurosciences, john radcliffe hospital, university of oxford, oxford, uk; wolfson card, king's college london, guy's campus, london, uk. schwann cells (scs) are the myelinating cells in peripheral nerve system and are important for normal peripheral nerve development and repair. rala and ralb are small gtpases that have been implicated in neural tube closure, neurite branching and have been described as upstream effectors of proteins involved in cell migration and membrane dynamics. due to their potential role in sc function here we investigated, by genetic ablation in transgenic animals of one or both gtpases, their importance in sc function in adulthood and in nerve repair. we showed that ral gtpases are dispensable for sc function in the naïve state. however ral signalling (provided by rala or ralb) is required for effective remyelination of axons following nerve injury. moreover, absent ral signalling produced defects in axon reinnervation of distal organs and a delay in motor function recovery after nerve injury. we also studied the ral dependent cellular mechanisms that may be responsible for impaired sc remyelination and noted abnormal sc lamellipodia formation that prevent normal axial and radial axon remyelination. this work demonstrates for the first time a novel mechanism for ralgtpases that controls sc lamellipodia formation and their importance in normal sc function during peripheral nerve repair. garcía-lareu b , , , ariza l , cobianchi s , , chillón m , , , , navarro x , , , bosch a , , . tumor necrosis factor (tnf) alpha has been implicated in the pathogenesis of diabetic peripheral neuropathy (dpn), among other inflammatory demyelinating diseases and neuropathic pain. tnf alpha is a pro-inflammatory cytokine that can act at several stages in the demyelination process. it is produced by schwann cells (scs) in the peripheral nervous system (pns) after nerve injury and released into the local environment to attract and activate macrophages at the site of injury, contributing to wallerian degeneration. in vivo studies demonstrated a local inflammation in the sciatic nerve of rats after injection of tnf alpha, followed by demyelination and axonal degeneration. furthermore, the application of tnf alpha resulted in acute mechanical hiperalgesia, a main characteristic of neuropathic pain and therefore tnf alpha is postulated as a biomarker for painful changes after nerve injury. with the aim to characterize tnf alpha effects in chronic neuropathic pain and in diabetic neuropathy, a transgenic mouse model overexpressing tnf alpha cdna under the peripheral myelin protein p promoter was generated. here we characterized the overexpression of tnf alpha in myelinated scs at different stages of myelination (postnatal days , and ) showing that high levels of tnf alpha in sciatic nerve leads to the downregulation of the major pns myelin proteins (p , mbp, pmp , mag) compared to wild type mice, correlating with the loss of structured myelin and an increase in p ntr, a marker for immature and non-myelinated scs in the sciatic nerve. iba staining showed high levels of macrophage infiltration in both sciatic nerve and spinal cord tissues, compared with wild type animals. stress conditions were induced by sciatic nerve crush surgery after which recovery and subsequent remyelination were delayed in the transgenic mice, as evaluated by the sciatic functional index (sfi) and electrophysiological tests. on the other hand, mechanical and thermal nociception seemed to be unaltered, with or without lesion. this model could be helpful in the characterization of the role tnf alpha in pain development, injury and dpn as well as in developing efficient therapeutic strategies to modulate such pathological conditions. garcía-sobrino t , , blanco-arias patricia , , vidal-lijó mp , quintáns bea , , sobrido mj , , pardo j , . department of neurology, hospital clínico, santiago de compostela, spain; neurogenetics research group, instituto de investigaciones sanitarias (idis), santiago de compostela, spain; genomic medicine group (u ), centre for biomedical network research on rare diseases (ciberer), spain; department of neurophysiology, hospital clínico, santiago de compostela, spain. charcot-marie-tooth (cmt) disease is a genetically heterogeneous group of hereditary motor and sensory neuropathies. more than genes were involved in the disease pathogenesis. the objective of this study was to assess the genetic distribution of cmt disease in galicia (northwestern spain). patients were diagnosed as cmt if they had a consistent neurological and/or neurophysiological examination or if they had sensory motor neuropathy with a positive family history. a total of cmt adult patients ( % females) were evaluated with a median age of [ - ] years. the molecular diagnosis was achieved in patients ( %) with a higher success in cmt ( %) than cmt ( %). globally, pmp duplication was the most frequent finding ( %), followed by mutations in mpz ( %), mfn ( %), gjb ( %) and gdap ( %). in cmt , with expception of the pmp duplication, pathogenic variants in egr , nefl and mpz gene were most common. pathogenic variants in mfn and gdap accounted for % of cmt patients. several patients referred to our institution as cmt were diagnosed as hereditary motor neuropathy (hmn) and pathogenic variants in the bscl gene were the most frequent. pathogenic variants not previously related to cmt were identified in mpz, mfn , gjb , egr , nefl and sh tc genes. sporadic or autosomal recessive (ar) cmt accounted for the % of all diagnoses. the genetic epidemiology of cmt in galicia follows a similar pattern to other populations, although some remarkable features are axonal gdap and demyalinating egr pathogenic variants as well a seemingly elevated proportion of ar cases. mutations in mpz gene are associated to a wide range of phenotypes. the r c, in particular, is associated to a severe and early onset disease. some cmt patients respond to immunosuppressive or immunomodulatory treatment, some of them harbouring mpz mutations. we present the case of a -year-old brazilian female with a severe cmt b (r c) that presented an acute episode of worsening after a febrile exanthematic disease. she complained of tingling, stopped walking without support, and could not raise her arms. her emg showed an asymmetrical reduction of motor cv, reduced amplitudes, severe temporal dispersion and possibly conduction block. csf protein was mg%, with normal cell count. diagnosed as gbs, she received ivig. curiously, she markedly improved, regaining the functional status of youth, recovering functions that she had long lost. on emg, motor amplitudes improved significantly, but cv remained the same. reviewing carefully her past history, we identified some motor fluctuations along her adult life. at age , while pregnant, she developed positive sensory symptoms, became unable to get up from the chair and could not walk without assistance. after delivery, she improved, but did not return to baseline. one year later, she presented a new transitory functional worsening after an influenza illness. her parents were healthy but two out her children have a severe cmt. her first daughter never walked and died at the age of six. six months after treatment with ivig, she faced a new relapse. we pulsed with corticosteroids, with great response. she remains stable to date, four months after treatment. the episodic fluctuations and the evident response to treatment clearly suggest an associated immunomediated process. the fast improvement of the amplitude with maintenance of cv suggests that another mechanism in addition to demyelination and axonal degeneration is involved. we propose the existence of an associated channelopathy. funded by cnpq, faepa, pronas (ministry of healthy) gbs is an acute, immune-mediated neuropathy that comprises three major subtypes: aidp, aman, and amsan. on clinical grounds, this distinction is based on nerve conduction studies (ncs): aidp is a demyelinating neuropathy, aman is an axonal motor neuropathy, and amsan, an axonal sensory and motor neuropathy. recently, it has been demonstrated that axonal gbs, in addition to axonal degeneration, may present conduction block (cb) that can progress to axonal regeneration or revert, characterising a reversible conduction failure (rcf), a finding usually associated to good prognosis. patients presenting axonal cb are frequently diagnosed as having aidp, a mistake that can be avoided with two sets of ncs, one as early as possible and the second after three weeks of disease. following these recommendations, we retrospectively classified a brazilian group with gbs followed in our institution that had been submitted to two ncs sets. from september to january , patients fulfilled clinical criteria for gbs at clinical hospital of ribeirão preto and had more than one ncs accomplished. at least four motor nerves (median, ulnar, peroneal, and posterior tibial) and five sensory nerves (medial, ulnar, radial, sural, and peroneal superficial) were evaluated in each examination, according to routine procedures. first ncs revealed patients with aidp ( %), cases of axonal gbs ( aman, amsan, %), and patients with equivocal results ( %). at follow-up study, patients ( %) had their classification changed. the main shifts were from aidp to axonal group and from equivocal results to aidp. aidp increased to % (n = ), axonal gbs increased to % (n = , aman, amsan), and there were no more equivocal patients. although the majority of studies have shown that aidp is more frequent in western countries while axonal gbs predominates in eastern countries, we found a different pattern of distribution in our population, with predominance of the axonal subtypes. the considerable increase of axonal gbs at follow-up studies reinforces that serial emg are mandatory for accurate diagnosis of gbs subtypes. prospective studies are now being carried out in order to confirm these results. in amyloid light chain (al) amyloidosis, misfolded light chain (lc) accumulates and causes progressive peripheral neuropathy (pn) and progressive failure of critical organs such as the heart and kidneys. progressive ascending sensorimotor neuropathy is often a related clinical finding. the deposition of toxic lc amyloid in peripheral nerves is associated with paresthesias, pain, muscle weaknesss, orthostatic hypotension, and diarrhea or constipation in approximately % of patients with al amyloidosis. there are no approved treatments for al amyloidosis. current therapeutic approaches for al amyloidosis are intended to limit lc production but do not directly target misfolded lc deposited as amyloid in organs. we report phase / trial results of neod , an investigational monoclonal antibody that targets misfolded lc and may neutralize circulating lc aggregates and clear insoluble deposits. trial inclusion criteria were one or more plasma cell-directed treatment completed before enrollment, partial or greater hematologic response to any previous therapy, and persistent organ dysfunction. neod was administered intravenously every days during a dose-escalation phase ( patients; . , , , , , , or mg/kg in a + study design) and an expansion phase ( patients; mg/kg). we assessed safety, tolerability, pharmacokinetics, immunogenicity, organ responses based on consensus criteria, and pn responses using the neuropathy impairment score-lower limb (nis-ll). neod treatment was not associated with dose-limiting toxicities or serious adverse events, drug-related discontinuations, or antidrug antibody development in any patient (n = ). of patients with measurable pn at baseline (n = ), % achieved pn response based on the nis-ll score after months of treatment, which resulted in a median % nis-ll score reduction (mean baseline nis-ll, . ). in a best response analysis, % of cardiac-evaluable patients (n = ) and % of renal-evaluable patients (n = ) met respective criteria for organ response. improvements in neuropathy have not been previously shown in al amyloidosis. these results demonstrated that monthly neod infusions were safe, well tolerated, and associated with responses across three different organ systems. gervais cbl , ross ma , goodman bp , khoury ja , muzyka i , smith be . mayo clinic in arizona, scottsdale, az, usa. this work describes clinical, examination and electrophysiologic findings in a cohort of patients with length dependent sensorimotor peripheral neuropathy (ldsmpn) with ventral abdominal sensory loss. ldsmpn affects the longest nerve fibers, namely those innervating structures in the feet and hands. what is less well appreciated is that length dependent involvement of sensory nerve fibers in ldsmpn from the thoracic segments gives rise to ventral abdominal sensory loss. consecutive patients seen for ldsmpn (n= ) were evaluated for the presence or absence of ventral abdominal sensory loss. demographic variables, symptoms and quantitative neurologic findings (neuropathy impairment score [nis]) were examined using descriptive statistics. final diagnoses were noted. ventral abdominal sensory loss to pinprick (which was asymptomatic in all patients tested) was documented in / ldsmpn patients ( . %), mean age was . years (range - ), m:f gender ratio was . ( : ), mean nis was . (range - ), ncs/emg abnormalities were found in / patients (the remaining showing objective evidence of small fiber sensory involvement). ldsmpn patients without ventral abdominal sensory loss (n= ) had a mean age of . (range - ), m:f of . ( : ), and mean nis of . (range - ). no patient ( / ) had dorsal torso sensory loss between the shoulder and buttock levels on either side. diagnoses of the ldsmpn patients with vs. without ventral abdominal sensory loss included charcot marie tooth or other hereditary neuropathy (n= vs. ), abnormal carbohydrate metabolism (n= vs. ), idiopathic (n= vs. ), hypothyroidism (n= vs. ), inflammatory (n= vs. ) and other (n= vs. ). ventral abdominal sensory loss appears to be common in patients diagnosed with ldsmpn of a variety of causes including inherited neuropathies; in addition to those innervating distal limb territories, distal sensory fibers from the thoracic region represent another category of length dependent involvement in ldsmpn; ) the clinical examination of ldsmpn should include the ventral abdomen. gibbons c , garcia j , casasola m , freeman r . beth israel deaconess medical center, harvard medical school, boston, usa. objective: to determine the relationship between measures of autonomic function, electrochemical sweat conductance (esc) and intra-epidermal (ienfd) and sudomotor nerve fiber density (sgnfd). background: structural and functional measures of small fiber neuropathy have been studied in patients with diabetes, but little information comparing these techniques exists. design/methods: we studied patients with diabetes (ages ± yrs, gender f) and healthy control subjects (age ± yrs, gender f). subjects underwent examination scores (nis-ll), quantitative sensory testing, autonomic testing (heart rate variability, valsalva maneuver, tilt test), esc, and millimeter punch skin biopsies at the distal leg and distal thigh for ienfd & sgnfd. results: there were strong correlations between exam scores (nis-ll) and biopsy ienfd (r=− . , p< . distal leg; r=− . , p< . distal thigh), and sgnfd (r=− . , p< . distal leg; r=− . , p< . distal thigh). moderate correlations were noted between exam scores and qst (r values . - . , p< . ), ienfd and qst (r= . - . ). modest correlations were noted between esc and parasympathetic function (r= . - . , p< . ). modest correlations were noted between esc and ienfd (r= . , p< . , but only at the distal leg) and esc and nis-ll (− . , p< . ). no correlations between exam scores and autonomic function were noted. no correlations were detected between esc and sgnfd (r=− . - . ) or esc and sympathetic adrenergic function (r= . ). conclusions: differences between tests are expected based on our understanding of the pathophysiology and natural history of diabetic neuropathy. exam scores, qst results and biopsy results do correlate, and are consistent with prior studies. in contrast, there was little relationship between tests of autonomic function and exam scores, similar to prior studies and our understanding of autonomic function and differences in p. esc had little correlation with either exam scores or biopsy ienfd or sgnfd. there was a modest correlation between esc and parasympathetic function. the exact relationship between esc and diabetic peripheral neuropathy is not clear, and further research studies are needed to determine the role this technique has in clinical practice. objective: to determine the optimal tissue thickness of skin biopsy sections for studies of cutaneous nerve fibers. background: although analysis of intra-epidermal nerve fiber density (ienfd) is routinely reported using μm thick tissue sections, many recent studies of peripheral alpha-synuclein deposition use or μm frozen sections, or μm paraffin embedded tissue sections. design/methods: we compared the results of biopsies from patients with parkinson's disease (pd), using tissue sections each of , and μm thickness. tissues were stained with pgp . and stained for phosphorylated alpha-synuclein (p-syn). the total number of dermal structures (hair follicles, sweat glands, pilomotor muscles) were quantified, nerve densities analyzed, and the frequency of p-syn positive results. we also studied μm paraffin embedded tissue samples from patients with pd. results: in the biopsies of patients with pd there were no differences in the number of sweat glands, hair follicles or pilomotor muscles in , or μm sections. there were significantly fewer blood vessels noted in and μm sections compared to μm sections (p< . ). ienfd and sgnfd declined with tissue thickness (p. . , all) and there was increased variability in results in thinner tissue sections. there was a highly significant reduction in p-syn positive sections in thinner tissue sections (p< . , all tissues compared to μm sections). paraffin embedded tissue sections had significantly lower nerve densities and positive p-syn results (p< . ) compared to all frozen tissue sections of , and μm thickness. thinner tissue sections carried a greater risk of false positive result or indeterminate results due to difficulty interpreting overlap with pgp . . conclusions: tissue sample thickness plays a critical role in interpretation of skin biopsy results. thinner tissue sections, or paraffin embedded tissue sections, do not provide equivalent data and significantly underestimate nerve densities and positive alpha-synuclein results with increase false positive results despite similar numbers of dermal tissue structures. ace- is an investigational protein therapeutic that acts as a localized ligand trap for myostatin and other negative regulators of muscle growth. local injection of ace- into the gastrocnemius muscle of wild-type, mdx, and sod mice produced dose-dependent increases in muscle mass and force without systemic effects. in a phase single-center, double-blind, placebo-controlled dose escalation study in post-menopausal women, unilateral injections of ace- into the rectus femoris (rf) or tibialis anterior (ta) muscle were generally safe and well tolerated. mean percent changes from baseline in muscle volume of the injected muscle were + . % in the rf and + . % in the ta at the highest dose administered with minimal changes observed in the contralateral side and placebo-treated subjects. frequent related aes (≥ %) included injection site pain, pain in extremity, injection site discomfort, and muscle twitching, with similar incidence in ace- and placebo-treated groups. all aes were grade - and reversible. together, these preclinical and clinical results support further studies of ace- in myogenic and/or neurogenic diseases with focal loss of muscle strength and function, including cmt. a phase study is ongoing in facioscapulohumeral muscular dystrophy (fshd). study a - is an ongoing multicenter, two-part, phase study to evaluate the safety, tolerability, pharmacodynamics, efficacy, and pharmacokinetics of ace- in patients with cmt and cmtx. part is open-label and will enroll up to dose-escalating cohorts ( patients per cohort); part is randomized, double-blind, and placebo-controlled, and will enroll an additional patients. ace- will be administered bilaterally to the ta muscle once every three weeks for up to five doses. a safety review team will meet periodically throughout the study to review safety data and make dosing recommendations, including the recommended dose level for part . eligible patients must have genetically confirmed cmt or cmtx with mild-moderate weakness in ankle dorsiflexion. safety and tolerability will serve as the primary outcome for part , muscle volume evaluated by mri for part . additional outcome measures of interest include strength by quantitative muscle testing, function by motor tests, and quality of life by the cmt-health index questionnaire. our study was aimed to evaluate the functional and morphological consequences of cellular and humoral responses in chronic inflammatory demyelinating neuropathy (cidp), using extensive standardized high-resolution sonography (hrus) and nerve conduction study (ncs) protocols in incident treatment-naive patients. we enrolled consecutive, newly diagnosed, treatment naive patients with cidp. in addition to all relevant clinical examinations, all patients underwent a standardized ncs and extensive hrus protocol, of median, ulnar, tibial, fibular and sural nerves. we assessed standard nerve and fascicle size, and echogenicity. we found focal sonographic enlargements in multiple nerves and nerve segments with and without ncs abnormalities. the degree of nerve hypertrophy was not associated with presence of ncs features of demyelination, i.e. / ( %) of median nerve segments showed enlargement without strong decrease in motor conduction velocity and / ( %) hypertrophic median nerve segments revealed no conduction block. a lower distal cmap of median nerve was related with lower mrc sums-scores (p < . ). we found no correlation between age, disease duration or mrc sum-score and nerve size. cellular and humoral responses in cidp may lead to nerve enlargement along the length of nerves, that can be detected by hrus, whereas ncs allows identification of its' specific focal disruption in nerve function. the most common complication of diabetes is peripheral neuropathy, which has prevalence as high as % and is characterized by damage to neurons, schwann cells and blood vessels within the nerve. the concept of schwannopathy as an integral factor in the pathogenesis of diabetic neuropathy is re-emerging, and it is now known that schwann cells cultured in hyperglycemic environments underproduce neurotrophins and exhibit loss of axonal associations, further indicating a non-optimal glial cell activation and function. furthermore, the increased expression of p ntr in myelin sheaths around fibers that are susceptible to axonal degeneration in diabetic neuropathy suggest an important role for this molecule in disease progression. with this project, it is our main goal to evaluate how disruption of p ntr signaling in the schwann cells affects the pathophysiology of diabetic neuropathy. by using a fluorescent live/death cell viability assay, our preliminary data indicate that wild-type schwann cell cultures present increased cell death rate h after stimulation with high levels of glucose. the p ntr has a highly recognized role in the activation of death signals and when absent, we observed that schwann cells are significantly more resistant to apoptosis in hyperglycemic conditions. the role of p ntr receptor signaling in neuron-schwann cell communication and myelination under in vitro diabetic conditions was investigated with primary schwann cell-sensory neuron co-cultures. after eight days of ascorbic acid stimulation, both under euglycemic and hyperglycemic conditions, myelination was assessed by confocal microscopy using specific markers for neurons and myelin. results highlight a compromised ability of wild-type schwann cells to myelinate axons when exposed to a hyperglycemic environment, which was even intensified in co-cultures with schwann cells lacking the p ntr . to complement the in vitro studies, we are modeling type diabetes in a p ntr schwann cell conditional ko mouse model and plan to investigate nerve mrna expression profile to disclose genetic regulation depending on this receptor signaling and its modulatory role in endoneurial hypoxia and neuroinflammation. the results from this project will provide an integrated vision of how impaired schwann cell activity guides neuropathy progression. gondim faa , barreira aa , cruz mw , cunha fmb , de freitas m , frança mc jr , marques w jr , nascimento ojm , oliveira asb , pereira rc , pupe c , rotta ft , schestatsky p . panelists on behalf of the scientific department of peripheral neuropathy, brazilian academy of neurology, brazil. neuropathy is one of the most common neurological manifestations of several diseases and sfn has been progressively receiving more attention in the medical literature. the aim of this study is to generate a set of recommendations to define and diagnose sfn in brazil. a group of neurologists, members of the scientific department of peripheral neuropathy from the brazilian academy of neurology reviewed a preliminary draft prepared by the first author that was distributed by email. the panelists got together on . . at the city of fortaleza, brazil, to discuss and finish the text for the first submission of the manuscript. sfn can be defined as a subtype of neuropathy characterized by selective involvement of unmyelinated or thinly myelinated sensory (sometimes also autonomic) fibers. it is usually characterized by sensory (pain/dysesthesias/pruritus) or combined sensory and autonomic complaints, associated with an almost entirely normal neurological examination (except for sensory changes). electromyography is normal. a growing list of medical conditions has been linked to sfn, although there is no evidence-based literature to support the use of any specific set of screening tests to diagnose the etiology of sfn (the panelists will suggest a basic screening panel). sfn may also serve as a fallacious but useful terminology to uncover discrepancies in the normal values from different neurophysiology laboratories. in brazil, skin biopsy is not usually performed and initial forms of leprosy may have predominant small fiber involvement. there are several tests to demonstrate involvement of small sensory and autonomic fibers. skin wrinkling test, sympathetic skin responses & heart rate variability (conducted on emg machines) and thermoregulatory sweat test may be low-cost screening alternatives. after the final meeting on . . , we finished the first draft for submission to arquivos de neuropsiquiatria (together with a translation to portuguese as supplementary material), the official journal of the brazilian academy of neurology to serve as a source for the definition and diagnosis of sfn in brazil. the final draft will be submitted after presentation at the pns meeting in barcelona on . . the lower cranial nerves (lcn) include the paired glossopharyngeal, the vagal, the accessory and the hypoglossal nerves. these are involved in the execution of swallowing, speech, phonation, tasting, as well as sensory and autonomic functions. lcn can be affected in central and peripheral nervous system diseases. this study investigates the use of ultrasound (us) to detect lesions of the peripheral course of lcn. in addition mri can be used in unilateral lcn local lesions to demonstrate indirect signs of nerve lesions such as muscle atrophy. the patients were examined in supine position with the neck in maximum extension with either a regular or a portable us system (logiq e and logiq e ge healthcare, milwaukee, wisconsin) and high frequency transducers ( to mhz). according to the detected pathology longitudinal or transversal images were recorded. a series of exemplary observations are demonstrated. in one case a paraganglioma in the glossopharyngeal nerve at the carotid sinus was found. in another patient an us of the thyroid gland revealed a nerve tumor, which was later identified to be a schwannoma. in another case multiple neurofibroma were identified in the vagal nerve during a routine neurofibromatosis screening. us has become also important in investigations of lesions of the accessory nerve, following damage by surgery. nerve continuity and scar formation can be distinguished. lesions of the hypoglossal nerve can be caused by tumor infiltration. in one patient an infiltration of the nerve by a squamous cell carcinoma, and in another case by a low grade sarcoma were detected. in addition mri demonstrated an atrophy of the tongue. identification and assessment of the lcn from the point of exit of the skull to their endpoint can be pursued by us techniques, which have been described for the individual nerves. us is easily applicable and -in addition to the "static" image of the mri -allows assessing nerve positions during different movements, as well as detecting muscle movements as fasciculations. mri can also be used to detect not only more proximal lesions, but also changes of the skeletal muscle by denervation. grümme l , hattenhauer st , wolffram k , kleinschnitz c , mausberg ak , stettner m . university hospital essen, essen, germany; heinrich-heine-university, düsseldorf, germany. in chronic inflammatory demyelinating polyneuropathy (cidp), t cells are suspected to play a crucial role in myelin destruction. cd and cd t cells contribute to the inflammatory process, while the exact mechanism of myelin damage is still under debate. to elucidate the molecular interaction of t cells and myelin sheets, we compared neuritogenic and control cells in a live imaging in vitro model. the myelinated fibres of rat dorsal root ganglia (drg) served as model for the peripheral nervous system. drgs of embryonic (e ) lewis rats were cultured; myelination was initiated after one week in vitro and continued for two additional weeks. lewis rats immunized with p and neuritogenic t cells of the lymph nodes were obtained ten days after immunization. control t cells were prepared from healthy rats. for vital tracking, neuritogenic t cells were stained with orange cell tracker, while the control cells were labelled with cfse. myelin detection in vital cultures was assessed by incorporation of c fatty acids conjugated with a fluorophore into the myelin layer. experiments were performed in a conditioned microscope chamber, the two t cell populations were added simultaneously to the drg culture and migration was tracked using live cell imaging. we observed differing migration patterns for neuritogenic and control t cells. the velocity as well as the directionality was altered. after initial contact, the non-neuriotgenic t cells in close proximity to myelin subsequently decreased over time, while the numbers of neuritogenic t cells close to myelin remained stable. long term incubation with neuritogenic t cells affected the myelin integrity in regard to the intermodal length as well as the myelin ratio. further experiments will elucidate the specific effects of neuritogenic t cells to decipher the role of t cells during inflammation in the pns, which could be useful in developing targeted therapies. gundapaneni b , sultan mb , keohane dj , schwartz j . inventiv health inc., burlington, ma, usa; pfizer inc., new york, ny, usa. transthyretin familial amyloid polyneuropathy (ttr-fap) is a rare, life-threatening disorder caused by protein destabilizing ttr gene mutations, broadly classified as val met (the most common mutation worldwide) and non-val met genotypes. tafamidis, a highly-specific ttr-stabilizer, is the only medicine approved to delay neurologic progression in ttr-fap. the objective of this post-hoc analysis was to compare the effects of tafamidis on neuropathy progression in patients with val met and non-val met genotypes. val met patients were participants in a randomized, double-blind, placebo-controlled clinical trial of tafamidis, while non-val met patients were participants in an open-label tafamidis study. patients were grouped into three cohorts: val met tafamidis (n= ); val met placebo (n= ); and non-val met tafamidis (n= ). baseline disease severity and change in disease severity from baseline to month was assessed using the neuropathy impairment score-lower limbs (nis-ll). the effect of tafamidis in the val met and non-val met cohorts versus the val met placebo cohort was determined using a mixed-effects model for repeated measures (mmrm). at baseline, patients in the non-val met cohort were older, had longer symptom duration, and more advanced neurologic impairment than the val met cohorts. at month , the baseline-adjusted mean ± standard error change in nis-ll was comparable between the val met tafamidis and non-val met tafamidis cohorts ( . ± . and . ± . , respectively). these changes were smaller than that observed in the val met placebo cohort ( . ± . ; p= . vs val met and p= . vs non-val met) indicating less disease progression. based on predicted values from the mmrm analysis, the size of the change in nis-ll across the full range of baseline nis-ll scores was remarkably similar in the val met tafamidis and non-val met tafamidis cohorts and was consistently smaller than that observed in the val met placebo cohort. moreover, in all three cohorts, as baseline nis-ll increased, the predicted level of disease progression also increased. in conclusion, while controlling for baseline disease severity, tafamidis delayed disease progression to a comparable extent in val met and non-val met patients. the similar trajectories of disease progression across val met and non-val met patients suggest that these two genotype groups may be more similar than previously considered. clin-icaltrials.gov identifiers: nct ; nct . gutmann l , shy m . university of iowa, iowa city, ia, usa. post tetanic potentiation (ptp) is a physiological phenonmenon seen with disorders of the neuromuscular junction (nmj). it is caused by the influx of ca++ into the terminal axon during the tetanus resulting in an increased number of acetylcholine (ach) vescicles released by each axonal action potential. in myasthenic syndromes it results in improved nmj function by increasing the probability of achieving a large enough end plate potential to generate a muscle action potential. ptp has different appearances depending on whether the nmj defect is pre-synaptic or post-synaptic. in pre-synaptic defects (the most common being lambert-eaton syndrome) there is an increased amplitude of the muscle action potential after a tetanus that persists up to minutes. prolonged ptp has now been reported in disorders, one on a genetically determined neuropathy/myasthenic basis and the other on an acquired toxic origin. prolonged ptp up to minutes was reported in families with a motor neuropathy (having leg weakness and foot deformites) and a congenital myasthenic syndrome caused by a heterozygous mutation of the synaptotamin ii (syt ) gene (c t>g p.pro [p.asp ala] and c g>a [p.pro leu]). electrophysiological testing showed features of a presynaptic defect with prolonged ptp persisting up to minutes. the same phenomenon has been noted in the acquired pre-synaptic defect caused by botululinum toxin. the ptp continued up to minutes. syt is the synaptic vescicle calcium sensor in the terminal axon, allowing for fusion of ach containing vescicles with the presynaptic membrane and the synchronous release of ach. the fusion requires a complex assembly process involving snare proteins. both the syt gene mutation and botulinum toxin affect normal syt function, the mutation by altering amino acids in the calcium-binding domain and the toxin by binding to syt as well as gangliosides gd a and gt b on the neural membrane. the mechanism for the ptp prolongation remains unknown. prolonged ptp appears to be a unique physiological abnormality resulting from altered syt . this phenomenon has been decribed to occur in syt mutations causing congenital motor neuropathy/myasthenic syndrome and botulism. the abnormality may represent a physiological marker for a presynaptic nmj defect involving altered syt . hachisuka a , , senger j , curran m , chan km , . division of physical medicine and rehabilitation, university of alberta, edmonton, canada; department of rehabilitation and medicine, university of occupational and environmental health, kitakyushu, japan; division of plastic surgery, university of alberta, edmonton, canada. background: motor unit number estimation (mune) techniques are valuable tools in neuromuscular disease. among them, the multiple point stimulation (mps) is one of the most common used. contamination by distant single motor unit potentials (smups) generated by neighboring muscles is a potential confounding factor. this is particularly problematic in ulnar neuropathy, one of the most common neuropathies in humans. reason being that the ulnar nerve innervates the majority of hand muscles. the goals of this study are to test the hypotheses that ) distant smups all have an initial positive deflection and ) elimination of smups generated by distal muscles will significantly lower the mune results in the hypothenar muscles. methods: to address the first hypothesis, we tested subjects by stimulating their median nerve while recording smups simultaneously over the hypothenar and thenar muscles. for the second hypothesis, we carried out mps mune of the hypothenar muscles using multi-channel recordings placed over ulnar innervated intrinsic muscles across the hand. when a smup with an initial positive deflection was detected at the hypothenar electrodes, its original was systemically tracked through all the recording channels. results: in the first series of experiments, in accordance with the dipole theory, all smups recorded at the hypothenar recording electrodes had an initial positive polarity. in the second series of experiments, of the studies carried out in subjects, distant smups generated by muscles other than those in the hypothenar eminence represented ± . % (mean±sd) of the overall sample. mune calculated using only smups generated by the hypothenar muscles was ± , compared to ± if all smups were included (p < . ). the extent of increase in mune was highly correlated with the proportion of distant smups found in each study (r = . , p < . ). conclusion: in contrary to some studies suggesting that smups from distant muscles could have an initial negative deflection, we found all smups from distant muscles had a positive deflection. exclusion of those smups from the sample had a significant impact on the mune results. hagiwara w , konno s , kihara inoue m , fujioka t . toho university, tokyo, japan. matrix metalloproteinase (mmp) plays crucial roles in developing immune-mediated neuritis as guillain-barré syndrome (gbs) and its animal model experimental autoimmune neuritis (ean). to investigate the intraneural expression of mmps during ean and the effect of a phosphodiesterase- inhibitor cilostazol (clz) on it, ean rats were treated with either mg/kg/day of clz or vehicle from one day post immunization (dpi). to induce ean female lewis rats were immunized with synthetic peptide from bovine p protein. cauda equina (ce) were removed in several time points, total rna was extracted and reverse-transcribed to obtain cdna that was subjected to real-time pcr analysis for expression of mmp- , mmp- and tissue inhibitor of matrix metalloproteinase- (timp- ) messages. mmp- and mmp- messages peaked at dpi, that is presymptomatic phase of ean. all rats developed motor paralysis at dpi, mmp- and mmp- messages subsided at this moment. however, timp- message reciprocally increased at dpi, persisted through dpi. treatment of clz suppressed motor paralysis of ean significantly. mmp- message peaked at dpi and mmp- message peaked at dpi. on the other hand, both messages at dpi were suppressed compared to untreated ean rats. timp- message in clz treated rats peaked at dpi coincided with motor paralysis peak. both mmp- and mmp- messages might result in subsequent upregulation of timp- that finally downregulates mmps activity and inflammatory process. clz treatment suppressed and delayed expression of mmps and facilitate timp- expression, resulting suppression of ean. the precise mechanism of expression of mmps and timp- remain unclear, however, that mmp- and mmp- messages peaked at dpi suggests involvement of pro-inflammatory cytokines such as tumor necrosis factor-alpha or interleukin- . clz might suppress these cytokines resulted in mmps down regulation. mmp- less affected by clz and thus stimulated timp- expression at dpi. clz might rational treatment for immune-mediated neuropathy via mmps modulation although further investigation especially in-vivo study is needed. haidar m , de winter v , asselbergh b , bouhy d , timmerman v . peripheral neuropathy research group, vib, university of antwerp, antwerp, belgium. the small heat shock protein hspb (hsp ) gene is ubiquitously expressed and encodes for a chaperone protein with essential cellular functions. our lab was the first to identify missense mutations in hspb responsible for axonal charcot-marie-tooth neuropathy (cmt f). since then we became interested in understanding the physiological functions of hspb and its association with cmt neuropathies. we demonstrated the involvement of hspb in microtubule stability. because of the link between autophagosome formation and its intracellular transport, and microtubules stability, we believed that the macro-autophagy process could be regulated by hspb . macro-autophagy is a cellular housekeeping process during which autophagosomes target, envelop and degrade aberrant protein aggregates and damaged organelles. there is strong evidence for an essential role for autophagy in the maintenance of neuronal homeostasis; hence its impairment can lead to a neuropathic condition. our data indicate that macro-autophagy is disrupted by hspb cmt-causing mutations. combining novel microscopy and interactomics techniques we unravelled the way different cmt-causing mutations in hspb impair the autophagic pathway. our data present the impairment of autophagy as a possible pathomechanism for cmt-causing hspb mutations. hajjar h , gautier b , berthelot j , gonzalez e , gess b , young p , tricaud n . institute of neurosciences of montpelllier, inserm, university of montpellier, montpellier, france; universitätsklinikum münster, klinik für schlafmedizin und neuromuskuläre erkrankungen, münster, germany. cmt a, the most common of charcot-marie-tooth diseases, results from the duplication of peripheral myelin protein (pmp ) gene. this gene encodes for a small protein of kda, pmp , mainly produced by schwann cells and the excess of pmp leads to demyelination. there is no cure for this disease but one approach for a treatment is gene therapy. a transgenic rat model exists for cmt a, which possesses copies of the mouse pmp gene. our goal is to provide a proof of principle for gene therapy in peripheral nerves using this rat model of cmt a. our strategy is to reduce the overexpression of mouse pmp protein in rats schwann cells using short hairpin rnas (shrnas). shrnas are small non-coding rnas that specifically bind to targeted mrnas resulting in their degradation. we tested for the efficiency of several shrnas targeting mouse pmp in vitro to find two shrnas that reduce pmp levels. the shrnas have been cloned in an adeno-associated serotype (aav ) viral vector together with green fluorescent protein in order to detect infected cells. aav was selected for its high transduction rate of myelinating schwann cells, for its good diffusion and low immunogenicity. we plan bilateral injections in the sciatic nerve of control and diseased rats. the efficiency of this gene therapy will be checked by assessing muscle strength (grip test), way of walking (catwalk), mobility (rotarod) and nerve conduction velocity of treated cmt a rats versus non-treated. the process of myelination and myelin maintenance in schwann cells will be analyzed by biochemistry and electron microscopy. biochemical tests include western blot for pmp protein expression in sciatic nerve, immunohistochemistry for pmp protein expression in myelinating schwann cells and pcr for mrna pmp expression. if the therapy is successful in rats, it could possibly be later on used in clinical trials. ultrasound is a widely-used tool in diagnosing carpal tunnel syndrome (cts). several different methods for sonographical evaluation of median nerve damage exist, such as calculating the ratio of cross sectional areas (csa) of median nerves in various sites. this enables detection of actual nerve swelling proximal to the carpal tunnel as an expression of median nerve damage inside the carpal tunnel (as seen in cts). in comparison to other diagnostic methods no data exist about the prevalence of cts like changes in an unselected population. a series of non-selected fresh cadavers were examined. arms of fresh, non-embalmed whole body cadavers were examined. the medical record did not allow to obtain conclusive information on the peripheral nerves. using a regular ultrasound system with a mhz transducer, median nerves were identified and tracked along their course in the forearms. csa measurements of the median nerves were performed at two sites in each arm: .) halfway between the elbow joint and wrist, .) directly proximal to the carpal tunnel. csa ratio was calculated with the following formula: csa ratio = csa wrist (cm ) csa forearm (cm ) csa ratio < . was found in ( . %) arms, csa ratio ≥ . in ( . %) arms and csa ratio ≥ in ( %) arms. csa ratio ≥ . was detected in . % of women and % of men. the overall mean (± sd) age was . ± . years. men ( . ± . ) were significantly younger than women ( . ± . ; p = . ). a weak but significant correlation between age and csa ratio was found in women (spearman -− . ; p= . ), but not in men (p= . ). the mean bmi for csa ratio ≥ . was . ± sd . . based on a csa ratio ≥ . as a criterion for cts, the present ultrasound results are consistent with the average cts prevalence reported in previous studies, which were obtained with electrophysiological methods. this study on a large unselected series of cadavers confirms the comparability of both methods. instrumental activities of daily living (badl and iadl), falls and gait patterns in the large, prospective, population based rotterdam study. in total, participants of this study (mean age years, % women) underwent a polyneuropathy screening involving a symptom questionnaire, neurological examination and nerve conduction studies. screening yielded four groups: no, possible, probable and definite polyneuropathy. participants were interviewed about badl (stanford health assessment questionnaire), iadl (instrumental activities of daily living scale) and frequency of falling in the previous year. in a random subset of participants, gait was assessed with an electronic walkway (gaitrite). associations of polyneuropathy with badl and iadl were analyzed continuously with linear regression, and dichotomously with logistic regression. history of falling was evaluated with logistic regression and gait changes were evaluated with linear regression. we found that participants with definite polyneuropathy had more difficulty in performing badl and iadl than participants without polyneuropathy. polyneuropathy related to worse scores of all badl (especially walking) and three iadl components (housekeeping, traveling, and shopping). participants with definite polyneuropathy were two times more likely to fall, and these falls more often resulted in injury. participants with polyneuropathy had worse gait parameters on the walkway, including lower walking speed and cadence, and more errors in tandem walking. in summary, chronic polyneuropathy is strongly associated with significant impairment in daily life. recognition of polyneuropathy and related disability is very important in order to inform, support and possibly treat patients, and to prevent future falls and dependence in daily functioning. patients with polyneuropathy often suffer from tingling sensations, numbness, weakness and pain. these symptoms are used in several screening questionnaires, most of which were developed for high-risk patient groups, such as individuals with diabetes mellitus. in most tools equal weights are applied to all symptoms, while some might be more informative than others. we evaluated the diagnostic value and frequency of occurrence of individual symptoms of chronic polyneuropathy and constructed and validated a simple screening questionnaire that can reliably help to diagnose polyneuropathy in low-risk patient groups. in a multi-step procedure, we initially compiled a twelve-item questionnaire concerning symptoms of polyneuropathy. the questionnaire was completed by polyneuropathy patients and controls (headache, transient ischemic attack, multiple sclerosis). we calculated the sensitivity, specificity and likelihood ratios of each individual symptom. next, stepwise multivariable logistic regression was used to create a compact model, able to discriminate cases from controls using only the most informative symptoms. a simple scoring system was subsequently developed based on the regression coefficients of this reduced model. external validation was subsequently conducted in a population of cases with chronic idiopathic axonal polyneuropathy and controls without polyneuropathy. performance was assessed with discrimination (area under the curve, auc), and calibration. numbness and tingling feet were most frequently reported by polyneuropathy patients and had the highest sensitivity. feeling as if walking on cotton wool and allodynia of the feet had the highest specificity. multivariable logistic regression yielded a model that contained these four symptoms, complemented with balance problems and tingling hands. based on this regression analysis, the erasmus polyneuropathy symptom score (e-pss) was created, a score ranging from to . this polyneuropathy symptom score had a good performance (auc . ) in de derivation set and proved to be valid in the external population (auc . ). in this study, we created a simple, validated polyneuropathy symptom score (e-pss) that takes both the individual value of only six different symptoms and its frequency into account. this tool can be helpful as screening instrument in clinical practice and for future studies on polyneuropathy. multiple sclerosis (ms) is traditionally viewed as a central nervous system disease. to date, there is no unequivocal evidence implicating involvement of the peripheral nervous system (pns). this study aims to prove whether the pns is additionally affected and if so, to detect, localize and quantify these peripheral nerve lesions in patients with multiple sclerosis (ms) by applying high-resolution mr-neurography (mrn) with large anatomical coverage in combination with standard electrophysiological and neurological tests. we prospectively enrolled patients with confirmed ms (> years), two patients with clinically isolated syndrome (cis), and age-/sex-matched healthy volunteers. any other potential causes for a concomitant polyneuropathy were excluded. all ms patients underwent detailed neurological and electrophysiological testing. tesla mrn with large anatomical coverage from lumbar plexus and spinal nerves down to ankle level was performed in all participants by using fat-saturated, t -weighted turbo-spin-echo (tse) sequences (tr/te / ms) and a dual echo tse sequence for t -relaxometry (tr ms; te /te / ms). a d t -weighted, fat-saturated space sequence (tr ms; effective te ms) was used for imaging of the lumbar plexus. manual segmentation of spinal/sciatic/tibial/peroneal nerves was performed on a total of , axial slices. besides evaluation of nerve t w-signal, detailed quantification of nerve lesions by analyzing morphometric (nerve caliber) and microstructural markers (proton-spin-density and t -relaxation-time) was conducted. mean lesion load at thigh level was higher in ms ( . ± . ) vs. controls ( . ± . ;p< . ). nerve proton-spin-density was also higher in ms (tibial/peroneal: . ± . / . ± . ) vs. controls (tibial/peroneal: . ± . / . ± . ;p< . ). in contrast, t -relaxation time was significantly higher in controls (tibial/peroneal: . ± . / . ± . ) vs. ms (tibial/peroneal: . ± . / . ± . ;p< . ). proximal tibial and fibular nerve caliber was also significantly higher in ms (tibial: p< . ; fibular: p= . ). for the first time, pns lesions in ms patients could be visualized and quantified in vivo by high-resolution mrn. lesions are indicated by an increase of proton-spin-density and a decrease of t -relaxation-time. nerve caliber as a morphometric criterion also significantly increased. this proof-of-concept study may offer new insights into the pathomechanism of ms and might have future implications on therapeutic approaches. immunoglobulin g (igg) fc-gammars confer diverse effector functions by linking the cellular and humoral arms of the immune system that has been involved in the pathogenesis of guillain-barré syndrome (gbs). in the post-polio era, the polymorphisms of fc-gammar and their relevant knowledge have become one of the main targets for new therapeutic strategies for the treatment of gbs patients. differences in severity and frequency of gbs subtypes found between south-asian and western populations can be attributed to their genetic susceptibility. therefore, we aimed to determine fc-gammar polymorphic alleles (fc-gammariia: h /r ; fc-gammariiia: v /f ; fc-gammariiib: na /na ) and their possible link with gbs on the currently available large gbs cohort in bangladesh. fc-gamma r polymorphisms of gbs patients and healthy controls were genotyped using sequence-specific pcr. for validation, we carried out the sequencing of some samples for fc-gammariia and fc-gammariiia alleles. no significant differences were found regarding the distribution of fc-gammar genotypes and allele frequencies in gbs patients and controls. fc-gammar-h/h- genotype was significantly predominant in patients with severe disease compared to patients with mild disease (p= . , or, . ; % ci, . - . ). no other significant associations were found in gbs patients for candidate alleles and disease severity. fc-gammariiia-f/f- was found to be significantly predominant in anti-gm antibody positive gbs patients compared to anti-gm antibody negative patients (p= . , or, . , % ci, . - . ). fc-gammariiia-v alleles were significantly higher in patients with poor prognosis when compared to patients with good outcome (p= . , or, . , % ci, . - . ). no significant association of fc-gammariiib genotypes and alleles were found with gbs patients, disease severity and disease outcome. extensive subgroup analysis revealed no significant association in genotype and allele frequencies between aman and aidp subtype. in conclusion, igg fc-gammar polymorphisms do not constitute significant risk markers for susceptibility to gbs, however homozygous fc-gammariia-h might be involved in the severe form of gbs. in addition, fc-gammariiia-f/f- might play an important role in the molecular mimicry against nerve gangliosides in gbs. further studies that enroll a large number of patients (e,g. igos) are required to confirm the present findings from different geographical areas. hayes jm , o'brien pd , backus c , feldman el . department of neurology, university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common complication observed in patients with impaired glucose tolerance and type diabetes. male mice fed a high fat diet (hfd) develop metabolic impairments and pn serving as an appropriate animal model to study pn development and progression. it is well documented that female mice fed a hfd display a degree of protection against hfd-induced metabolic changes with mice retaining relatively normal insulin sensitivity. this protection is attributed to differences in fat accumulation and to the anti-diabetic effects of estrogen. based on these sex-dimorphisms we hypothesized that hfd-fed female mice would also exhibit resistance to developing pn. in the present study male and female c bl /j mice were fed either a standard diet ( % kcal fat; sd) or a high fat diet ( % kcal fat; hfd) from wk. at wk, wk and wk, neuropathy phenotyping was performed on all groups complemented with longitudinal metabolic assessments including insulin tolerance testing (itt). neuropathy phenotyping consisted of hindpaw latency to heat stimulus, motor and sensory nerve conduction velocities (ncvs), and terminal intraepidermal nerve fiber (ienf) counts. assessment of insulin resistance through itt demonstrated that during early hfd feeding, female hfd-fed mice exhibited relatively normal insulin responsiveness, while male hfd mice exhibited insulin resistance. despite this finding, wk female hfd mice displayed a similar pattern of pn to that of their male counterparts, with similar fold-changes in hindpaw latency and sensory and motor ncvs. therefore, although female hfd-fed mice exhibit resistance to hfd-induced metabolic changes, they display a pn comparable to male hfd-fed mice suggesting that systemic insulin resistance does not mediate pn. further studies are underway investigating the role of insulin signaling in the peripheral nerves of female hfd-fed mice. anti-mag (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal immunoglobulin m (igm) autoantibodies that recognize the carbohydrate epitope hnk- (human natural killer- ). this glycoepitope is highly expressed on adhesion molecules, such as mag, present in myelinated nerve fibers. since the pathogenicity and demyelinating properties of anti-mag autoantibodies are well established, current treatments aim at a reduction of autoantibody levels. however, the therapies applied so far are primarily immunosuppressive and lack selectivity and efficacy. we therefore hypothesized that a significant improvement of the disease condition could be achieved by selectively neutralizing the pathogenic anti-mag antibodies with carbohydrate-based ligands mimicking the natural hnk- glycoepitope. in an inhibition assay, a mimetic (mimhnk- ) of the natural hnk- epitope inhibited mag-binding by pathogenic igm antibodies from patient sera, however only with micromolar affinity. therefore, considering the multivalent nature of the mag-igm interaction, polylysine polymers of different sizes were substituted with the mimetic. with the most promising polylysine glycopolymer pl (mimhnk- ) the inhibitory effect on patient sera was improved by a factor of up to , per epitope, consequently leading to a low nanomolar inhibitory potency. since clinical studies indicate a correlation between the reduction of anti-mag igm levels and clinical improvement, an immunological surrogate mouse model for anti-mag neuropathy, producing high levels of anti-mag igm, was developed. the observed efficient removal of these antibodies with the glycopolymer pl (mimhnk- ) represents a first step towards an antigen-specific therapy for anti-mag neuropathy. hinder lm , mendelson f , backus c , feldman el . university of michigan, ann arbor, mi, usa. in the united states % of children and young adults are obese and at risk of developing prediabetes. prediabetic patients largely develop the same macro-and microvascular complications as patients with type diabetes, including peripheral neuropathy (pn). moreover, recent clinical data suggest normoglycemic obese patients develop pn. central obesity, characterized by excess fat storage in visceral white adipose tissue, leads to systemic metabolic dysfunction largely due to an imbalance between pro-inflammatory/anti-inflammatory adipokine production. subcutaneous adipose tissue is considered 'benign', but adopts a visceral-like phenotype in response to metabolic stress, with reduced thermogenicity, reduced brown adipose identity, and increased pro-inflammatory gene expression. the popliteal adipose tissue (pat) depot, corresponding to subcutaneous adipose, is adjacent to the peripheral nerve affected in pn, contains the lymph node for lymphatic drainage of the hind limb, and expands following local, sterile hind paw inflammation. the aim of the current study was to characterize pat changes in the high fat diet (hfd) mouse model of obesity and pn, and consider its contribution to peripheral nerve dysfunction. we previously reported c bl /j mice fed % hfd from - wk develop obesity, 'prediabetes' and pn; and switching mice back to a standard diet from - wk improves metabolic and pn phenotypes. at and wk pat was bilaterally dissected and the lymph node removed. the left pat was processed for histomorphometry, and the right pat for rt-qpcr. at wk hfd was associated with a significant shift in adipocyte size-frequency distribution, with a greater number of larger adipocytes. switching the hfd mice back to standard chow from - wk restored the size-frequency distribution towards age-matched controls. rt-qpcr was performed to assess changes in thermogenicity (ucp ), brown adipose identify (cidea) and sterile inflammation (saa ). at and wk hfd pat had reduced thermogenicity and brown adipose identify, and increased sterile inflammation. this switch towards a visceral-like phenotype was reversed in the hfd mice switched back to standard chow. in summary, hfd-induced changes in pat histomorphometry and adipose identity closely associate with pn phenotype. these preliminary data suggest a potential role for pat-nerve signaling in pn. hinder lm , backus c , hayes jm , feldman el . university of michigan, ann arbor, mi, usa. peripheral neuropathy (pn) is a common and debilitating complication of obesity and diabetes that triggers pain and loss of sensation. substantial nerve damage occurs in many patients prior to noticeable symptoms and no treatments are currently available; therefore, there is a critical need to identify treatment strategies that impact the underlying disease pathogenesis. our in vivo fluxomics data in the bks-db/db mouse model of type diabetes (t dm) and pn suggest that 'metabolic reprogramming' occurs in the t dm nerve to downregulate mitochondrial oxidative phosphorylation of substrates derived from glycolysis and fatty acid beta-oxidation. therefore, we hypothesize that distinct systemic metabolic alterations occur in obesity and diabetes which induce tissue-specific metabolic reprogramming within the peripheral nerve, altering fuel utilization and ultimately leading to tissue dysfunction. we contend that identifying conserved bioenergetic profiles across mouse models of pn will provide insight into key pn mechanisms. the current study utilized two mouse models of pn: the % high fat diet (hfd) mouse model of obesity and prediabetes at wk of age ( wk hfd), and the leptin receptor-deficient bks-db/db model of t dm at wk of age. mitochondrial function was determined in primary dorsal root ganglia (drg) neurons and sural nerve tissue from both models using the seahorse xf analyzer. resting mitochondrial oxidative metabolism was upregulated in drg neurons from mice with pn, with increased resting atp production and maintained mitochondrial coupling. in contrast, resting atp generation was decreased in sural nerve from mice with pn, with decreased coupling efficiency. relative spare respiratory capacity was attenuated in both drg neurons and sural nerve from mice with pn, indicating that mitochondria were less able to increase respiration in response to an energetic challenge. moreover, mitochondrial copy number was unchanged in drg neurons, but decreased in sural nerve tissue of mice with pn compared with respective controls. these data suggest a change in absolute number and function of sural nerve mitochondria, and a conserved cross-model proximal-distal bioenergetic profile in pn. we are currently exploring the relationship between these changes and pn pathogenesis. hoang ttn , umapathi t . hospital, ho chi minh city, vietnam; national neuroscience institute, singapore. ho chi minh city (hcmc) is the biggest metropolitan city in southern vietnam. its population is more than ten million. adult patients with neurological disorders are seen at six city public hospitals, including , hospital. there has been no systematic study of guillain barré syndrome (gbs) at hcmc or in vietnam in general. we are in the process of starting a prospective gbs database that we hope to expand to the other public hospitals at hcmc. here we describe our experience from . we saw gbs patients at , hospital. most of the cases were admitted in the rainy season, from late april to november, when mosquito-borne flavivirus infections are more common. patients were seen in the first week of illness and reported antecedent fever. two patients had diarrhea. diagnoses were made largely on clinical features, cerebrospinal fluid analysis and nerve conduction study. clinical findings include limb weakness, numbness and vii cranial nerve palsy. extraocular eye movements were affected in one patient. none had respiratory involvement severe enough to require artificial ventilation or intensive care. there were no pure miller-fisher syndrome cases; we suspect this might be related to the mild deficits that did not prompt hospitalization. nerve conduction studies showed typical features such as loss of f waves and abnormal blink reflex. the electrophysiology of patients' was dominated by demyelinating changes, one case was largely axonal and the remaining patient had normal electrodiagnostic study. repeat nerve conduction studies were not feasible because of limited resources. neurologists use corticosteroids as the main treatment. intravenous immunoglobulin and plasma exchange are costly and not reimbursed by medical insurance. we are currently preparing to systematically study gbs in southern vietnam, specifically with regards to possible role of antecedent flavivirus infections. we are also exploring the possibility of using low volume plasma exchange as a feasible cost-effective therapeutic modality. about % of patients with guillain-barré syndrome (gbs) treated with intravenous immunoglobulin (ivig) or plasma exchange deteriorate after initial improvement or stabilization -a phenomenon that is termed treatment-related fluctuation (trf). it is important to distinguish acute onset cidp (a-cidp) from gbs-trf during early course of the disease, because their therapeutic strategies and prognoses are different. herein, we describe a patient with gbs-trf, but with an extended progression phase that exceeds weeks. a -year-old woman was admitted due to acute onset progressive leg weakness and diplopia that had developed weeks prior (onset, d ). on neurological examination, right facial palsy was also observed. lower extremity weakness was moderate in proximal and distal muscles (mrc grade iv) with absent knee and ankle jerks. sensory examination revealed no abnormality in all modalities. she denied any recent diarrhea or upper respiratory infection, and vaccination. albumino-cytologic dissociation was noted in csf analysis; white blood cell count of / l and protein level of . mg/dl. nerve conduction study revealed demyelinating sensorimotor polyneuropathy with prolonged distal latency and conduction blocks. anti-ganglioside antibodies (gm igm/g, gd b igm/g, and gq b igm/g) were all negative. following ivig treatment, she was discharged with considerable improvement (d ). days later, she was re-admitted due to deterioration of leg weakness and hand clumsiness (d ). after another ivig treatment, she was discharged with clinical improvement (d ). days later, however, she was admitted again (d ) due to another considerable deterioration with four extremity weakness being worst at this time (mrc grade ii-iv in upper extremity, grade ii in lower extremity). a-cidp was considered given the progression phase exceeding weeks, but, we decided to give another treatment with ivig instead of a switch to corticosteroids because of uncertainty regarding distinction between a-cidp and gbs-trf. she was significantly improved following ivig treatment, and finally discharged (d ). thereafter, there has been no further deterioration during long-term follow-up of year. conclusively, this is a rare case of gbs with extended progression phase and trf. we propose that this could be referred to as subacute inflammatory demyelinating polyradiculoneuropathy (sidp) with trf. hsieh s , chao c . national taiwan university hospital, taipei, taiwan. transthyretin (ttr)-related familial amyloid polyneuropathy (fap) constitutes a major etiology of adult-onset hereditary neuropathies worldwide, in particular, a mutant ttr of ala ser (ttr-a s) in taiwan, the most common cause of acquired genetic neuropathy with adult onset (> years of age) of taiwanese patients. fap is a pan-modality neuropathy involving motor, sensory, and autonomic components of the peripheral nervous system with early involvement of small fibers as a major symptom. the early symptoms of fap are sometimes minimal and difficult to ascertain, mainly related to the fact that conventional electrophysiological examinations were not sensitive enough to detect small fiber neuropathy. skin biopsy with quantification of intraepidermal nerve fibers (ienf) has become one of the standard approaches to diagnose small fiber sensory neuropathy based on pathological documentation of nociceptive nerve degeneration. to explore the issue of early biomarkers in fap, we performed skin biopsy and compared ienf density with parameters of nerve conduction studies (ncs) and quantitative sensory testing (qst) on subjects ( men, aged . ± . years) with genetic confirmation of ttr-a s: patients and carriers. the ienf densities were significantly reduced compared to the age-and gender-matched controls in carriers ( . ± . vs. . ± . fibers/mm, p = . ) and patients ( . ± . vs. . ± . fibers/mm, p = . ). the latter was consistent with our previous report (neurology, : - , ) . the abnormal rate of ienf density was significantly higher than that of ncs and qst, respectively. in conclusion, there was significant skin nerve degeneration in carriers with ttr-a s. compared with qst and ncs, ienf density assessment had the highest abnormal rate and highest sensitivity to detect neuropathic changes in the early stage of fap. charcot-marie-tooth type p (cmt p) has been associated with frame-shift mutations in the ring domain of lrsam (an e ligase). this study describes families with a novel missense mutation of lrsam gene and explores pathogenic mechanisms of cmt p. this american family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-us families. the affected members in our family co-segregated with a novel missense mutation cys arg that alters a highly conserved cysteine in the ring domain. this mutation leads to axonal degeneration in the in vitro neuronal cell-line. moreover, using protein mass spectrometry, we identified a group of rna binding proteins (including fus, a protein critically involved in motor neuron degeneration) that interacted with lrsam . the interactions were disrupted by the cys arg mutation, which resulted in reduction of intranuclear rna-binding proteins. a knockin mouse of cys arg has been created for further explorations of cmt p mechanisms and therapeutic development. together, our findings suggest that the mutant lrsam may aberrantly affect the formation of transcription machinery. given a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of rna/rna-binding protein complex may play a role in the neuronal degeneration of cmt p. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr ). this double-blind, multicenter, parallel-group trial randomized ( : ) adult participants (n= ) with chronic inflammatory demyelinating polyradiculoneuropathy being treated with intravenous immunoglobulin (ivig) or corticosteroids to . mg fingolimod (n= ) or placebo (n= ) once-daily. previous treatment was discontinued (ivig) or tapered (corticosteroids). in the total trial population, there was no significant difference between the groups in the primary outcome, time-to-first confirmed worsening (≥ -point increase on the adjusted inflammatory neuropathy cause and treatment [incat] scale vs. baseline), or time-to-first any worsening (confirmed on incat assessment or unconfirmed). no significant difference between the two treatment groups was shown on secondary outcomes; change from baseline in grip strength and rasch-built overall disability scale (r-ods) at six months or trial end. analyses of pre-specified subgroups were performed for primary and secondary outcomes. this trial evaluated the efficacy and safety of fingolimod in chronic inflammatory demyelinating polyradiculoneuropathy (cidp). corticosteroids, intravenous immunoglobulin (ivig) and plasma exchange are recognized treatment options but no other immunomodulators demonstrated efficacy in a controlled trial. fingolimod has been shown to be efficacious and is approved for the treatment of relapsing multiple sclerosis. results from experimental autoimmune neuritis in rats suggested that it might show an effect in cidp. in this double-blind, multicenter, parallel-group trial, cidp participants receiving ivig or corticosteroids were randomized to once-daily fingolimod . mg or placebo ( : ). participants were stratified by inflammatory neuropathy cause and treatment disability (incat) scores and prior treatment. previous ivig treatment was discontinued after one final course before randomization. previous corticosteroid treatment was tapered over weeks. the primary outcome was time-to-first confirmed worsening (≥ -point increase on the adjusted incat score versus baseline). secondary outcomes included change in grip strength and rasch-built overall disability scale (r-ods) score from baseline to month and at trial end. the trial was stopped for futility by an independent data monitoring committee after a pre-planned interim analysis based on pre-specified criteria. in all, participants received fingolimod (ivig: , corticosteroids: ; age: . ± . years [mean±standard deviation]; male: . %); received placebo (ivig: , corticosteroids: ; age: . ± . years; male: . %). the percentage ( % confidence interval) of participants free from confirmed worsening at the trial end was not significantly different between fingolimod ( . % [ . %- . %]) and placebo ( . % ( . %- . %); p= . ). in the first days, approximately % participants experienced worsening. at trial end, approximately % participants had no worsening. there was no significant difference after six months or at the trial end (whichever occurred earlier) in the secondary endpoints. adverse events were reported in / and / participants in the fingolimod and placebo group, respectively. there were no deaths. nine participants in the fingolimod group and in the placebo group had serious adverse events. adverse events leading to trial drug discontinuation occurred in ( %) participants on fingolimod and none on placebo. no new safety signals emerged in this trial. acknowledgment: the authors consulted for or were employed by the study sponsor novartis pharma ag, basel, switzerland. myelin sheath enwraps non-nociceptive mechanoselective abeta-afferents transmitting touch/vibration sense. a prominent reduction in the mechanical stimulus required to evoke a withdrawal response in rodents, a phenomena interpreted as mechanical allodynia, arises due to peripheral nerve/myelin damage. evidence has emerged that nerve injury-induced mechanical allodynia depends on the adaptive immune/t cell activity in female but not male rodents. having previously demonstrated both the release of the cryptic - peptide regions of myelin basic protein (mbp - ) following sciatic nerve chronic constriction injury (cci) and the direct, robust and t cell-dependent ability of the pure mbp - peptides to induce mechanical allodynia after injection into the intact sciatic nerve, we hypothesized that mbp - contributes to sexual dimorphism in mechanical allodynia. the pure mbp - wild-type (wt), its histidine (his) mutant or scramble peptides were administered into an intact sciatic nerve fascicle in male and female rats or mice, followed by von frey testing. intra-sciatic mbp - -wt peptide induced robust and lasting allodynia in females. in contrast, males responded with a brief and mild decline in mechanical sensitivity for one day post-injection of both wildtype and control peptides. the algesic ability of mbp - -wt was diminished in the his mutant. we here present the molecular changes in the sciatic nerve, drg and the spinal cord after the intra-sciatic mbp - injection in male and female animals. in addition, using the biotin-labeled mbp - peptide and the hrp-labeled goat anti-rat igg/igm antibodies, we developed an elisa to quantitatively assess seropositivity for the specific anti-mbp - peptide igm/igg autoantibodies in female and male rats post-cci. human serum from female patients with multiple sclerosis was used for control. our work corroborates the findings of sexual dimorphism of mechanical hyperpathia and suggests its potentially autoimmune nature in females. iijima m , nishi r , ikeda s , kawagashira y , koike h , sobue g , katsuno m . nagoya university, nagoya, japan. non-obesity diabetic (nod) b - knockout (ko) mice are characterized by chronic and progressive neuritis and expected as models of immune-mediated neuropathies, especially cidp. hindlimb-predominant weakness due to inflammatory demyelination followed by axonal degeneration begins from around twenty week-age in all female mice until thirty week-age. to clarify the efficacy of immunoglobulins as immune-regulating therapeutics and the similarity of pathogenesis of human cidp, we injected intraperitoneally human-derived immunoglobulins (ipig, mg/mg bw/week) and saline as a control to totally forty female mice. clinical and pathological estimations in sciatic nerves were performed in time series. as a result, the ipig-treated group was protected from weight loss which could be related to axon loss followed by muscle atrophy as well as inflammatory demyelination between twenty-five week-age and thirty week-age compared to the control. in addition, the pathological findings in sciatic nerves showed that ipig apparently suppressed inflammatory infiltrates. about the subsets of inflammatory infiltrates, while macrophages (cd +) and lymphocytes (cd +) highly existed and suggested to play a main role in the neuritis until thirty week-age, only macrophages naturally disappeared after thirty week-age without any therapeutic induction. immunoglobulins effectively suppressed only macrophages although that did not suppress cd + lymphocytes. in conclusion, nod b - ko mice respond to immunoglobulins in a similar manner to human cidp and this efficacy is due to the suppression of macrophage-dominant pathogenesis. therefore, macrophage-derived pathogenesis is for the main target of immunoglobulin therapy and we should focus on the lymphocyte-derived pathogenesis which might plays an important role in non-responders to immunoglobulins. ikeda s , nishi r , kawagashira y , iijima m , koike h , katsuno m , sobue g , . department of neurology, nagoya university graduate school of medicine, nagoya, japan; research division of dementia and neurodegenerative disease, nagoya university graduate school of medicine, nagoya, japan. chronic inflammatory demyelinating polyneuropathy (cidp) is an acquired immune-mediated polyradiculoneuropathy that is characterized by heterogeneous clinical manifestations. typical cidp is defined as neuropathy manifesting in a progressive manner, stepwise manner, or with recurrent symmetrical proximal and distal weakness and sensory impairment in all four limbs. although they occur at a lower proportion than the so-called typical cidp, atypical forms, such as multifocal acquired demyelinating sensory and motor (madsam), distal acquired demyelinating symmetric (dads), pure sensory, pure motor, and focal, are considered to cidp subtypes. thus far, pathological features characterizing each clinical subtype have not been fully elucidated. we analyzed clinical and pathological correlations in consecutive cidp patients who underwent sural nerve biopsy and fulfilled the definite or probable efns/pns criteria. there were male and female patients. the age at biopsy was . ± . (mean ± sd) years, and the duration from the onset of neuropathy to biopsy was ± months. fifty-five percent (n = ) of the patients were classified as having typical cidp. regarding atypical cidp, madsam (n = , %), dads (n = , %), and pure sensory (n = , %) subtypes were the major subtypes, while pure motor (n= , %) and focal (n= , %) subtypes were rare. no significant difference was found among these subtypes in terms of sex, age at biopsy, and disease duration. sural nerve biopsy specimens revealed that the densities of large myelinated fibers significantly decreased in the madsam subtype than in the other subtypes (p = . ). in addition, the variation in nerve fibers among fascicles was more conspicuous in the madsam subtype than in typical cidp (p= . ). patients with the dads subtype tended to show the formation of onion-bulbs. in conclusion, pathological findings of sural nerve biopsy specimens were different among the cidp subtypes. further studies are needed to clarify mechanisms leading to different pathological features. small volume plasma exchange (svpe) can be an affordable and potentially effective alternative form of plasma exchange. svpe is the repeated removal of small volumes of supernatant plasma over several days via sedimentation of patient whole blood. the aim of this study is to assess the clinical feasibility and safety of svpe in patients with gbs in low-income countries. twenty adult patients with gbs diagnosed as per the criteria for gbs of the national institute of neurological and communicative disorders and stroke (ninds) were enrolled for svpe at a centre in bangladesh. serious adverse events (sae) were defined as the number of patients developing severe sepsis associated with the central venous catheter (cvc) or deep venous thrombosis in the limb where the cvc is placed for svpe. the svpe procedure was considered safe if less than of svpe-treated gbs patients have a sae, and feasible if eight litres of plasma could be removed in at least of svpe-treated gbs patients. among the cases who received svpe, ( %) patients were male and the age range between to yrs. all the patients were quadriplegic and bedbound at enrolment for svpe with a median mrc score of (iqr, - ). cranial nerve involvement, autonomic dysfunction and requirement for assisted ventilation were observed in ( %), ( %) and ( %) patients respectively. electro physiologically ( %) patients were motor axonal and ( %) patients were sensory-motor demyelinating type. during the svpe none of our patients experienced sae and one patient experienced central line associated blood stream infection. common adverse effects were transient intravenous fluid responsive hypotension during the svpe sessions in ( %), cv catheter insertion site hemorrhage in ( %) and hypersensitivity reaction to fresh frozen plasma in ( %) patients. there was no hypo-albuminemia, anemia or electrolyte imbalance observed in most patients ( %) treated with svpe. improvement in one or more grade of the gbs disability score at four weeks after the onset of svpe was observed in ( %) patients. in conclusion svpe can be a safe, feasible and cost effective alternative to standard pe in the developing countries. guillain-barré syndrome (gbs) is a descriptive disease entity defined by a set of clinical, electrophysiological and laboratory criteria. various clinical phenotypes exist that may be triggered by different antecedent infectious events. although the disease appears to affect primarily the elderly in developed countries, but, scenario is different in developing countries. bangladesh has made an impressive progress towards the eradication of poliomyelitis, and no new cases have been reported since . gbs, an acute polyradiculoneuropathy, is the most frequent cause of acute flaccid paralyis. the crude incidence rate of gbs in < years of age reported here appears to be . × to × higher than that reported in the literature. we conducted a hospital based observational study including patients fulfilling the national institute of neurological disorders and stroke (ninds) criteria for gbs patients between and in dhaka medical college hospital, dhaka, bangladesh. detailed clinical, electrophysiological, serologic and microbiological data were obtained. gbs affected predominantly in young adults males (m/f= : ) living in rural areas. antecedent events were recorded in > % of patients; frequent events being gastroenteritis (> %) and upper respiratory tract infection ( %). more than % of the patients were bed-bound (gbs disability score ) at entry and about % patients required mechanical ventilator. about % patients did not receive specific treatment either intravenous immunoglobulin (ivig) or plasmapheresis due to high expensive treatment cost. % patients had died during hospitalization. % of patients had an axonal variant of gbs and evidence for a recent c. jejuni infection ( %). c. jejuni infection was significantly associated with serum antibodies to the gangliosides gm and gd a, axonal neuropathy, and greater disability. in conclusion, the majority of the patients do not receive standard treatment with ivig in view of its high price. therefore, we developed low-cost treatment strategies and conducted a safety and feasibility trials for small volume plasma exchange (svpe) on gbs patients in bangladesh. in future, it is essential to conduct a phase ii clinical trial to assess the efficacy of svpe for low-in-come countries. prognosis of guillain-barré syndrome (gbs) has not improved in last two decades. current therapies (intravenous immunoglobulin, ivig and plasma exchange, pe) had been proved to be effective on two third of patients in developed world. unpredictable and poorly understood clinical course of gbs hamper treatment development. in bangladesh, most patients affected by gbs cannot afford specific treatments with ivig or pe instead most of them receive only supportive care. therefore, we aimed to compare the outcome of ivig treated patients with supportive care patients in improvement of gbs disability score and mrc sum score by using world's largest gbs cohort in bangladesh. we conducted a prospective observational study enrolling gbs patients between and from dhaka medical college hospital and national institute of neuroscience and hospital, dhaka, bangladesh. only gbs patients ( %) received standard ivig treatment. in current analysis, ivig treated patients and age, sex and severity matched controls (supportive care only) were considered. outcome of both groups were compared using fisher's exact or chi square test and survival analysis were performed by kaplan meier method using log rank test. among patients (cases and controls), male/female ( / ), median age years, % patients were bed-bound, one-fourth patients required mechanical ventilation and % were axonal. we did not found any significant differences of treatment outcome in both cases and control groups in gbs disability score (week : p= . , months: p= . ) and mrc sum score (week : p= . , months: p= . ). survival analysis revealed, the differences of time required for independent locomotion, improvement of one gbs disability score and improvement of mrc score were not statistically significant between treatments (ivig) and supportive care patients. in conclusion, our analysis showed that standard dose of ivig use has no considerable advantage to improve specific outcome measures among gbs patients in bangladesh. as the phenotype of gbs in bangladesh is different from developed world; therefore, an efficacy trial for ivig is needed for developing countries like bangladesh or new targeted therapeutic strategies can append beneficial effects for gbs patients. isose s , , watanabe k , omori s , sekiguchi y , beppu m , shibuya k , amino h , suichi t , misawa s , kuwabara s . graduate school of medicine, chiba university, chiba, japan; national hospital organization chiba east national hospital, chiba, japan. diabetic neuropathy is a frequent cause of neuropathic pain, suggesting the small-fiber involvement. additionally, persistent peripheral pain-related inputs could cause neuronal hyperexcitability and complex interactions of the nociceptive pathways, i.e., central sensitization. to investigate the pathophysiology of neuropathic pain in diabetic neuropathy, we studied pain-related evoked potentials (preps) after selective intraepidermal electrical stimulation (ies) to a-deltaand c-fibers in diabetes patients with neuropathic pain (n= ) and without neuropathic pain (n= ). we also conducted a longitudinal study to assess changes in preps and pain profiles in patients with neuropathic pain months after the start of treatment with duloxetine. this study is registered with the umin clinical trials registry, umin . ies was applied in the hand and foot, and preps were recorded from the cz electrode referenced to the linked earlobes. we evaluated prep latencies, amplitudes, and amplitude ratios of preps after c/a-delta -fiber stimulation. in the conventional nerve conduction studies, patients with neuropathic pain significantly showed conduction slowing and decreased snap amplitudes in the median and sural nerves compared with those in patients without neuropathic pain. in pain-related sep studies, there were no significant differences in prep amplitudes and latencies after a-delta -or cfiber stimulation between the patients with neuropathic pain and without it. prep amplitude ratios after c/a-delta -fiber stimulation tended to increase in patients with neuropathic pain compared to patients without pain. after the treatment with duloxetine, c/a-delta -prep amplitude ratios were significantly decreased after both hand and foot stimulation, and as for numerical rating scale (nrs) scores as the intensity of pain. patients with less pain relief showed the tendency of higher c/a-delta prep amplitude ratios before treatment compared to patients with better pain relief. the correlation between reduction of c/a-delta prep amplitude ratios and nrs reduction did not reach statistical significance. this pain-related sep study demonstrated that abnormal cortical response in patients with neuropathic pain could improve after the treatment with duloxetine, this might reflect the cortical hyperexcitability as a central sensitization. this study is funded by shionogi & co., ltd. the sponsors played no role in the design and management of the study, collection and analysis of data, interpretation of the results, or the writing of the writing of the report. peripheral nerve injury is commonly associated with traumatic injury which is often amenable to surgery. despite improved methods in surgical repair, functional recovery remains a challenging clinical problem that often leads to significant morbidity in patients. alternative therapies that could augment surgical repair may be beneficial in functional outcomes. neuroinflammation is a complex pathway with different cellular components and cytokines that are activated following peripheral nerve injury. macrophages are responsible for the breakdown of debris following injury as well as promotion of regenerative signals. macrophage polarization is the process by which macrophages take on phenotypically distinct functions based on the local environment and signaling cues. exercise has been shown to drive macrophage polarization from a pro-inflammatory m phenotype towards an anti-inflammatory m phenotype in numerous tissues, but remains uninvestigated in the peripheral nervous system. the purpose of our study was to identify how exercise affects macrophage polarization, motor and sensory function, and neuroregeneration following sciatic nerve crush. c bl/ mice underwent sciatic nerve crush injury and were then given access to running wheels (exercised) or not given access to running wheels (sedentary) for weeks. exercised mice ran an average of . km per night. injured exercised mice were protected from the development of thermal hyperalgesia when compared to injured sedentary mice. exercised mice had fewer paw slips on beam walk testing compared to sedentary mice. no differences were measured in mechanical sensitivity or motor coordination and balance assessed by rotarod. while motor nerve conduction velocities were significantly reduced for injured mice compared to uninjured controls, motor nerve conduction velocities from injured exercised animals were significantly higher than injured sedentary animals suggesting improved nerve recovery with exercise. injured sciatic nerves from exercised mice demonstrated increased m macrophages compared to sciatic nerves from injured sedentary mice. the behavioral changes and altered macrophage polarization correlated with increased epidermal nerve fiber density, improved myelination, and increased in vitro neurite outgrowth from injured exercised animals. therefore, exercise alters macrophage polarization towards an anti-inflammatory phenotype which improves repair and recovery of the injured peripheral nerve. jack mm , shah k , everist b , reyna j , hylton p . university of kansas medical center, kansas city, ks, usa. diffusion tensor imaging (dti) has long been used to evaluate the location and integrity of white matter tracts in the brain. dti uses quantitative data and directionality of water diffusion to determine axonal connectivity of the nervous system. the technology has only recently been utilized in limited settings in the peripheral nervous system due to challenging technical factors and lack of widespread availability. magnetic resonance (mr) neurography or peripheral neurography is a technique which uses diffusion to differentiate between intraneural and perineural tissues. it allows for fascicle patterns to be visualized particularly in the setting of peripheral nerve sheath tumors. peripheral nerve sheath tumors of various pathologies cause surrounding nerves to be involved or displaced in a range of directions. this technique helps determine the anatomic location of these nerve fibers in relation to the mass, which is particularly helpful at distinguishing neuromas from schwannomas. this data is invaluable to the surgeon to ensure a safe and low morbidity operation. while this technology has benefit particularly with surgical planning, it has been underutilized due to the challenges of requiring complex software to produce fiber tracks and the inability to translate these images into the operating room. here, we utilized brainlab software that is commonly available and utilized in surgical suites to produce images of the radial nerve fiber tracts with an associated peripheral nerve sheath tumor prior to surgical resection. while the software is commonly used in the central nervous system, it has not been reported to have been used in the peripheral nervous system. this software offers a high usability and produces anatomically correct and reliable fiber tracts. this technique overcomes the reliance on highly specialized software and extensive training required for use that most other tractography software has. utilizing peripheral neurography in this case allowed for complete surgical resection without postoperative deficits. this data offers clinicians an option to investigate peripheral nerve fibers in various pathologic states, to plan appropriate operative trajectories to peripheral nerve pathology, and to improve surgical outcomes for patients with peripheral nerve sheath tumors. jacobsen b , parry g , allen j , walk d , muley s , ortega e . barrow neurological institute, phoenix, az, usa; university of minnesota, minneapolis, mn, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is an immune mediated neuropathy that is responsive to immunomodulatory agents such as glucocorticoids, intravenous immunoglobulin (ivig) and plasma exchange (pe). the specific immunopathogenic mechanisms of cidp remain unclear but there is increasing interest in nodal proteins as a site of the immune attack. even though the majority of patients respond to one of the aforementioned immunomodulatory agents there are some who are unresponsive or incompletely responsive to these first line agents and other more aggressive treatments may be necessary. cyclophosphamide and stem cell transplantation may be effective but are associated with considerable morbidity. anecdotal reports suggest that rituximab may be beneficial for some patients that fail first-line therapy, especially if they have antibodies to nodal proteins. we present four patients with intractable cidp who responded to rituximab. one of the three patients had diabetes. disease duration prior to starting rituxan was short ( months) in two patients and longer and months in the other two. all patients had failed treatment with glucocorticoids and ivig, and in two, plasma exchange and ivig-pe were also ineffective. two of the four patients were quadriparetic and non-ambulatory. two gm doses two weeks apart of intravenous rituximab were instituted in all patients. all patient tolerated the treatments well without adverse effects. all patients responded within four weeks and continued to improve at six months. other immunomodulatory agents were successfully tapered but not totally discontinued. it remains unclear whether antibodies to nodal proteins were present in these patients. in conclusion, although rituximab efficacy remains uncertain on the basis of randomized controlled clinical trials, it may be beneficial in selected patients otherwise intractable to first-line treatments. further studies are necessary to better understand which patients may benefit most from rituximab and where in the treatment algorithm rituximab should be applied. neuropathic pain is a chronic condition seen in patients suffering a direct injury to the peripheral or central nervous system or an indirect injury due to, e.g., diabetes or multiple sclerosis. current treatment options fall short of preventing or completely relieving patients of their pain. for years, research has focused on understanding the role of neurons in neuropathic pain pathogenesis while overlooking the role of supportive cells in general and satellite glial cells (sgcs) in the dorsal root ganglion in particular. these cells not only buffer the neuronal microenvironment they are also involved in controlling the electrical activity flowing through the neurons and in neuropathic pain pathogenesis. the aim of this project is to understand the role of sgcs in neuropathic pain development and thereby aid the identification of new drug targets. to purify the sgcs from adult mice we optimized a fluorescently activated cell sorting (facs) protocol. the success of our purification method was confirmed using qrt-pcr and visual inspection of the sorted cells. finally, we are running rna sequencing on sgcs after peripheral nerve injury to compare their transcriptome to that of uninjured cells at different time points. the results from our study are likely to deepen our understanding of how sgcs contribute to the development and maintenance of neuropathic pain. guillain-barrésyndrome (gbs) is an immunemediated disorder in the peripheral nervous system (pns) triggered by molecular mimicry against nerve gangliosides. one of the cell surface receptors (fas)-ligand (fasl) interaction transmits apoptotic signal to eliminate the auto-reactive b and t-cells, which generates cross-reactive antibody against nerve cells. host genetic polymorphism of fas and fasl may alter their expression and induce aberrant apoptotic response to develop gbs. therefore, we determined the single nucleotide polymorphisms (snps) of both fas receptor (− g/a and - a/g) and fasl ligand (− c/t) in gbs patients (n= ) as well as healthy controls (n= ) using the lightcycler technique. serum level of soluble form of fas and fasl was measured using commercially available sandwich elisa kit. comparison of genotype, allele and haplotype frequencies was done with the gbs subgroups based on the clinical and serological data. ag heterozygote (p= . , or= . , % ci= . - . ) and polymorphic g-allele (p= . , or= . , % ci= . - . ) of fas receptor - a/g promoter snps were significantly associated with anti-ganglioside (gm ) antibody positive gbs patients. in addition, − g-allele (p= . , or= . , % ci= . - . ) and - g/- g haplotype (p= . , or= . , % ci= . - . ) were predominantly associated with the axonal variant of gbs patients. serum soluble form of sfas (median levels pg/ml vs. pg/ml, p= . ) and sfasl (median levels pg/ml vs. pg/ml, p= . ) were found to be elevated in anti-gm antibody positive gbs patients compared to anti-gm negative patients. no significant association was found in genotypic distribution between gbs patients and healthy controls. in conclusion, fas/fasl promoter snps are not a susceptible factor for gbs but could be one of the influencing factors to develop cross-reactive anti-ganglioside antibodies in gbs patients in bangladesh. furthermore, functional studies with a larger sample size (using cohort like international gbs outcome studies-igos) are required to explain the immune pathogenic role of these snps for gbs patients. jang sy , yoon ba , shin yk , yun sh , jo yr , park ji , shin kj , kim jk , park ht . dong-a university, busan, south korea; inje university, busan, south korea. myelination is essential for the proper function of the nervous system. schwann cells, which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. during schwann cell dedifferentiation-associated demyelination in wallerian degeneration after axonal injury, schwann cells exhibit myelin and junctional instability, down-regulation of myelin gene expression and autophagic myelin decomposition. however, in inflammatory demyelinating neuropathy, it is still unclear how schwann cells react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for myelin clearance. here, we show that schwann cell dedifferentiation-associated demyelination is a mechanism involved in the initial demyelination observed in a mouse model of inflammatory demyelinating neuropathy using ultrastructural, biochemical and microarray analyses. myelin uncompaction and myelin membrane instability generated by dedifferentiated schwann cells lead to autophagolysosome-dependent cytoplasmic amputation between the axon-containing myelin sheath and the schwann cell body, resulting in the formation of the "myelin corpse", thereby allowing macrophages to phagocytose the myelin corpse in the end stage of segmental demyelination. we found myelin corpse formation in inflammatory demyelination to be a process similar to the myelin rejection during wallerian degeneration, which appeared to be dependent on schwann cell autophagolysosome activation since schwann cell-specific atg knockout mice exhibited delayed myelin rejection following nerve injury. finally, lysosome inhibition in schwann cells not only prevented segmental demyelination but also delayed the progression of clinical stages by suppressing the myelin corpse formation in inflammatory demyelinating neuropathy. thus, our findings indicate that demyelination by schwann cells and macrophages might be part of a process that includes sequential divisions of labor with respect to myelin rejection and digestion, respectively. in conjunction with previous studies showing schwann cell dedifferentiation and autophagy in toxic and hereditary neuropathies, the concept of "schwann cell dedifferentiation-associated demyelination" provides insight into the development of possible therapeutic strategies to prevent schwann cell demyelination in peripheral demyelinating neuropathies. methods: electrophysiological data, the charcot marie tooth exam score (cmtes), and bmi from patients with known cmt a were obtained and analyzed. results: when controlled for age, bmi does not affect studies of ulnar motor nerve conduction in ctm a patients, but rather specific components of the cmt exam scores (cmtes, loss of pinprick sensation and motor strength in the lower extremities). discussion: bmi and clinical components of the cmtes are correlated, but it is uncertain which is the primary effect -i.e., whether the reductions in pinprick sensation and motor strength in the lower extremities lead to a higher bmi, or higher bmi results in these signs. introduction: charcot-marie-tooth disease type c (cmt c) is a rare, dominantly inherited neuropathy caused by mutations in the lipopolysaccharide-induced tumor necrosis factor (litaf) or small integral membrane protein of the lysosome/late endosome (simple) gene. methods: we present a case series comprised of patients in whom cmt c is caused by a gly ser substitution in the encoded protein. we focus on clinical presentation, electrodiagnostic analyses, and our findings in the context of previously described cases. results: the gly ser mutation causing cmt c is a mild form of cmt, as patients walked on time, had less weakness than those with charcot-marie-tooth disease type a (cmt a), had a charcot marie tooth neuropathy score (cmtns) indicative of mild disease, and had faster ulnar and median motor nerve conduction velocities compared to those with cmt a. discussion: the g s mutation in litaf seems to be clinically indistinguishable from a mild presentation of cmt a. jha mk , russell k , lee y , rothstein jd , morrison bm . johns hopkins university, baltimore, md, usa. peripheral nerves are highly dependent on metabolic energy to maintain both basic cellular functions such as axon transport, na+/k+ ion gradients, and myelination, as well as to support regeneration following injury. though glucose certainly provides some metabolic support, our recent studies have shown that monocarboxylates, such as lactate, pyruvate, and ketone bodies, also contribute to recovery from peripheral nerve injury. monocarboxylate transporters, particularly mct , are the predominate transporters for monocarboxylates in the peripheral nerve. in a recent publication, we found that mct heterozygous null mice, which express % less mct in all cells, have slowed nerve regeneration and reduced myelination following sciatic nerve crush. this study was limited by the global reduction of mct , which is widely expressed in schwann cells (sc), dorsal root ganglia (drg) neurons, endothelial cells, macrophages, and perineurial cells within the regenerating peripheral nerve. to better understand the mechanism by which mct contributes to normal nerve function and nerve regeneration, we produced and validated conditional mct null (mct loxp) mice that allow selective deletion of mct from scs, drg neurons, endothelial cells, or macrophages through mating to cell-specific cre lines. we are currently quantifying peripheral nerve development, aging, and regeneration in each of these mouse lines. following sc-, but not drg-, specific mct knockdown, sensory peripheral nerves develop demyelination by months of age, manifest by reduced myelin thickness, increased g-ratio, and reduced conduction velocity. studies are ongoing in cultured scs to determine the mechanism for demyelination. neither sc nor drg knockdown of mct impairs nerve regeneration following sciatic nerve crush. these results suggest that sc-specific mct is critical for maintaining myelin in sensory, but not motor, peripheral nerves as they age. they also suggest that mct expression in peripheral nerve cell types, other than sc and drg, is important for nerve regeneration. ongoing studies are determining the contribution of mct in other peripheral nerve cell types, particularly endothelial cells and macrophages, to normal development and regeneration following injury. our results will clarify the role of lactate and its transporter, mct , in peripheral nerve function, potentially suggesting novel targets for demyelinating neuropathies or nerve injuries. small fiber neuropathy (sfn) is a condition in which the smallest nerve fibers are affected, characterized by neuropathic pain and autonomic dysfunction. according to international criteria, sfn diagnosis is based on clinical symptoms in combination with abnormal temperature threshold testing (ttt) and/or reduced intraepidermal nerve fiber density (ienfd) in skin biopsy. skin biopsy is moderately sensitive, invasive and the process is time consuming and expensive. ttt is a widely available diagnostic tool, but lacks specificity. previous studies introduced stimulated skin wrinkling (ssw) as an objective, non-invasive diagnostic tool to detect sympathetic nerve dysfunction in sfn by means of a categorical assessment. however, our unpublished data has shown that inter-observer reliability of categorically assessed ssw is quite low. in this current study we will use a new digital method for ssw quantification: the digit wrinkle scan© (dws©). the primary study objective is to define normative values for dws© expressed as total wrinkle length per fingertip surface (mm/mm ). subsequently we investigate the applicability of dws© in patients with definite sfn, based on abnormal iefnd and/or ttt, determining the dws© sensitivity and specificity, as well as its validity. for this cross-sectional study, we will include healthy participants and patients diagnosed with sfn. eligibility is based on meeting the inclusion and exclusion criteria and providing written informed consent. skin wrinkling is induced by emla (eutectic mixture of local anesthetics) cream© application and captured by taking pictures. the primary outcome measure is total length of wrinkles per mm as shown on the photographs, which will be calculated by a new software program. patients are stratified according to age and gender. based on the results of healthy participants, normative values will be defined. inter-and intra-observer reliability will be determined. in the sfn group, additional correlation analysis will be conducted to determine the correlation between dws© and different outcome measures (sfn-symptom inventory questionnaire, visual analogue pain scale, neuropathic pain scale, sfn-rasch-built overall disability scale, ienfd and ttt). we expect to provide digitally quantified ssw (dssw©) normative values that can be used in clinical practice in the diagnostic workup for sfn. cmt , characterized by axonal degeneration, is an inherited motor and sensory neuropathy accounting for about % of total cmt patients. the cmt subtype shows on its own, a vast genetic heterogeneity with more than mutations in known genes rendering the identification of relevant drug targets and therapies very challenging. so far, only hdac inhibitors have shown promising results in mouse models for hspb mediated axonal cmt (cmt f), albeit such a single gene approach may have a limited relevance at clinical levels owing to the limited number of patients per genotype. in this study, we investigated common causative molecular players of cmt associated axonal degeneration. for this, an itraq based proteome analysis was performed on five patient's derived lymphoblasts bearing different cmt causal mutations alongwith respective age and gender matched unaffected controls. software-assisted interpretations of the obtained data led us to identify two proteins which were significantly downregulated in cmt patients compared to controls. these two proteins were then validated using western blotting and qpcr on patient derived lymphoblasts and fibroblasts. our results prompted us to unveil whether these two proteins can be used as potential biomarkers for identifying cmt patients. therefore, through a europe-wide collaboration, we constructed a cohort of cmt patients and healthy controls. these two proteins exhibited significant downregulation in this cohort suggesting a potential new role of these proteins as cmt biomarkers. remarkably, we were also able to validate the significant decrease in ineurons (neurons differentiated from patient derived ipscs) strengthening the importance of our finding and also suggesting the relevance of these proteins in the pathogensis of axonal cmt. this will be the first study involving multiple cmt causal genes at once, thereby holding the potential to offer new drug targets and potential biomarkers with wider application both clinically and pharmaceutically. mesenchymal stem cells (mscs) represents a valuable source of stem cell therapy, can differentiate into various cell types. we investigated of the neuromuscular potential of human tonsil-derived mscs (t-mscs) for neuromuscular regeneration in trembler-j mice that is considered to be a model for charcot-marie-tooth disease type a (cmt a diseases), which is involving hereditary motor and sensory peripheral neuropathies. the t-mscs differentiated toward skeletal myocytes, as evidenced by increased expression of skeletal muscle-related markers (including troponin i type , and myogenin) and the formation of myotubes in vitro. in situ transplantation of t-msc-derived myocytes (t-myocytes) into gastrocnemius in trembler-j mice, a mouse model of cmt a, enhanced motor function, as identified with recovery by a compound muscle action potential (cmap) amplitude. and the regenerated shape of the sciatic nerve and skeletal muscle by immunochemistry, without the formation of teratomas. furthermore, the expression levels of nerve growth factor (ngf) and glial cell line-derived neurotrophic factor (gdnf) were significantly increased in t-myocyte compared with t-mscs in vitro. these results indicate that the transplantation of t-myocyte can be a therapeutic option of cell therapy for the neuromuscular regeneration in hereditary peripheral neuropathy, comprising cmt a disease. kagiava a , karaiskos c , richter j , tryfonos c , lapathitis g , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory, the cyprus institute of neurology and genetics, nicosia, cyprus; department of molecular virology, the cyprus institute of neurology and genetics, nicosia, cyprus; neurology clinics, cyprus school of molecular medicine, the cyprus institute of neurology and genetics, nicosia, cyprus. x-linked charcot-marie-tooth disease (cmt x) is a common form of inherited demyelinating peripheral neuropathy resulting from mutations affecting the gap junction protein connexin (cx ). using a cx knockout (ko) mouse model of the disease, we have shown that targeted expression of virally delivered cx results in morphological and functional improvement. since patients with cmt x express mutant forms of cx in schwann cells, that could potentially interact with virally delivered wild type (wt) cx through dominant-negative effects, we also treated mutant mice expressing the t i, r w and n d mutations associated with cmt x on a cx ko background. all three mutants were localized in the perinuclear compartment of myelinating schwann cells consistent with retention in the er (t i) or golgi (r w, n d) with loss of physiological expression in non-compact myelin areas. following intrathecal delivery of the human gjb gene we could detect the virally delivered wt cx correctly localized in the non-compact myelin areas only in t i/cx ko mutant mice, but not in the other two mutants, suggesting dominant effects of the r w and n d mutant but not of the t i mutant. gjb treated t i/cx ko mice showed improved motor performance, along with lower ratios of abnormally myelinated fibers and reduced numbers of inflammatory cells in all tissues examined compared to mock-treated animals. in contrast, gjb treated r w and n d mutant mice showed only slight but not statistically significant improvement. this study provides additional proof of principle for a clinically translatable gene therapy to treat cmt x even in the presence of endogenously expressed cx mutants, since at least one er-retained cx mutant did not interfere with the expression of virally delivered cx allowing a therapeutic benefit similar to cx ko mice. however, golgi-retained mutants may interfere with virally delivered wt cx and other approaches besides gene addition may be needed for effective treatment. funding: muscular dystrophy association (grant mda to kak). kaida k , kadoya m , koike h , iijima m , takazaki h , ogata h , moriguchi k , shimizu j , nagata e , takizawa s , chiba a , yamasaki r , kira j-i , sobue g , ikewaki k . national defense medical college, tokorozawa, japan; nagoya university graduate school of medicine, nagoya, japan; kyushu university, fukuoka, japan; university of tokyo, tokyo, japan; tokai university school of medicine, isehara, japan; kyorin university, tokyo, japan. antibodies to a glial protein, neurofascin (nf) have recently been identified in approximately % of patients with chronic inflammatory demyelinating polyneuropathy (cidp), which are igg -predominant. igg anti-nf -associated cidp may be a distinct subtype from typical cidp in terms of clinical features and response to immunotherapy. however, a diagnostic criterion of anti-nf -associated cidp has not established yet. to develop optimal criteria and design the best treatment plan for the anti-nf -associated cidp, procedures for determining anti-nf antibodies should be simplified, prevalent, and reproducible, as well as being accurate. cell-based assay (cba) has hitherto been utilized for determining antibodies to nf in sera from patients, results of which have been confirmed by immunohistochemistry (ihc) using teased nerve fibers from rodents. these methods are the most reliable techniques, while not necessarily easy-to-use and easy to maintain in most laboratories. in the present study, we aimed to validate the diagnostic utility of a conventional enzyme-linked immunosorbent assay (elisa) for determination of anti-nf antibodies and the igg subclass. sera from patients with efns/pns criteria-met cidp were examined with elisa using human recombinant nf . to verify elisa results, ihc on rat sciatic nerves, western blot (wb) and cba using nf -transfected and naive hek cells were conducted. the human nf -based elisa clearly distinguished between anti-nf antibody-positive and -negative sera. fifteen cidp patients ( %) were igg anti-nf antibody-positive, which were confirmed by wb, ihc and cba studies. none of disease controls or healthy subjects had positive results. twenty-five sera randomly selected from anti-nf -negative cidp sera were also negative on cba. the anti-nf activities on elisa were significantly positively-correlated with those on cba (p < . ). analyses of clinical and laboratory findings showed that anti-nf -associated cidp was characterized by younger onset, distal dominant phenotype, tremor, sensory ataxia, higher protein levels in cerebrospinal fluid, and poor response to ivig, which were consistent with those in previous studies. this elisa combined with determination of the igg subclass is a simple and reliable method for initial screening for anti-nf antibodies. the genetic abnormality responsible for x-linked charcot-marie-toothy neuropathy subtype cmtx was recently identified by whole genome sequencing to be a kb insertion into chromosome xq . . the clinical profile of cmtx in childhood is not well described. we reviewed the clinical characteristics, neurophysiological profile and cmt pediatric scale (cmtpeds) assessments of children with genetically confirmed cmtx . cmtx was characterized by early onset, and early and progressive hand weakness. most affected children were symptomatic within the first two years of life. the most common presentation was with equinovarus foot deformity in the first year of life. cmtpeds analysis in these children revealed that cmtx progressed more rapidly ( . ± . points/ year, n= ) than cmt a and cmtx . grip strength in the second decade of life in most affected males was two standard deviations below age-and sex-matched normative reference values. the most severely affected individual was wheelchair bound at years of age and two individuals had no movement in the small muscles of the hand in the second decade of life. there was only a single symptomatic female identified and she had mild signs. nerve conduction studies showed a demyelinating sensorimotor neuropathy with motor conduction velocity in eight children while one child had a length-dependent sensorimotor axonal neuropathy. understanding the unique phenotype of cmtx is essential for directing genetic testing, as the cmtx insertion will not be detected on the snp microarrays, multi-gene panels or whole-exome sequencing currently used for the diagnosis of cmt. the early onset of disease coupled with rapid progression means that many children with cmtx will have severe disability within the first two decades of life and hence early diagnosis is needed for early commencement of rehabilitation. kapoor m , catania s , sarri-gonzales s , lunn mp , manji h , reilly mm , carr as . centre for neuromuscular diseases, national hospital for neurology and neurosurgery, london, uk; department of neurophysiology, national hospital for neurology and neurosurgery, london, uk. the conventional dosing of ivig in cidp and mmn is based on treatment trials that used bolus and maintenance dosing of ivig between - . g/kg. there are rare published articles reporting the efficacy of higher maintenance ivig doses. we present three cases of inflammatory neuropathies, who are currently stabilized on ivig doses of mg- mg/kg of ivig per month, refractory to standard dose ivig and other immunosuppressants. the first case is a year-old-lady with cidp who presented with episodes of ascending sensory disturbance, weakness, and diplopia. she had activity related fluctuations and pre-dose deterioration on g/kg/month ivig. she then had an acute deterioration with mrc sum score dropping from to even with additional plasma exchange. her bilateral foot drop (mrc grade - ) and fluctuations persisted with an increase of ivig to . g/kg/month. she is now clinically stable (ankle dorsiflexion mrc grade - , mrc sum score ) on mycophenolate and g ivig weekly ( . g/kg/month). case is a -year-old male fitness instructor with mmn and sjogren's syndrome. he presented with recurrent proximal and distal weakness that responded to g/kg of ivig and deteriorated with iv methylprednisolone. he had peri-dose fluctuations, intermitted proximal weakness, and persistent foot drop (ankle dorsiflexion mrc grade - ) at . mg/kg/month, worsening to mrc grade - on . g/kg/month and fluctuating between mrc grade - on . g/kg/month. an increase of ivig to g weekly ( . g/kg/month), has resulted in mmn rods scores of / , improved distal power and return to full capacity at work. case is a -year-old man with predominantly upper limb cidp. he received g/kg ivig without any benefit, had no response to doses of plasma exchange, doses of cyclophosphamide or dose of rituximab between and . since , he has received g/kg/month ivig with improvement of mrc sum score from to . these cases highlight that some patients require a much higher than conventionally prescribed dose of ivig, and that these doses are tolerated over years without serious adverse events. idiopathic rapid eye movement sleep behaviour disorder (irbd) has been identified as a precursor of alpha-synucleinopathies, such as parkinson's disease, dementia with lewy bodies, multiple system atrophy. several studies linked changes in cutaneous innervation with central nervous system pathology in neurodegenerative disorders. recently small fiber neuropathy and alpha-synuclein deposition in the skin found to be a potential biomarker in parkinson's disease. we evaluated the epidermal innervation of irbd patients and age and sex-matched controls from skin punch biopsies from the distal leg using pgp . immunohistochemistry. furthermore, a battery of clinical examinations were performed on patients and controls alike, including structured interviews, clinical motor and non-motor questionnaires and rating scales (e.g. unified parkinson's disease rating scale [updrs], non-motor symptoms questionnaire [nms-quest] and beck depression inventory, epworth sleepiness scale, evaluation of cognitive and olfactory functioning as well as blood samples. irbd patients, compared to controls, showed a significant reduction in intraepidermal nerve fiber density (p = . ), whereas the axon swelling ratio, did not differ between groups. patients with irbd reported non-motor symptoms more frequently than controls (updrs i, nms-quest). olfaction and daytime sleepiness differed between both groups, whereas there were no differences regarding cognition. these in vivo findings demonstrate small fiber neuropathy in irbd patients that are associated with non-motor symptoms indicating that peripheral abnormalities may occur early in irbd. they warrant larger scale longitudinal studies in order to investigate their prognostic value. katz j , lewis r , spatafora d . california pacific med center, san francisco, usa; cedars-sinai medical center, los angeles, usa; neuropathy action foundation (naf), santa ana, usa. multifocal motor neuropathy (mmn) is a rare condition that affects . in every , individuals worldwide and is associated with motor dysfunction and moderate to severe disability. the neuropathy action foundation conducted a global survey to determine the impact of mmn on patient quality of life (qol) and gaps in patient/provider educational needs. the first global mmn qol survey was an item internet questionnaire available between january and july , . the survey focused on three primary areas: timely and accurate diagnosis, the efficacy of treatment, and the impact of the disease on patients qol. the survey was completed by patients from countries. the majority of respondents said they were diagnosed between the ages of and years ( . %), more than % reported that it took more than one year to be diagnosed and more than % reported that it took - years or longer to be accurately diagnosed. with respect to treatment options : . % reported receiving intravenous immune globulin and . % reported receiving subcutaneous immune globulin therapy. other therapies being used to treat mmn were gabapentin ( . %), and pregabalin ( %). almost half ( . %) said that mmn often impacts their overall schedule. half of the participants reported that mmn often or always interferes with their employment; % had difficulty typing on a computer or using a telephone, . % had trouble concentrating, and . % said they had to work really hard to pay attention or else they would make a mistake. this is the first assessment of mmn from a patient's perspective. the survey highlighted critical issues relating to the diagnosis, management, and impact on the qol of individuals with mmn. the data also identified gaps and insights in provider education relating to proper diagnoses and management of the condition from a patient's perspective. katz j , levine t , dimachke m , barohn r . forbes norris center, san francisco, ca, usa; phoenix neurological institute, phoenix, az, usa; kansas university medical center, kansas city, ks, usa. in the united states, cidp cases are submitted to insurance companies to determine whether ivig therapy is appropriate. this is done using specified diagnostic criteria, which reduce diagnosis to a boolean analysis, where a disease can only be present or absent. this leaves no room for uncertainty, even when it truly exists. boolean criteria are useful for clinical trials, but fall short where real decisions are made under uncertainty and based on perceived cost/benefit analysis. this project attempts to elucidate root causes of uncertainty and to find solutions to this dilemma. we asked cidp experts to select a single diagnosis in cases where ivig was approved using the submitted case records. while there was agreement on many cases, in the five most "uncertain" cases no more than reviewers agreed on a single condition, who chose up to four separate entities. among these, at least three reviewers diagnosed an immune neuropathy in all five. the root cause of the disagreement, to a large degree was unclear documentation (aunts) which consisted of pasted, missing, and disorganized data. reviewers missed useful information, admitting it was too difficult to fully parse records. to resolve uncertainty, reviewers admitted to discounting certain reported datum to help fit the entity they suspected, such as reported therapeutic responses or certain electrodiagnostic/exam findings. other disagreement, however, reflected the complexity of neuropathy diagnosis, such as knowing if improvement was due to natural history or treatment, unawareness of rare presentations, or analyzing a true uncle (complex case). reviewers used bayesian (select most likely diagnoses from a list) and fuzzy logic (compare best fits to base cases). when the "best" diagnosis did not fit the base case, they had to re-interpret the data. improving review procedures requires eliminating aunts by collecting all key information and simplifying how records are presented. it also needs more advanced data methods to analyze common and rare borderline presentations (uncles like mama v pma, cidp v cspn, etc..), developing diagnostic algorithms that address real uncertainty, educating prescribers and patients on process, and creating systems that measure outcomes longitudinally after induction or tapering of therapy. keisuke y , miyuki m , motoi k , susumu k . department of neurology, faculty of medicine, kinki university, osaka, japan. anti-ganglioside antibodies are closely associated with clinical phenotype and specific symptoms in acute immune-mediated neuropathies. igg anti-gq b antibodies are specifically associated with miller fisher syndrome (mfs), bickerstaff brainstem encephalitis (bbe) and guillain-barré syndrome (gbs) with opthalmoplegia (gbs-op). in addition, ganglioside complexes (gscs) containing gq b also can be targets in such diseases, and might be associated with the clinical features. however, factors regulating clinical phenotype in those gq b-associated antibodies-positive diseases have not yet been known. for investigating the differences of antibody reactivities among those diseases, we examined, using combinatorial glycoarray, igg antibodies to ten individual glycolipids [gm , gm , gm , gd a, gd b, gq b, galactocerebroside (gal-c), lactosylceramide (laccer), ga , sulfatide] and glycolipid complexes consisting of two of the glycolipids listed above in sera from patients with gbs-op who were positive for anti-gq b antibody by elisa (gbs-op-gq b), patients with mfs with the clinical triad (opthalmoplegia, ataxia, and areflexia), and patients with bbe. by combinatorial glycoarray, overall sensitivity of antibodies to gq b and gscs containing gq b was . % ( / ) in gbs-op-gq b, . % ( / ) in mfs, and . % ( / ) in bbe, respectively. there were no significant differences in antibody reactivities between mfs and bbe. it is notable that antibodies to gscs containing gd b were more frequently found in gbs-op-gq b patients than in mfs or bbe patients (e.g., gd b/sulfatide: p= . and p< . , respectively). presence of the antibody reactivities to gscs containing gd b may possibly be related with clinical features of gbs-op-gq b, including frequent need of artificial ventilation. kennedy r , , , carroll k , , paterson k , ryan mm , , , mcginley jl , . royal children's hospital, parkville, australia; university of melbourne, parkville, australia; murdoch childrens research institute, parkville, australia. problems with walking and footwear fit are often reported by children and adolescents with charcot-marie-tooth disease (cmt). a cross-sectional, case controlled study of gait was conducted in children with cmt and typically developing (td) children. gait was assessed barefoot and in two types of the participants' own typical footwear; optimal (e.g. athletic shoes) and suboptimal (e.g. slip-on footwear). the aims were to determine differences in spatio-temporal (s-t) gait variables between children with cmt and td children; and to investigate the effect of footwear choices. twenty-nine independently ambulant children aged - years with confirmed genetic or clinical diagnoses of cmt, and age and gender matched td children participated (mean age . years; males). exclusion criteria included developmental disorders, other neuromuscular conditions or musculo-skeletal diseases that could affect gait, and lower limb injury or surgery in the preceding months. assessment included s-t gait patterns, footwear characteristics, metre run, and cmtpeds. gait was assessed at self-selected speed with an electronic walkway (gaitrite™), with trials for each condition. the primary gait variable assessed was speed; other variables included step length, step time, cadence, base of support width and step-to-step gait variability. across all footwear conditions children with cmt walked more slowly ( regeneration of cutaneous unmyelinated axons is known to be slowed in dm-patients and after -months, the density of intraepidermal nerve fibers (ienf) does not return to baseline levels after chemical or mechanical axotomy. however, the long-term outcome of regeneration in dm or control subjects is not known. additionally, it is not clear if the regeneration of sensory distal axons is length-dependent. here we measured the rate of axonal regeneration -months after chemical denervation using a capsaicin model in dm patients (n= / dm /dm ) without neuropathy, and controls. dm skin punches were performed at distal thigh at baseline, -hours post-capsaicin, and at , , and days. blood glucose and hgba c were measured at baseline, , and days. healthy controls had skin punches at both distal leg and proximal thigh at baseline, after capsaicin chemical axotomy, and days , , and . regeneration rate was significantly higher at the thigh in healthy controls ( . fibers/mm/day ( % ci: . - . fibers/mm/day) compared to dm (p= . ), but no difference between dm ( . fibers/mm/day % ci: . - . fibers/mm/day) or dm ( . fibers/mm/day % ci: . - . fibers/mm/day) (p= . ). comparing regeneration rate at different time intervals, showed that regeneration was significantly slowed between day and dm patients, while it continued with the same rate in controls. blood glucose or hga c had no effect on regeneration rate. ienfd returned to baseline in controls by -months ( % of baseline) while it is did not in dm subjects, %/ % (dm /dm ) of ienfd baseline, (p= . dm vs. controls). there was no difference in regeneration rate ienfd %-baseline by -months at distal leg and proximal thigh in controls (p= . ). these results suggest that the rate and outcome of regeneration is independent of the length of the axon. additionally diabetic patient have incomplete nerve regeneration after months regardless of diabetes type or the level of glycemic control. regeneration of axons slowed down over time in patients with dm and reached a plateau after days. kiessling p , lledo-garcia r , watanabe s , langdon g , tran d , bari m , christodoulou l , price g , smith b , byrnes w , brock m , jolles s . ucb pharma, monheim, germany; ucb pharma, slough, uk; ucb pharma, braine-l' alleud, belgium; ptx solutions ltd, london, uk; ucb pharma, raleigh, durham, usa; university hospital of wales, cardiff, uk. ucb is a humanised high-affinity monoclonal igg antibody developed to bind human neonatal fc receptor (fcrn), selectively inhibiting igg salvage and recycling. conditions such as myasthenia gravis (mg) are characterised by pathogenic igg autoantibodies; inhibition of fcrn may provide a suitable therapeutic approach. this phase i, double-blind, dose-escalating, first-in-human study (nct ) evaluated the safety and pharmacology of ucb . forty-nine healthy adults (mean age years, range - ) were randomised and received a single dose of intravenous (iv) or subcutaneous (sc) placebo (n= and n= , respectively), or a single dose of iv or sc ucb ( , or mg/kg; n= per dose, per administration). subjects were followed up until day . one placebo iv subject did not complete the study. twenty-seven of subjects ( %) receiving ucb , and / ( %) receiving placebo, reported ≥ treatment-emergent adverse event (teae) of mild/moderate intensity. severe teaes occurred in four subjects, all in the ucb mg/kg iv group (headache [n= ], back pain [n= ]). no serious aes occurred. incidence of infections was similar with ucb and placebo. the most frequently reported infection was nasopharyngitis. treatment-related teaes were reported by % of subjects receiving ucb and % receiving placebo: the most common in the ucb -treated groups were headache ( / ; %) and vomiting ( / ; %); these occurred more frequently with the iv than sc route. non-linear increases in ucb plasma concentration-time profile with increasing dose were observed with ucb . serum igg was reduced in a dose-depended manner with ucb iv and sc: decreases from baseline to day with ucb iv were . %, . % and . % for , and mg/kg doses, respectively, and . %, . % and . %, with ucb sc doses, respectively. these data indicated that the fcrn inhibitor ucb effectively reduced serum igg, with sc administration generally better tolerated than iv. further to these observations, the efficacy, safety and tolerability of ucb sc for chronic-intermittent treatment of moderate-to-severe mg are being evaluated in an ongoing phase ii, multi-centre, randomised, double-blind, placebo-controlled study (eudract - - middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, very little information has become available on this syndrome since it first erupted in . this study aimed to evaluate the frequency of neurological complications and their clinical presentations in mers. we reviewed the medical records of all patients who were diagnosed with laboratory-confirmed mers coronavirus (cov) infections and subsequently admitted to a single reference center for mers treatment during the outbreak in korea. in total, patients ( . %) reported neurological symptoms during or after mers-cov infection. the potential diagnoses in these cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, critical illness polyneuropathy or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, but were instead delayed by - weeks. neuromuscular complications were not rare in mers-cov-infected patients, and they may have previously been underdiagnosed. understanding neurological manifestations is important in an infectious disease like mers, because evaluation is frequently limited during treatment, but it can interfere with prognosis and sometimes require modification of treatment. kim hj , hyun jk , kim tu . dankook university, cheonan, south korea. the diagnosis of carpal tunnel syndrome (cts) is based on clinical symptoms, physical examinations and supported by nerve conduction study (ncs). ultrasonographic examinations can be performed to assess peripheral nerves with less discomfort and the surrounding anatomic structures. while the usefulness of ultrasonography (usg) in the cts has been reported, no study to date has compared the diagnostic utility of various usg findings for cts. we investigated the correlation of various usg findings to the clinical symptoms/signs and ncs findings in patients with cts. twenty-eight hands ( patients) with cts based on electrodiagnostic criteria and clinical symptoms such as tingling sensation or pain in the first to third fingers, burning sensation, paresthesia and weakness of hand grip power. all subjects were examined with usg. cross-sectional area (csa) and flattening ratio (fr) of the median nerve was calculated at level of radio-ulnar joint, pisiform and hamate. swelling ratio of the median nerve and palmar displacement of the flexor retinaculum was also calculated. clinical assessment was conducted using the boston carpal tunnel questionnaire (bctq) scale and historical-objective (hi-ob) scale. the analysis of correlation between usg findings and clinical symptom scales/ncs findings was performed using correlation analysis. the csa of the median nerve at level of radio-ulnar joint was significant correlated with bctq scale, hi-ob scale, distal motor latency, and conduction velocity (cv). the csa of the median nerve at level of pisiform was significantly correlated with hi-ob scale, distal motor latency, and cv. the fr of the median nerve at level of radio-ulnar joint was significantly correlated with bctq scale, hi-ob scale, distal motor latency, and cv. the swelling ratio of the median nerve was also significantly correlated with distal motor latency and cv. in patients with cts, csa of the median nerve at level of radio-ulnar joint was most closely related to ncs findings and clinical symptoms. so, csa of the median nerve at radio-ulnar joint might be a complementary tool for the diagnosis of cts. kitaoji t , tsuji y , ashida s , yamada t , ishii r , tanaka a , mizuno t . department of neurology, graduate school of medical science, kyoto prefectural university of medicine, kyoto, japan. the case was a -year-old woman. first, she noticed paresthesia in the right plantar eight months before admission and in the left plantar four months before admission. three months before admission, she developed muscle weakness in her feet. the muscle weakness and paresthesia extended to the lower legs in a few months. twenty days before admission, she experience difficulty in walking. on admission, the muscle weakness was observed in the legs, especially in the right tibial anterior muscle (ta). there was severe sensory disturbance and loss of deep tendon reflex in the legs. she had trouble walking due to the weakness and sensory aphasia. in nerve conduction study (ncs), conduction block was observed between the ankle and popliteal in both tibial nerves. the blood level of angiotensin converting enzyme (ace) was elevated. cerebrospinal fluid analysis was normal. there was no enhancement in the lumbar nerve roots shown on mri. gallium- scintigraphy showed hot spots on bilateral hilar lymph nodes and mediastinal nodes and biopsy of mediastinal nodes showed non-caseating epithelioid granuloma. therefore, we diagnosed her with sarcoid peripheral neuropathy by sarcoidosis. by using the nerve ultrasound, partial spindle-shaped nerve enlargement was observed at the part of conduction block in the left tibial nerve. we started the treatment with methyl prednisolone ( mg, days) and oral prednisolone therapy ( mg/kg/day). after treatment, the paresthesia and muscle weakness in the legs had gradually improved. the partial enlargement in the left tibial nerve also improved on the -hospital day. in ncs, the conduction block improved, however, the compound muscle nerve potential of tibial nerve decreased because of axonal damage. this partial spindle-shaped nerve enlargement by using ultrasound has never been reported in sarcoid peripheral neuropathy before. the nerve ultrasound may be useful for evaluation of therapeutic effect of sarcoid peripheral neuropathy. peripheral nerve injuries are still debating problems in the world because of poor recovery. there is absolutely a need for new therapeutic agents to improve outcome by altering nerve regeneration. there are some studies in the literature about some therapeutic agents that used in the cases of peripheral nerve injuries. despite these studies, an agent with clinical use has not been presented yet. in this experimental study, we aimed to analyze the effects of curcumin (cur) in the cases of peripheral nerve injuries. forty rats were randomly and equally divided into four groups. the first group was control group. rats in this group were not operated. right sciatic nerve injuries were performed to the other groups. the second group was operation group with no therapeutic agent. the third group was operation and local cur applied group. the fourth group was operation and systemic cur applied group. electrophysiological evaluations were performed with electroneurography (enog) before and after the surgeries. systemic use of cur although caused improvement in the enog values but could not make a positive contribution to the nerve regeneration statistically. additionally local use of cur made negative effect to the nerve regeneration statistically. according to our statistical results we could not recommend cur as a nerve protective agent. klein cj , wu y , jentoft me , mer g , spinner rj , dyck pjb , dyck pj , mauermann ml . mayo clinic, rochester, usa. intraneural perineurioma is a hypertrophic peripheral nerve tumor having immunoreactivity to epithelial membrane-antigen, negative for s- . the origin of perineurial cells is debated to be similar to meningeal cells. ip does not metastasize, but motor deficits accumulate over time from tumor growth in nerve and plexus. after schwannomas and neurofibromas, perineuriomas are the most common nerve tumor of young adults. a chromosome q deletion has been reported in one patient. we identified ip cases from our previously published clinical cohort with available flash frozen ip tissue for dna isolation. wes with cnv analysis and cgh microarray analysis (agilent x k superprintg ) were performed on extracted dna; had available germline dna (lymphocytes and buccal tissue). we compared the exome data against online and in-house control data (∼ , ) examining variants less than . frequencies, predicted damaging or nonsynonymous. wes identified three novel, heterozygous, damaging mutations in tumor necrosis factor receptor-associated factor (traf ) in of ( %) cases; p.l p (n= ), p.h r (n= ) and p.s r (n= ). mutations were within the wd domain, p.l p, p.h r within exon and p.s r within exon , and mapped to a limited region of traf with protein structure modeling. two of cases ( . %) showed macroduplications/deletions on multiple chromosomes, including chromosome , confirmed with cgh microarray analysis and cnv results from exome data analysis. four of ( %) had no discovered mutation. age of onset or severity did not correlate with type of mutations. this study provides strong evidence that traf is a specific tumor driver of ip. mutations in traf are also linked to benign intracranial meningiomas suggesting a shared pathogenesis and close origins of perineurial and meningeal cells. study supported by: mayo foundation and the mayo center of individualized medicine. klein i , , bobylev i , , lehmann hc , . university hospital cologne, cologne, germany; center for molecular medicine cologne (cmmc), cologne, germany. peripheral neuropathy is a common side effect of paclitaxel. clinical evidence suggests that the delivery mechanism of paclitaxel formulations influence time course and severity of paclitaxel induced peripheral neuropathy. in a preclinical model we studied access, distribution and toxicity of two paclitaxel formulations (nanoparticle albumin-bound paclitaxel (nab) and solvent-based paclitaxel) in the peripheral nervous system (pns). c bl/ mice were treated with mg/kg or mg/kg of nab-paclitaxel or solvent based paclitaxel. kinetics of paclitaxel in neurons was assessed by a newly established immunostaining technique. neurotoxicity was evaluated by functional assays and nerve morphology. paclitaxel accumulated mostly in dorsal root ganglia, whereas distal nerve segments showed only low uptake of paclitaxel. treatment of mice with the two paclitaxel formulations resulted in paclitaxel uptake mostly in nf + larger fiber neurons. in ib +, and cgrp+ small fiber neurons, paclitaxel was less frequently detected. nab-paclitaxel was incorporated more rapidly compared to solvent-based paclitaxel but neurons also showed a faster clearance of nab-paclitaxel compared to solvent based paclitaxel. functional assays and nerve conduction studies indicated that nab-paclitaxel was less neurotoxic compared to solvent-based paclitaxel. this is the first study that characterizes in detail the access of nab-paclitaxel and solvent based paclitaxel into the pns. our findings have important implications to understand the pathomechanisms of paclitaxel induced neurotoxicity and to develop neuroprotective strategies by preventing access of paclitaxel to the pns. the aim of our study was to investigate the etiology of neuropathy in patients with rheumatoid arthritis (ra). subjects were neuropathy patients with ra admitted to our department. laboratory investigations, nerve conduction studies (ncs) and sural nerve biopsy were performed. mean patient age was . years (range, - years), and mean disease duration was . years (range, - years). clinical diagnosis for neuropathy was rheumatoid vasculitis (rv) in patients, rv with acute motor axonal neuropathy (rv-aman) in and chronic inflammatory demyelinating polyneuropathy with ra (ra-cidp) in . rheumatoid factor ( / ) and rheumatoid arthritis particle agglutination ( / ) was high and c tended to be lower in the rv group (rv and rv-aman). anti-ganglioside antibodies were examined in patients, with positive results in . an rv-aman case was diagnosed with motor-dominant clinical presentation and the presence of anti-galnac-gd a immunoglobulin (ig)g antibody. no other cases with rv were examined for anti-ganglioside antibodies. positive results for anti-gm and gm igg antibody were seen in one ra-cidp patient. we evaluated sural/median (s/m) ratio) for sensory nerve action potential (snap). s/m ratio was low in rv cases ( / ) and high ( / ) in immune-mediated cases, suggesting a so-called normal sural abnormal median pattern in immune-mediated neuropathies. the rv-aman case showed a moderate value in s/m ratio. nerve biopsy revealed thinly myelinated nerve fibers in ra-cidp cases compatible with demyelination, while the rv group showed the typical pathology for necrotizing vasculitis. rv cases were treated with prednisolone (psl), intravenous methylprednisolone, intravenous cyclophosphamide and increased psl dose. ra-cidp and rv-aman were treated with intravenous ig. in conclusion, neuropathy in ra can be divided into vasculitic and immune-mediated groups. ncs, and the s/m ratio of snap with some laboratory parameters in particular, may be of use in differential diagnosis and deciding treatment strategies. koh s , wong shj , loh kw , chng ysk , pawa c , ei ma , lee bjh , subramaniam t , umapathi t . national neuroscience institute, singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university singapore, singapore; tan tock seng hospital, singapore; khoo teck puat hospital, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful autonomic neuropathy that develops with abrupt improvement in glycaemic control. typically, type or dm patients on insulin or oral hypoglycaemic agents (ohga), present with painful neuropathy and autonomic dysfunction within weeks of rapid improvement in glucose control. current emphasis to achieve good glycaemic control rapidly may inadvertently increase incidence of tind, hence the impetus to understand risk of over-zealous glycaemic control. we therefore set out to study the occurrence of tind in a dm cohort of a tertiary hospital. we screened all patients who had two hba c measurements between and . during this period, approximately patients were seen per year. we found patient-encounters that showed hba c decrease of ≥ % over months or ≥ % over months. we then used a structured checklist of tind symptoms to shortlist cases. these case-encounters were scrutinised and classified as; 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or uncertain temporal relationship to decrease in hba c; unlikely tind: alternative explanation exists for symptoms. only one case was deemed 'probable tind'-a middle-aged man with newly diagnosed type dm who presented to emergency department with palpitations and worsening 'frozen feet' sensation that disturbed sleep. his hba c decreased by . % in weeks. his symptoms improved within a month with neuropathic pain medications and resolved months later. he also developed maculopathy and proliferative retinopathy. ten months later, he developed significant proteinuria. four other cases were classified as 'possible tind' while the remaining were unlikely tind. our study is limited by retrospective design and reliance on hospital records. nevertheless, our findings suggest that tind is uncommon in a general cohort of dm patients. on the other hand, the number of patients with painful neuropathy and acute dysautonomia symptoms contemporaneous with rapid decline in hba c raises the intriguing possibility that forme fruste of tind exists and one should interrogate the rate of hba c decline in dm patients with these symptoms. koike h , kadoya m , kaida k , ikeda s , kawagashira y , iijima m , kato d , ogata h , yamasaki r , matsukawa n , kira ji , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; national defense medical college, tokorozawa, japan; nagoya city university graduate school of medical sciences, nagoya, japan; kyushu university, fukuoka, japan. we investigated the morphological features of chronic inflammatory demyelinating polyneuropathy (cidp) with autoantibodies directed against paranodal junctional molecules, particularly focusing on the fine structures of the paranodes. sural nerve biopsy specimens obtained from cidp patients with anti-neurofascin antibodies and patient with anti-contactin antibodies were assessed. these antibodies were examined using sera obtained from patients with cidp who fulfilled the criteria of the european federation of neurological societies/peripheral nerve society. thirteen cidp patients without these antibodies were also examined to compare pathological findings. characteristic light and electron microscopy findings in transverse sections from patients with anti-neurofascin and anti-contactin antibodies indicated a slight reduction in myelinated fiber density, with scattered myelin ovoids, and the absence of macrophage-mediated demyelination or onion bulbs. teased-fiber preparations revealed that segmental demyelination tended to be found in patients with relatively high frequencies of axonal degeneration and was tandemly found at consecutive nodes of ranvier in a single fiber. assessment of longitudinal sections by electron microscopy revealed that detachment of terminal myelin loops from the axolemma was frequently found at the paranode in both anti-neurofascin and anti-contactin antibody-positive cidp patients compared with antibody-negative cidp patients. patients with anti-neurofascin antibodies showed a positive correlation between the frequencies of axo-glial detachment at the paranode and axonal degeneration, as assessed by teased-fiber preparations (p < . ). in conclusion, paranodal dissection without classical macrophage-mediated demyelination is the characteristic feature of patients with cidp with autoantibodies to paranodal axo-glial junctional molecules. koike h , ikeda s , takahashi m , kawagashira y , iijima m , misumi y , ando y , ikeda si , katsuno m , sobue g . nagoya university graduate school of medicine, nagoya, japan; kumamoto university, kumamoto, japan; shinshu university hospital, matsumoto, japan. peripheral neuropathy is the cardinal feature of familial amyloid polyneuropathy (fap), but its mechanism has not been fully elucidated. we used electron microscopy to examine schwann cells and endoneurial microvessels. sural nerve biopsy specimens from fap patients with transthyretin val met mutation were assessed. patients were consisted of early onset cases from endemic foci and late onset cases from non-endemic areas. loss of nerve fibers with or without neighboring amyloid deposition was a common feature. the amount of amyloid deposition was greater relative to the extent of nerve fiber loss in early onset cases than in late onset cases. the atrophy of schwann cells, particularly nonmyelinating cells, that were apposed to amyloid fibrils was more conspicuous in early onset cases than in late onset cases. the numbers of endothelial cell nuclei, endothelial cell profiles, and occluded microvessels were significantly increased in the fap patients compared with patients with nutritional/alcoholic neuropathies (p < . , . , and . , respectively). findings suggestive of the disruption of blood-nerve barriers, such as the loss of tight junctions and the fenestration of endothelial cells, were also more frequently found in the fap patients (p < . ), irrespective of the presence or absence of amyloid deposition. in conclusion, these findings suggest that direct insult of amyloid fibrils causes schwann cell damage resulting in the predominant loss of small-fiber axons characteristic of early onset cases. in addition, vasculopathy may also participate in the pathogenesis of neuropathy, particularly in late onset cases. kolb n , smith ag , singleton jr , beck s , howard d , dittus k , karafiath s , mooney k . university of vermont, burlington, vt, usa; university of utah health, slc, ut, usa. chemotherapy induced peripheral neuropathy (cipn) is a major cause of morbidity due to numbness, pain, and gait instability. this prospective study compares the current standard care for cipn symptom management to a new care delivery model which utilizes an automated symptom tracking program paired with a nurse practitioner led intervention triggered by moderate to severe symptoms. all participants beginning taxane or platin based chemotherapy called a telephone based automated symptom tracking program daily (symptom care at home -sch) to report chemotherapy related numbness and tingling. sch tracked the presence and severity of neuropathic symptoms and their interference with activities of daily living (adls) on a - scale. participants were randomized to two groups. the usual care (uc) group was advised to call their oncology provider for recommendations on symptom management. in the nurse practitioner (np) group, when symptom severity was ≥ participants received automated self care strategies and a call from a nurse practitioner to provide treatment recommendations based on consensus guidelines. patients participated in the study. mean duration of follow up was . ± . days with . ± . calls. the np group had fewer days with any neuropathic symptom ( . % ± . vs. . % ± . , p= . ), with moderate to severe neuropathic symptoms ( . % ± . vs. . % ± . , p< . ) or days of distress from neuropathic symptoms ( . % ± . vs. . % ± . , p= . ). on days with moderate to severe symptoms participants also reported burning ( . ± . %), weakness ( . ± . %), balance problems, ( . ± . %), and tripping ( . ± . %). there was no significant difference between groups in the interference in adls (np . ± . vs. uc . ± . , p= . ). overall the automated telephone system effectively identified neuropathy symptoms and their severity. compared to usual care in which patients must independently reach out to their care team for symptom management, sch is effective in decreasing symptom prevalence, severity and distress. kouton l , kremer l , tard c , morales r , kuntzer t , attarian s , boucraut j , delmont e . referral centre for als and neuromuscular diseases, marseille, france; neurology department, strasbourg, france; neurology department, lille, france; neurology department, montpellier, france; neurology department, lausanne, switzerland; immunology laboratory, marseille, france. antibodies against proteins of the node of ranvier have been recently described in severe chronic inflammatory demyelinating polyradiculoneuropathies (cidp). they target paranodal proteins, namely contactin (cntn ) and neurofascin (nf ). cell-based assay and elisa are available in research, but no gold standard technic is admitted for the detection in routine of these antibodies. our objective was to evaluate if flow cytometry analysis is an efficient technic to detect antibodies against ctn and nf in a large cohort of cidp patients. flow cytometry analysis were performed on a bd facs-diva. human embryonic kidney (hek) cells were transfected either with nf or cntn . sera were diluted / . antibodies anti-cntn or nf were revealed using fitc conjugated anti human igg antibodies. delta mfi (mean fluorescence intensity) was calculated as mfi of transfected cells less mfi of non-transfected cells. measures were normalized using positive controls and negative controls from healthy blood donors. sera of cidp patients from different french neuromuscular referral centres were analysed with flow cytometry. respective delta mfi were (standard deviation ) and (standard deviation ) for antibodies against nf and cntn in cidp antibodies negative patients. antibodies against nf were found in patients (respective mfi , , ) and against cntn in two other patients (respective mfi and ). isotype of these antibodies was igg in patients and igg and igg in the remaining patient. all the patient had severe cidp. four patients had poor response to intravenous immunoglobulins (ivig) and have been treated with immunosuppressive drugs. as usually reported, the patient with anti-nf antibodies had postural tremor. flow cytometry seems effective to detect antibodies against nf and cntn . compared to other assays, benefits of flow cytometry are: to analyse a large number of sera in the same time and to give objective numerical results expressed in mfi that can be compared to the results of other samples. further studies are needed to confirm that flow cytometry can be the best test to assess antibodies against cntn and nf in routine. the mechanism by which intravenous immunoglobulins (ivig) improves peripheral nerve function in multifocal motor neuropathy (mmn) is unknown. the rapid clinical improvement following ivig could be related to blocking complement deposition on gm epitopes, change in ion-channel properties of affected motor axons, or both. the present study investigated median nerve motor excitability parameters at ∘ c just before ivig administration as well as at the peak of clinical improvement in patients with mmn. the investigated nerves were characterized either by conduction block (n= ), demyelinative slowing without block (n= ), or motor axon loss (n= ). the results of motor excitability testing in mmn showed no difference between pre-and post ivig recordings. clinical assessment of apb muscle showed increase in mrc score in patients and decrease in patient after ivig administration. in patients mrc score of the apb remained the same. those findings indicate that clinical changes following ivig administration are not related to excitability parameters of affected motor axons in mmn. where impulse conduction is blocked or markedly slowed in motor axons but is normal in sensory axons. sensory symptoms or signs are usually absent but have occasionally been reported in skin areas innervated by nerves with prominent motor axon loss. although the mechanism of selective motor involvement in mmn is unresolved, it may be related to differences in antigenic properties between motor and sensory axons or differences in biophysical properties. the objective of the present study was to compare ion-channel activity in both motor and sensory axons of nerves affected by mmn. affected nerves had to have motor conduction block, demyelinative slowing on motor ncs, or motor axon loss, whereas sensory ncs had to be normal. we performed excitability tests of motor and sensory axons in affected median nerves of mmn patients and healthy controls at ∘ c. conditioning and test stimuli were delivered at the median nerve at the wrist; cmaps were recorded from the thenar muscle and snaps from the rd digit. results of motor excitability testing in mmn showed fanning-out of threshold electrotonus, decreased i/v slope, and increased superexcitability, all compatible with persistent hyperpolarization of resting membrane potential in motor axons. sensory excitability testing in mmn showed decreased subexcitability but was otherwise normal. this may indicate minimal involvement of sensory axons in mmn. krarup c , , moldovan m , , alvarez s , ciano c , pisciotta c , pareyson d . university of copenhagen, copenhagen, denmark; rigshospitalet, copenhagen, denmark; fondazione irccs istituto neurologico c. besta, (incb), milan, italy. mutations in the gene coding for myelin protein zero (mpz, p ) are associated with different forms of charcot-marie-tooth (cmt) disease. we describe a family harboring a frameshift mutation (c. dela / p.asp thrfster ) in the p gene, predicted to result in a nonfunctional p truncated very early in the extracellular domain. this offered the rare opportunity to assess the consequences p deficiency in absence of the potential gain-of-function effects of the mutations itself. conventional conduction studies and multiple measures of nerve excitability by "threshold tracking" were carried out in heterozygote parents (aged and ) and their two homozygote sons (aged and ). in the homozygous patients, all distal limb cmaps and snaps were absent. for neurophysiological assessment, the spinal accessory nerve was stimulated at the neck and cmap was recorded over the upper trapezius muscle. eight normal subjects, mean age , were used as control. the two sibs showed a severe phenotype with early onset, severe scoliosis, complete loss of distal movements and relevant proximal weakness, cmt examination score (cmtes) - / ; both heterozygous parents had very mild adult-onset neuropathy with cmtes < / . control subjects had a trapezius cmap with a latency of . ms and an amplitude of . mv. heterozygotes had a mild cmt type b phenotype, with a cmap latency of . ms and an amplitude of . mv whereas the homozygotes had a severe neuropathy with a cmap latency of . ms and an amplitude of . mv. consistently, the homozygotes had a more severe impairment in excitability with a rheobase of . ma as compared to . ma in the heterozygote and . ma in controls. deviations in excitability measures were similar to our previous reports in p +/− and p −/− mice. mathematical modeling, indicated both altered passive cable properties due to dysmyelination and depolarizing features with increased na+ currents. our data suggest that p deficiency is associated with impaired axonal na+ channel function, arguing for the translational value of na+ channel blocker treatments as found in p null mouse models. krishnarajah s , divino v , mallick r , dekoven m . csl behring, king of prussia, pa, usa; quintilesims, fairfax, va, usa. chronic inflammatory demyelinating polyneuropathy (cidp) is a rare neurological disorder of the peripheral nervous system. the objectives of this retrospective real-world study were to compare demographic and clinical characteristics among cidp cases and matched controls and to assess cidp treatment utilization. adults newly diagnosed with cidp between / / and / / were identified in the quintilesims pharmetrics plus health plan claims database (first diagnosis date termed the index date). eligibility requirements were: confirmation of cidp (second cidp diagnosis or initiation of cidp therapy) within year of initial diagnosis, continuous health plan enrollment in the months prior to diagnosis (the pre-index) and the years following diagnosis (the follow-up), and no cidp diagnosis or use of cidp therapy in the pre-index. a total of , cidp cases met the study eligibility criteria. cases were direct-matched to controls based on age, gender, region, health plan, and payer type at index, and pre-index charlson comorbidity index score. the final sample consisted of cases matched to controls (both: mean [sd] age . [ . ]; . % male; . % commercially-insured). alternative pre-index diagnoses among cases included inherited neuropathies ( . %) and chronic acquired polyneuropathies ( . %). in the pre-index, neuropathic pain ( . % vs. . %), back pain ( . % vs. . %), and use of opioids ( . % vs. . %) and anti-convulsants ( . % vs. . %) were significantly higher among cases compared to controls (p< . for all). median total pre-index healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . ). over the follow-up, median total healthcare costs were . x higher for cases than controls ($ , vs. $ , , p< . [mean $ , and $ , ] ). cidp-related therapy costs accounted for . % of total healthcare costs for cases. the majority of cases ( . %) initiated cidp therapy over the follow-up, in a mean of . ( . ) days from initial diagnosis. half ( . %) of cases initiated treatment with corticosteroids only, while . % initiated ivig only. over the follow-up, . % of cases used any corticosteroid, while . % used any ivig. our findings suggest a substantial clinical and economic burden of cidp compared to matched controls. corticosteroids and ivig were most commonly used to treat cidp. kronlage m , baeumer p, pitarokoili k , schwarz d , schwehr v , godel t , heiland s , gold r , bendszus m , yoon ms . department of neuroradiology, heidelberg university hospital, germany; department of neurology, st. josef hospital, ruhr university of bochum, germany. objective: to evaluate large coverage magnetic resonance neurography (mrn) in chronic inflammatory demyelinating polyneuropathy (cidp). methods: in this prospective study patients with cidp and healthy controls were examined by a standardized mrn protocol at tesla. lumbosacral plexus was imaged by a t -weighted d-sequence ( mm isotropic voxel size); peripheral nerves of the upper and lower extremity by axial t -weighted turbo-spin-echo sequences ( . x . mm in-plane resolution). lesions were characterized by nerve cross sectional area (csa) and t -weighted signal (nt ). additionally, t -relaxometry of the sciatic nerve was performed using a multi-spin-echo sequence. all patients received a complementary electrophysiological exam. results: patients with cidp exhibited increased nerve csa and nt compared to controls (p < . ) in a proximally predominating pattern. roc analysis revealed best diagnostic accuracy for csa of the lumbosacral plexus (auc = . ) and nt of the sciatic nerve (auc = . ). csa correlated with multiple electrophysiological parameters of demyelinating neuropathy (f-wave latency, nerve conduction velocity) of sciatic and median nerve, while nt only correlated with f-wave latency of sciatic and not median nerve. t -relaxometry indicated that mr-signal increase in cidp was due to increase in proton-spin-density (p < . ), and not increase in t -relaxation time. conclusion: both nt and csa might aid in diagnosis of cidp, but csa correlates more robustly with electrophysiological parameters. since best diagnostic accuracy was shown for proximal nerve locations, mrn may be a useful complementary tool in select cidp cases. kühnemund j , wetzel c , bégay v , moshourab r , lewin gr . mdc & bih, berlin, germany; mdc, berlin, germany; charité, berlin, germany. damage of peripheral sensory nerves due to diabetes, herpes zoster infection, chemotherapy or trauma can cause chronic neuropathic pain. common symptoms include increased pain sensation (hyperalgesia), touch-induced pain (allodynia), paresthesia and spontaneous pain. we currently have poor understanding about the underlying molecular mechanisms at the peripheral level and treatment of patients suffering from neuropathic pain is inadequate. recent meta-analysis studies show that common first-line medications only yield nnts (numbers needed to treat) between . to . . it is still unclear to what extent allodynia can be attributed to changes in the physiological properties of intact sensory afferents. in this study, we aimed to elucidate whether changes occur in intact sensory afferents that innervate the plantar skin of the hind-paw following induction of neuropathic symptoms. this question was of particular interest considering that blocking mechanotransduction in the skin can alleviate mechanical hypersensitivity in neuropathic pain models (wetzel et al nature neuroscience ( ): - ). we used the chronic constriction injury (cci) model in mice which is behaviourally characterized by robust mechanical hypersensitivity. we made electrophysiological recordings from primary afferent neurons which had intact axons passing through the constriction to innervate the plantar skin using an ex-vivo skin-nerve preparation. we were able to record from myelinated afferent fibers and unmyelinated c-fibers with receptive fields in the control uninjured plantar skin as well as plantar skin of cci mice. we will present evidence that changes in the mechanosensitivity of sensory fibers innervating the glabrous skin may contribute to the symptoms of neuropathic pain. neuropathology, national hospital for neurology and neurosurgery, london, uk; irccs foundation "carlo besta" neurological institute, milan, italy; neurogenetics unit, national hospital for neurology and neurosurgery, london, uk; nuffield department of clinical neurosciences, oxford, uk; department of clinical neurophysiology, norfolk and norwich university hospital, uk. hsn secondary to sptlc / is a rare slowly progressive neuropathy resulting in marked sensory loss, especially nociception and significant motor deficit. despite most of the patients having the same c w mutation in sptlc , there is marked heterogeneity in the phenotype. l-serine oral supplementation has been suggested as potential therapeutic candidate however the lack of outcome measures is a major limiting factor in the initiation of a clinical trial. we undertook a natural history study to identify outcome measures that are responsive enough to be used in a clinical trial. the assessments used were cmt neuropathy score (cmtns version and cmtns version rasch modified), mri of calves and thighs, computerised myometry, quantitative sensory testing (qst), comprehensive neurophysiological assessment, proximal thigh skin biopsy for intra-epidermal nerve fibre density (ienfd), plasma dsl levels and patient based questionnaires (neuropathic pain symptom inventory and sf- v ). standardised response mean, srm (mean change/standard deviation of change) was used to compare responsiveness between tests. patients were recruited: with sptlc (c w) and with sptlc mutations. when analysed as a whole cohort, proximal calf mri fat fractions showed the most significant change over months. ienfd, plasma dsl levels, npsi and sf- v showed minimal change or the change was not in the clinically expected direction. for subsets of the remaining assessments which showed the highest responsiveness, the cohort was sub-divided into mild-moderate (cmtns ≤ ) and severe (cmtns> ) subgroups. in the mild-moderate subgroup, the greatest improvement in responsiveness was seen in computerised myometry (ankle plantarflexion: srm=− . and ankle eversion: srm=− . ). in the severe subgroup, qst (vibration detection thresholds on hands: srm=− . and face: srm=− . and pressure pain threshold on the face: srm= . ) and proximal calf mri fat fractions (srm range= . - . ) showed the greatest improvement. focusing on subgroups classified according to disease severity improved the responsiveness of some tests into the highly responsive range with mri still being the best outcome measure. this will reduce the number of participants required to power a clinical trial and might be a possible solution for designing a clinical trial for a rare, slowly progressive disease with a heterogeneous phenotype. kugathasan u , clark aj , suriyanarayanan s , laurá m , wilson e , , kalmar b , greensmith l , , hornemann t , reilly mm * , bennett dlh * . hsn secondary to sptlc / mutation is a slowly progressive sensory motor neuropathy leading to profound sensory loss with variable but often severe motor deficit. the genes sptlc and encode for the essential enzyme serine palmitoyltransferase (spt) which catalyses the rate limiting step in the sphingolipid de-novo biosynthesis. mutations in these two genes alter the substrate specificity of spt leading to the synthesis and accumulation of atypical metabolites called -deoxysphingolipids ( -deoxysl). plasma levels of -deoxysl are raised in hsn patients. deoxysphingolipids have been shown to be toxic in avian and by our group, in mammalian primary drg and motor neuron cultures. firstly, this study looked at the effects of -deoxysl on the survival and neurite integrity of human ipsc derived sensory neurons following exposure to different concentrations of deoxysphingolipids. later, we determined if there was autonomous -deoxysl production in hsn patient ipsc derived sensory neurons. sensory neurons were differentiated from human ipscs using a combination of small molecular inhibitors. deoxysphingolipids were found to be neurotoxic in this model after hours of treatment. in control lines, there is a significant reduction in neuronal survival following treatment with both -deoxysphinganine ( -deoxysa) and -deoxymethylsphinganine ( -doxmethsa). a clear dose-dependent increase in the expression of the axonal injury marker, atf , is seen with both -deoxysphngoid bases. in both instances, -deoxysa is more neurotoxic than -doxmethsa, which is similar to the findings in avian and mammalian primary neuronal cultures. autonomous -deoxysl production is seen in ipsc derived neurons obtained from three different hsn patients with the levels being significantly greater than that seen in multiple control lines. this is the first study to demonstrate that human ipsc derived sensory neurons can be used as an in-vitro model for hsn , providing a great opportunity both to probe the pathomechanisms mediating deoxysphingolipid toxicity and to test potential therapeutic agents. recent studies have demonstrated an association between autoantibodies directed against antineurofascin- (anti-nfasc ) and a subpopulation of cidp patients characterized by sensory ataxia, tremor and poor response to ivig treatment. here we report on the clinical features of three patients who developed acute changes in their phenotype during the course of their neuropathy, a potential clue to recognize a neuropathy with predominantly humoral dysimmunity. case reports: our index patient had developed sensory changes over weeks, followed by irregular locomotion, and muscle weakness with general areflexia. a first run of ivig improved the patient in a week, but he relapsed within days. a second ivig course with prednisone again normalized deficits within days. ivig runs and rituximab were still necessary to treat a nd , and then a rd relapse before obtaining complete improvement. the course of the neuropathy was months. two other patients were encountered with more chronic courses but in whom periods of worsening or improvement suggested a relapsing-remitting neuropathy, either spontaneously or following immunomodulating treatments. in all three, extensive work-ups were negative, anti-nfasc igg were positive, and detailed repeat nerve conduction studies demonstrated fluctuating conduction blocks. discussion: our report underscores that in chronic cidp patients a relapsing-remitting course could be encountered as a key feature of anti-nfasc neuropathy. this not yet described characteristic course could be of value when deciding using rituximab instead of immunomodulating treatments. kusunoki s , morikawa m , kuwahara m , ueno r , samukawa m , hamada y . faculty of medicine, kindai university, osaka-sayama, japan. anti-glycolipid antibodies are often detected in sera from patients with autoimmune neuropathies, such as guillain-barré syndrome (gbs), chronic inflammatory demyelinating polyradiculoneuropathy (cidp), and multifocal motor neuropathy (mmn). not only individual glycolipid antigens but also mixtures of two different glycolipids (glycolipid complexes) are sometimes recognized by serum antibodies. to investigate antibody activities against large number of glycolipid complexes in serum samples from patients with gbs, mmn, and cidp, we examined igm and igg antibodies against glycolipids [gm , gm , gd a, gd b, gq b, galnac-gd a, lm , galactocerebroside (gal-c), asialo-gm (ga ), and sulfatide] and glycolipid complexes consisting of two different glycolipids listed above, by using combinatorial glycoarray. serum was obtained from patients with gbs, patients with cidp and patients with mmn, all in the acute or relapsing phase. serum was also obtained from healthy controls and patients with other neurological diseases. we investigated the relationships between the clinical features and presence of those antibodies. high titers of igg antibodies were detected almost exclusively in gbs patients. in contrast, igm antibodies were frequently present in mmn and gbs. among the anti-glycolipid complex antibodies in gbs, anti-gm /sulfatide, anti-ga /sulfatide, anti-gm /gd a, and anti-gq b/sulfatide igg antibodies were common ( , , , and patients, respectively). igg antibodies against antigens containing gm were significantly correlated with pure motor gbs (p < . ) and those against antigens containing gq b were significantly correlated with gbs with ophthalmoplegia (p < . ). in seven of the patients with anti-gq b/sulfatide complex antibodies, the antibodies were specific to the gq b/sulfatide complex rather than the individual gq b and suldfatide antigens. moreover, four patients did not have antibodies other than those to the anti-gq b/sulfatide complex. in patients with mmn, igm antibodies to antigens containing gm or galnac-gd a were present in % and . %, respectively. glycoarray is efficient for detecting antibodies against numerous glycolipid complexes in immune-mediated neuropathies. we need further investigations on other immune-mediated diseases using larger number of antigens. kuwabara s , misawa s , sekiguchi y , susumu kusunoki and the jet-gbs study group . department of neurology, chiba university, chiba, japan; department of neurology, kindai university, osaka, japan; japanese eculizumab trial for guillain-barré syndrome, chiba, japan. guillain-barré syndrome is a monophasic immune-mediated neuropathy, but a substantial number of patients with severe disease have poor recovery, even if treated with immunoglobulin. recent studies suggest that complement activation plays a pivotal role in gbs-associated axonal degeneration, and eculizumab is a monoclonal antibody that specifically binds to complement component and inhibits complement activation. jet-gbs is an investigator-led, phase , randomized, placebo-controlled trial conducted in hospitals, this trial aims to investigate the safety and efficacy of eculizumab for treatment of severe gbs. patients were randomly assigned ( : ) to treatment with immunoglobulin plus either eculizumab ( mg/day; n= ) or placebo (n= ) once weekly for weeks. the primary outcome measures are safety and efficacy (the proportion of subjects who regain their ability to walk independently at week ). the secondary outcome measures included the proportion of subjects who were able to walk independently at week , and other measures such as mrc sum scores, nerve conduction parameters. enrollment for the trial began in august , and follow-up of the last patient was completed in october . analyses will be made in april , and the results will be presented at this meeting. this trial is registered with clinicaltrials.gov identifier: nct , and funded by the japanese agency for medical research and development, and alexion pharmaceuticals inc. neurofascin , a paranodal protein in peripheral nerve, is a target antigen for autoantibodies in a subset of chronic inflammatory demyelinating polyneuropathy (cidp). anti-neurofascin antibody-positive cidp is characterized by onset at younger age, tremor, and refractoriness to ivig. we have treated four patients with anti-neurofascin antibody-positive cidp at kindai university hospital. anti-neurofascin antibody was detected by both elisa and cell-based assay in those patients. igg subclass was predominantly igg in all four cases. the median age of the patients at admission was . years [range: - years]. among the four patients, three had tremor, and two had severe cerebellar ataxia. cerebrospinal fluid protein levels were remarkably increased [median: . mg/dl, range: - mg/dl]. although ivig treatment was administered in all four patients, the responses were poor or partial. in contrast, plasma exchange (pe) was performed in all four patients and the clinical symptoms dramatically improved in two of them. corticosteroids were also effective in those two patients. sural nerve biopsy was performed in all four patients. although sensory nerve action potentials of the sural nerves from those patients were not evoked, the transverse semithin sections of sural nerves from three patients revealed only slight or mild loss of myelinated fibers. partial paranodal demyelination was observed in teased-nerve fibers from three patients. in addition, abnormal paranodal lesions such as loss of the transvers bands were observed by electron microscope in all four patients. those electron microscopic findings were not observed in control patients with anti-neurofascin antibody-negative cidp. anti-neurofascin igg antibody-positive cidp shows distinctive clinical and pathological features. labeyrie c , besson f , vandendries c , cauquil c , beaudonnet g , not a , durand e , adams d . neurologie adulte, chu bicêtre, le kremlin bicêtre, france; médecine nucléaire, chu bicêtre, le kremlin bicêtre, france; clinique bizet, paris, france; unité de neurophysiologie clinique, chu bicêtre, le kremlin bicêtre, france. study of the proximal portion of the peripheral nervous system (pns) is difficult because less accessible to electrophysiological exploration and biopsy. the indications of mri are increasing in proximal neuropathies to analyse morphology of the roots, plexus and proximal nerves: integrity of the nerve bundle, inflammation or infiltration of these structures. pet-computed tomography (pet-ct) can detect infiltration of the proximal segments of the pns, with hypermetabolism of roots, plexuses and large nerve trunks. pet-ct is also useful in detection of solid neoplasm which can infiltrates pns and primary nerve sheath tumors. however, spatial resolution of pet -ct scan is limited to explore the roots and plexuses for moderate hypermetabolisms. we believe that a mild hypermetabolism could be highlighted in inflammatory neuropathies or in mild tumor infiltrations. we performed a fusion of whole body pet (performed because of suspected neoplasia) and plexus mri (pet-mri) images in ten patients with a peripheral neuropathy for which we suspected proximal involvement. the pet-ct and mri were first read separately, then with merge of the images by a nuclear physician and a neuroradiologist. five out of ten patients presented with hypermetabolism of pns (hmpn+) on pet-mri: root (n= ), and/or dorsal root ganglia (n= ). median sul of lesions was . . among the hmpn+ patients: hypermetabolism was already apparent on pet-ct in cases, the merge invalidated abnormalities seen on pet-ct in patients and mri detected additional lesions, not visible on pet-ct, in one patient. all hmpn+ patients had hypersignal and or hypertrophy of pns on mri either diffuse (n= ), or multifocal (n= ). among hmpn-(no hypermetabolism) out of ( %) had abnormal mri ( multifocal and diffuse). gadolinium enhancement was found in all patients receiving gadolinium in the hmpn+ (n= ) and only in / patients in the hmpngroup. final diagnoses of hmpn+ patients were neurolymphoma in one ( %), and idiopathic cidp in the others ( %) with histological proof on nervous root biopsy in one of them. final diagnosis in hmpn-patients was cidp in out of and sequelae of neuropathy in relation to lymphoma without relapse for the last one. cidp was more disabling (onls≥ ) in hmpn+ than in hmpn-group % vs. %. pet-mri could be helpful to detect a proximal inflammation of the pns, and to plan further testing. further studies are needed to evaluate the prognostic value of hmpn+. lancaster e , li j , liem r , scherer ss . perelman school of medicine, university of pennsylvania, philadelphia, pa, usa; columbia university school of medicine, new york, ny, usa. heterozygous nefl n s/+ mutant mice are the first animal model of a charcot-marie-tooth disease e (cmt e). people with this mutation have a severe, early onset axonal neuropathy. axons in the mutant mice have reduced number of neurofilaments and decreased diameters. they also show early onset of tremor and abnormal hindlimb clasping behavior. we measured the compound action potentials (caps) from tails every weeks from the same cohort of nefl n s/+ mutant mice and their wt littermates from to weeks. even at the age of weeks, the amplitude of mutant caps was only ∼ % of wt caps ( ± . microv, n= vs ± . microv, n= ), and the caps stayed substantially smaller than those in wt mice for weeks ( ± . microv, n= vs ± . microv, n= ). the conduction velocity and the duration of caps in mutants were slower and wider compared to those of wt. separate cohorts of mice (n= mutants and n= wt) were sacrificed at different time points for analysis by light microscopy. in caudal nerve of mutant mice, the number of axons was significantly reduced compared to that of wt at weeks and, by weeks, it was reduced more than %. this model system may be useful for preclinical studies of treatments for cmt e since the animals show progressive neuropathy over weeks, which can be objectively measured electrophysiologically and anatomically. despite more than cmt genes identified today, at least % of cmt patients do not carry a mutation in any of these genes. progress in gene identification in recent years suggests that there are still many more cmt disease genes to be discovered; however, it has become rare to gather support from multiple large families that allow for conclusive linkage analysis. we have studied multiple extended dominant cmt families with linkage support for a gene at chromosome p . . originally a czech family yielded a two point lod score of . at this locus and a family from southern italy showed a lod score of . . whole exome sequencing of multiple family members identified missense mutations in the gene atpase na+/k+ transporting subunit alpha (atp a ). atp a has not been associated with human diseases thus far. in expression studies on teased fiber preparations we already confirmed predominant expression in the nodes of ranvier of peripheral nerves. through collaborative efforts in the inherited neuropathy consortium and beyond we identified five additional multigenerational families via exome or sanger sequencing resulting in a total of seven unique segregating missense changes: leu arg, ile thr, ala thr, asp phe, pro ala, pro thr and asp ala. five of these mutations fall into a remarkably narrow motif associated with the sodium binding structure of atp a , flanking the flexible hinge motif. functional studies in different model systems (mammalian cell lines, xenopus oocytes, patient fibroblast lines) are underway to determine whether a loss or a dominant gain-of-function represents the disease mechanism. taken together, we show strong support for a major new dominant cmt gene, atp a . this finding represents a new pathway and an attractive new target for therapy development in axonal cmt. laurá m , ramdharry g , , singh d , kozyra d , skorupinska m , reilly mm . mrc centre for neuromuscular diseases, ucl institute of neurology, london, uk; school of rehabilitation sciences, st george's university of london/kingston university, uk; royal national orthopaedic hospital, stanmore, uk. charcot-marie-tooth (cmt) disease is the most common inherited peripheral neuropathy. foot deformities are frequent complications and orthopaedic surgery is often required. however there are no evidence based guidelines on the type or timing of the surgery. only few studies have described the long-term results of surgical procedures and evidence regarding optimal surgical management of these patients is lacking. we prospectively studied surgical management of cmt patients attending our centre. we collected data and assessed cmt patients before and after surgery. data included: history of ankle instability, pain, skin condition, details of physiotherapy and orthotic management, assessment of lower limb strength, charcot-marie-tooth examination score (cmtes), foot posture index, ankle dorsiflexion range of movement and specific questionnaires (foot index and manchester-oxford foot questionnaire, modified fatigue severity scale and modified falls efficacy scale), details of surgical procedures. patients were assessed yearly after surgery. so far patients ( males and females, age range - ) have been evaluated prior to surgery. all patients but one had genetically confirmed cmt ( cmt a, cmtx, cmt a). patients have been assessed after year, patients after years, patients after years and patient after years from surgery. a wide range of surgical procedures were performed by one dedicated orthopaedic surgeon. preliminary results showed reduction of number of falls in / ( %) patients and improvement of callosities in / ( %) patients at year follow up. there was also significant improvement of alignment of the operated foot (p= . ) and pain (p= . ). there were no significant changes in measures of strength and ankle range of motion. further analysis on a larger number of patients will be important to determine the long-term outcome of surgery. data acquired from this study will help develop orthopaedic intervention guidelines and identify areas for further research. lavigne-moreira c , oliveira mf , marques vd , onofre ptbn , dos santos acj , nascimento ojm , barreira a , marques w jr . division of neuromuscular diseases and neurogenetics, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; department of neurology, fluminense federal university, rio de janeiro, brazil. fap associated to ttr mutations is defined as a length-dependent axonal sensory and motor polyneuropathy that at early stages affects mainly the small nerve fibers, associated to autonomic and thermo-algesic sensations. the expected emg pattern was that of an axonal sensory and motor polyneuropathy, but in fact several unexpected patterns may be found. in this study we present the results found in a brazilian population with ttr mutation. patients were divided in three groups: ttr-met of early onset, ttr-met of late onset and ttr non-met . in the first group (ttr-met of early onset), ( . %) examinations were normal, ( . %) were axonal, were demyelinating fulfilling pidc criteria, one suggested a predominately motor polyneuropathy and the final one presented a lower motor neuron disease pattern. in the second group (ttr-met of late onset), ( . %) had an axonal pattern, ( . %) had an intermediate cv, ( . %) had a demyelinating pattern, had a lumbossacral pattern and the final one had no definite pattern. among the non-ttr met , two patients had the ttr -asp tyr, one presented an axonal pattern, while the second presented initially an axonal pattern, that changed to a demyelinating pattern in the second examination. patients with ile val mutation presented an axonal neuropathy associated to cts. most patients with demyelinating or intermediate pattern were treated with corticosteroids or ivig, with no satisfactory results. this small series of patients shows clearly that fap-ttr is associated to several emg patterns in addition to the expected sensory and motor axonal polyneuropathy. this variability is present in the same family and in the same patient in different occasions. clinicians should be alert to these possibilities to do not delay diagnosis and treatment. identifying the mechanisms involved in this variability could improve our knowledge of this intriguing disease. lavin tm . greater manchester neurosciences centre, salford royal hospital, manchester, uk. subcutaneous immunoglobulin (scig) has evidence from small trials for its use following intravenous igg (ivig) loading in chronic inflammatory demyelinating polyneuropathy (cidp). potential advantages of scig include stable igg levels and reduced complication rates. scig de novo can be considered as igg levels gradually rise with subcutaneous delivery and maybe beneficial for patients for whom complications are a concern. we present cases of cidp who were initiated on subcutaneous immunoglobulin g ( g/kg/month, % scig, hizentra, csl behring) following corticosteroids; without intravenous igg loading. patient is year old with a history of ihd with a progressive lower limb sensory changes and sensory ataxia over years. neurophysiology confirmed sensory demyelinating changes. he did not respond to corticosteroids. concerns were raised about the potential risk orf adverse cardiac events with iv immunoglobulin. scig therapy was introduced at g weekly. initial therapy has been well tolerated and objective measures (rods-cidp score, jamar grip strength, hole peg) have remained stable through initiation, and at months. patient is yr old male with a past history of branch retinal vein thrombosis. he developed a sensorimotor polyneuropathy over years with neurophysiology fitting efns criteria for cidp. given concerns regarding previous thrombosis, a trial of scig was given at g/week, with resolution of sensory ataxia and improvement in objective markers (jamar grip strength, rods-cidp score, hole peg) at months. unfortunately an adverse event occurred of an urticarial skin reaction. patient is a yr old male who presented with motor predominant cidp. there was no response to corticosteroids but improvement following plasma exchange. due to a past history of transient ischaemic attack, scig was started. there has been a positive response after months with improvement in walking distances and stable objective markers (rods-cidp score, grip and pinch strength). our cohort remained neurologically stable during initiation of scig, with adverse reaction. our experience would support further trials in this area regarding the efficacy of scig compared to ivig in both the short and long term. diabetic peripheral neuropathy is the most common and debilitating complication of diabetes and it is associated to neuropathic pain and non-traumatic amputations. despite the economic burden and human costs, nowadays there is not a specific treatment to cure diabetic peripheral neuropathy. hyperglycaemia and dyslipidemia-mediated oxidative and endoplasmic reticulum stress has been linked to diabetic peripheral neuropathy, among other altered pathways. in order to study in more detail the molecular mechanisms that lead to the development of diabetic peripheral neuropathy we set up in vitro models of the disease using nsc- (motoneurons) and med . (sensory neurons) cell lines or primary cultures of dorsal root ganglia and motoneurons exposed to saturated fatty acids. it has been reported that palmitic acid is able to induce endoplasmic reticulum stress and oxidative stress in multiple cell types including schwann cells and myenteric neurons, resulting in a good model of dyslipidemia-mediated stress. here we found that palmitate induces upregulation of the molecular chaperone bip/grp and the ccaat-enhancer-binding protein homologous protein (chop) mrnas and a dose-dependent downregulation of the apoptosis marker bcl- in primary cultures. moreover, palmitic acid induces loss of neuron dendrites and cell death at high doses and upregulation of heme oxygenase (ho- ) and chop at lower doses in the nsc- cell line. we are currently characterizing multiple molecules implicated in oxidative stress, endoplasmic reticulum stress and inflammation pathways in these cell types to find new therapeutic targets to treat type diabetic sensorimotor polyneuropathy, that will be subsequently validated in the db/db mouse model by pharmacological or gene therapy strategies. lee bjh , ohnmar o , wong j , koh sj , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. treatment-induced neuropathy of diabetes mellitus (dm) (tind) is an acute painful peripheral neuropathy and autonomic dysfunction that occurs within weeks of tight glycaemic control. therapy with either insulin or oral hypoglycaemic agents (ohga) may result in tind. the quantum and rate of decline in hba c predicts development and severity of tind. both type i and ii dm patients are prone to this iatrogenic complication. besides the expected morbidity associated with painful somatic and autonomic neuropathy, tind patients also develop life-threatening eye complications such as maculopathy. with greater awareness, we picked up at least typical cases of tind in recent few months. however, there was a fourth possible tind case, whom we feel deserves special attention. the circumstances surrounding this case are common and may go under-recognised in acute hospitals. a year-old man with -year history of poorly controlled type ii dm was admitted with a partial left middle cerebral artery stroke. one month before admission, his hba c was . %. his hospitalization was prolonged because of his considerable disability that required rehabilitation. he stayed weeks. his blood sugar was difficult to control with hyperglycaemic episodes requiring ohgas and insulin. the highest capillary blood glucose recorded was mmol/l. he also had episodes of hypoglycaemia. three weeks into hospitalization, he developed severe orthostatic hypotension. he came to our attention two months later when he was sent for autonomic screening tests for severe postural hypotension, in spite of therapy with fludrocortisone and midodrine. his hba c was . %. he had marked orthostatic hypotension suggestive of sympathetic dysfunction. we were not able to discern if he had a painful neuropathy because of his aphasia. there was no recent ophthalmology review. we suggested a diagnosis of possible tind. he was discharged with lower doses of ohga. however, day after discharge, he attended the emergency department for a syncopal episode. this case raises intriguing questions on the safety of glucose control paradigms commonly employed in patients with acute stroke and myocardial infarction as well as the possible role of major fluctuations in blood glucose in the development of tind. lee hs , kim sm . presbyterian medical center, jeonju, korea; yonsei university college of medicine, seoul, korea. the occurrence of peripheral neuropathy by a tumor within or compressing a nerve in neurofibromatosis (nf) type and is relatively well known. however, nf presenting with demyelinating polyneuropathy unrelated to tumor masses is rarely reported. we report a rare presentation of an nf patient with demyelinating polyneuropathy of subacute onset. a -year-old woman was referred to our hospital with paresthesia on both limbs months before. she was healthy and had no family history. the symptoms worsened over the next months, tremor and weakness in both limbs occurred. although she treated as a chronic inflammatory demyelinating polyneuropathy (cidp) with intravenous steroid and immunosuppressant in another hospital, symptoms were getting worse. bifacial numbness and left eyeball pain occurred weeks ago and she visited our hospital. on neurologic examination, bilateral facial sensory hypoesthesia and symmetrical both limbs weakness (mrc grade iv+) were observed. decreased touch and pinprick sensation on both limbs were observed like stocking-glove distribution. the reflexes were sluggish. on nerve conduction study, sensorimotor demyelinating polyneuropathy with conduction blocks and temporal dispersions was observed. bilateral r , r responses were prolongation on facial blink test. mri of cervical spine and brain revealed contrast enhancing tumor-like enlargement of multiple nerve roots and cranial nerves. nerve biopsy was performed on right supraorbital nerve, neurofibromatous changes were observed. the symptom deterioration was stopped without treatment. gene test dose not revealed nf type and . on review of the literature, polyneuropathy in nf patient can result from tumor masses within the proximal nerve roots, or along the peripheral nerve, or in the extramedullary lesion affecting neighboring nerve roots. thus, axonal type polyneuropathy and focal amyotrophy have been reported in nf. demyelinating polyneuropathy has not been reported before in our knowledge. although the etiology of demyelinating polyneuropathy in nf requires further clarification, some authors claim that unknown local toxic or metabolic influences of the endoneurial pathological cells on adjacent nerve fibers. in this patient, atypical symptoms were observed in cidp such as cranial nerve involvement and stocking-glove sensory distribution. in this case, a nerve biopsy can be helpful the accurate diagnosis. lee jy , yoo jh , kang dk , bae js . department of neurology hallym university, seoul, korea. acute disseminated encephalomyelitis (adem) is an uncommon post-infectious inflammatory demyelinating disorder of central nerve system, while guillain-barre syndrome (gbs) is a prototype of acute post-infectious peripheral neuropathy. previous reports regarding the coexistence of these relatively rare diseases suggest that certain immunogenicity within central and peripheral nerves may share a common autoimmune process during the disease course. a previous healthy years old man was admitted because of fever, headache, nausea and myalgia in department of infectious disease. two weeks before admission, he suffered from watery diarrhea for days and spontaneously recovered. at initial presentation, he had high fever( ∘ c), headache and myalgia and intermittent horizontal diplopia. a few days after admission, he began to complain of drowsy mentality, bilateral extremities weakness, especially lower limbs, dysarthria, bilateral facial paralysis, urinary retention, dyspnea. on neurologic examination, he had mildly drowsy mentality, symmetric muscle weakness scoring of on bilateral hip, knee flexion and finger extension, but he had no sensory symptoms. he showed gazed evoked nystagmus with no extraocular muscle palsies. deep tendon reflexes were not present. pulmonary function test revealed a severe restrictive pattern. csf studies disclosed a dissociative increase of protein contents ( mg/dl) without pleocytosis. anti ganlioside antibody assay identified an anti-gt a igg positivity in his serum. nerve conduction study (ncs) showed prolonged motor terminal latencies and slow motor conduction velocity on multiple nerves. in contrast, sensory ncs revealed no abnormal findings. imaging studies unexpectedly revealed apparently symmetrical lesions across bilateral brainstem and basal ganglia suggesting a diagnosis of adem. after both ivig and high dose steroid treatment, he remarkably recovered from disturbed mental state and motor weakness. about month after the symptom onset, he could walk with assistant aid and discharge to other hospital for rehabilitation. our case suggest that certain component of autoimmunity simultaneously result in both cns and pns inflammation. specific immunological mechanism is remained to be elucidated. although we could not conclude whether cellular component or humoral component is dominant for our case, the presence of anti gt a antibody suggest a role of humoral mechanisms. the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. leitao amf , , araújo df , marques h , pamplona l , , souza mh , , nbraga ll , , gondim faa , . the aim of this study is to evaluate whether peripheral neuropathies and headaches affect the same subgroups of patients with ibd. since , we have established a cohort study to evaluate the prevalence and incidence of neurological diseases in patients with ibd. over a period of years, all patients with ibd (either crohńs disease or ulcerative colitis) were invited to participate in a study designed to evaluate the risk factors for the presence of headaches and peripheral neuropathy in ibd. a separate group of control patients (age-matched relatives of ibd patients) was also formed. after a clinical interview and neurological examination, patients were invited to undergo skin wrinkling test (swt) to evaluate small fiber function and/or electromyography. headaches were present in . % of the patients with ibd, and were more common in patients with ulcerative colitis than in control patients (p< . ). migraine comprised . % of all cases of headache and was more prevalent in patients with crohńs disease than control patients (p< . ). tensional headaches were also common affecting . % of the ibd patients. electromyography was abnormal in . % of the ibd patients tested ( / ). swt was abnormal in . % of the ibd patients tested ( / ). . % of the ibd patients had abnormal swt but had no neuropathy symptoms. patients with abnormal swt or emg were not more likely to have headaches (p= . and . , respectively). overall, patients with symptomatic polyneuropathy were not more likely to have headache (p= . ). patients with abnormal swt or emg were also not more likely to have migraine (p= . and . , respectively). patients with abnormal swt or emg were also not more likely to have tension-type headache (p= . and . , respectively). in summary, although highly prevalent in this population of brazilian ibd patients, primary headaches and neuropathy do not affect the same subgroups of ibd patients. further studies are necessary to understand the mechanisms of both conditions in ibd patients. the classic guillain barré syndrome (gbs) is characterized by motor weakness, hyporreflexia, but limited sensory deficits. sensory variants involving either small or large fibers or both are unusual and represent a diagnostic challenge. we described patients presenting with the sensory variant of gbs and retrospectively analyzed the clinical and electrophysiological findings of patients fulfilling the criteria for sensory gbs according to oh et al. criteria. six patients were identified (mean age years: range - years). four had a previous infection. they all consulted due to distal painful paresthesias and allodynia. on examination the patients presented normal strength and normal cranial nerves through the course of the disease with reduced knee and ankle reflexes in patients. distal hyperesthesia to pinprick was identified in and one of them additionally had hyperhidrosis and constipation. two additional patients presented hypoesthesia to pinprick and temperature. one patient had distal proprioceptive sensory loss with sensory ataxia. csf albumin cytological dissociation was present in patients. nerve conduction studies (ncs) identified a sensory motor demyelinating neuropathy in patients. among the with normal ncs, had abnormal cold and warm threshold in their qst evaluation. all patients received symptomatic treatment for the neuropathic pain and only two ivig therapies. longstanding pain, fatigue or both were persistent findings in patients after a mean follow up of months. in conclusion the sensory variant of gbs is both an infrequent presentation and a diagnostic challenge. longstanding pain and fatigue are common persisting findings. the epidemic of zika virus (zikv) throughout the americas and asia, and the subsequent rise in reported cases of guillain-barré syndrome (gbs) caused worldwide concern. as of january , countries have reported evidence of mosquito-borne zikv transmission and in of these countries, a sudden increase of gbs has been reported. moreover, case studies and a case-control study further indicate that zikv may trigger gbs. however, accurate diagnosis of both zikv and gbs in many of these studies is disputed, and a comprehensive description of the clinical phenotype of gbs related to zikv is lacking. the international gbs outcome study (igos) is a prospective observational study on the factors determining the onset, clinical course and outcome of gbs. at present, a research consortium of centers from countries has included patients in igos. our aim is to investigate zikv-related gbs in igos as is already occurring in colombia. in igos-zika, data on clinical features and ancillary investigations will be collected in zikv endemic areas according to the igos protocol with some modifications. first, igos-zika has a case-controlled study design to define the association between gbs and zikv and other arboviruses. second, urine samples will be collected and additional questions on preceding events will be asked, focusing on arbovirus infections. third, a more limited follow-up is required. our aim is to recruit additional centers via the inflammatory neuropathy consortium (inc) and centers in all arbovirus endemic regions that are willing to participate. the focus of igos-zika will be on the accuracy of the diagnosis of both gbs and zikv and on defining the associated clinical phenotype, course and outcome. igos-zika provides the opportunity to combine data and biobanks from various geographical regions using a standardized protocol and to compare these data with data and biosamples already collected in igos. studying these cases will help to optimize diagnostics and care for gbs patients in arbovirus endemic countries and provides a unique opportunity to further understand the pathogenesis of gbs. moreover, this study design and network can be used to adequately respond to other future viral epidemics related to gbs. lerat j , cintas p , dzugan h , , magdelaine c , , sturtz f , , lia as , . service de biochimie et génétique moléculaire -chu de limoges, limoges, france; service de neurologie et d'explorations fonctionnelles -chu de toulouse, toulouse, france; ea -université de limoges, limoges, france. pharc syndrome is an autosomal recessive neurodegenerative pathology leading to demyelinating polyneuropathy, hearing loss, cerebellar ataxia, retinis pigmentosa and early-onset cataract. these various symptoms can occur at different ages, so that pharc syndrome can be a differential diagnosis of charcot-marie-tooth disease (cmt) associated with deafness. only abhd mutations have been reported in patients. we described the th mutation and compared our results to the literature data. we analysed by next generation sequencing (ngs) strategy using a targeted cmt and associated neuropathies -gene panel the dna of a -year old male who has suffered from demyelinating sensory and motor polyneuropathy and ataxia since the age of . bilateral sensorineural deafness was diagnosed at the age of five. bilateral congenital cataracts were operated on at the age of . a new large complex homozygous mutation, with one deletion of seven base pairs and one insertion of base pairs, was detected. by analyzing our patient data and those of the literature, we evaluated that, in pharc syndrome, sensorineural deafness always occurs as the first feature in late teens. the ophthalmological symptoms are cataracts that occur at a mean age of yo and then retinis pigmentosa at a mean age of . demyelinating sensory-motor polyneuropathy is the most variable characteristics, which occurs in the thirties. we report the first large complex homozygous mutation in pharc syndrome, which is certainly under-diagnosed. therefore, it seems interesting to include abhd in the panels of the five symptoms, especially deafness ones. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a chronic disabling disease that often improves with immune therapy. to date, the most reliable diagnostic criteria for cidp are the efns/pns revised criteria, with a reported sensitivity of % and specificity of %. we implemented a web-based database to collect data from patients with cidp followed by italian centers with expertise on cidp to determine the frequency and characteristic of cidp and it variants, the diagnostic criteria used for their diagnosis, the possible evolution into typical cidp, the association with specific anti-nerve antibodies, and their response to therapy. all the patients were evaluated at the time of inclusion and will be followed for two years to monitor their outcome and response to therapy. by february we included patients with cidp and variants ( men, women), aged - years (median ) with a mean disease duration of . years (range . - years). based on clinical symptoms, cidp was defined as typical in % and atypical in %. the diagnosis of typical cidp fulfilled efns/pns criteria in % of the patients while nerve conduction studies were not diagnostic in % (grouped as clinical cidp) or not available in %. we analyzed the frequency of supportive criteria for the diagnosis of cidp in patients with clinical cidp and found that increased csf proteins, demyelination or cell infiltration on nerve biopsy and imaging abnormalities consisting with cidp on us or nmr were present in %, % and % of the patients, respectively. a relapsing course was present in % of patients with clinical cidp, increasing the reliability of the diagnosis for cidp. in addition an improvement after one or more therapies was reported by % of the patients, with a positive response to ivig in %, steroids in % and plasma exchange in %, similarly to what observed in patients fulfilling efns/pns criteria. in % of the patients with clinical cidp two or more supplementary criteria for cidp were present. this study on a large population of patients is providing useful information that may help to revise the current diagnostic criteria for cidp. li j , cannell m , suragani r , pearsall r , kumar r . acceleron pharma inc, cambridge, usa. charcot-marie-tooth (cmt) is the most common hereditary peripheral neuropathy and is characterized by demyelination and/or axonal damage of peripheral nerves and muscle weakness. foot drop, steppage gait, and foot deformities are a typically seen in cmt patients. consequently, falls are commonly reported in these patients. improvement of dorsiflexor muscle function to prevent falls may improve quality of life and activities of daily living in patients with cmt. ace- , a locally-acting ligand trap that binds growth and differentiation factors (gdfs) and activins, has previously been shown to increase muscle mass and force in both duchene muscle dystrophy (dmd) and amyotrophic lateral sclerosis (als) mouse models. in the current study, we evaluated the therapeutic effects of ace- to improve muscle strength in the trembler (tr-j) mouse model of cmt a. these mice harbor a mutation in the peripheral myelin protein (pmp ) known to cause cmt a. seven-month old (b .d -pmp tr-j /j) mice were administered ace- ( g, twice weekly) intramuscularly to one of the unilaterally tibialis anterior (ta) muscle for weeks. the contractility of the ta muscle was evaluated during isometric contraction. all data were compared to the uninjected contralateral control hind-limb. after weeks of ace- treatment, ta muscle mass was increased by % (p< . ) and its physiological cross-sectional area was increased by % (p< . ). the increase in muscle mass correlated with an increase in strength, with maximum tetanic force and twitch force improved by % (p< . ) and % (p< . ), respectively. in addition, temporal properties during isometric contraction, such as maximum rate of contraction and relaxation, were accelerated by % and % respectively (p< . ) in the ace- -treated ta muscle compared to its contralateral hind-limb. pathological and biochemical assessment of ace- -treated mice showed enlarged myocyte area (+ %, p< . ) and reduced atrogin- mrna expression (− %, p< . ). together, these results demonstrated that ace- attenuates the degree of muscle atrophy and also improves muscle function in a mouse model of cmt a. the current study provides proof of concept for the use of ace- as a therapy for cmt to improve dorsiflexor muscle function and alleviate foot drop. we have generated a rat model of charcot-marie-tooth disease a (cmt a) harboring the p.arg trp mfn mutation, whose human counterpart results in a severe, early-onset axonal neuropathy. the mutation was made using zinc finger nuclease-mediated genome editing in fertilized rat eggs. a large cohort of mutant and wt littermates were characterized behaviorally and found to develop multiple motor deficits that worsened over time. nerve conductions of the tail (caudal nerve) was performed on a separate cohort of mutant (n= ) and wt littermates (n= ) every weeks from to weeks. mutant rats showed a progressively decline in the amplitude of the compound action potential after weeks, whereas the amplitude progressively increased in their wt littermates. separate cohorts of rats were sacrificed at , , and weeks and analyzed by light microscopy. in mutant rats, there was a reduced density of myelinated axons and active axonal degeneration in distal but not proximal nerves, and in the fasciculus gracilis of the cervical spinal cord at and weeks. these findings were not present in the -week-old cohort of mutant rats, or in wt rats at or weeks. a genetically authentic animal model of cmt a that develops a progressive, length-dependent axonal neuropathy will be a valuable tool for examining the pathogenesis and treatment of cmt a. lindborg ja , niemi, jp , defrancesco a , zigmond re . case western reserve university, cleveland, usa. traditionally the role of immune cells in nerve degeneration and regeneration has focused on the infiltration of inflammatory monocytes into the distal nerve after nerve injury and the phagocytosis by the resulting macrophages of myelin and axonal debris, thereby clearing a path for regenerating axons. therefore, it was surprising when we discovered that in ccr knockout (ko) animals, in which the entry of these inflammatory monocytes does not occur, that wallerian degeneration precedes normally. we now report that the reason for this is that neutrophils and schwann cells compensate for the decrease in macrophage accumulation. furthermore, nearly complete depletion of circulating neutrophils by systemic injection of an antibody to ly g leads to an inhibition of myelin clearance both in ccr ko and in wild type animals. on the other hand, we have demonstrated a second site of macrophage accumulation in wild type animals, namely around axotomized sensory neurons in dorsal root ganglia (drgs). blockade of that accumulation, for example as occurs in ccr ko animals, leads to a dramatic impairment of nerve regeneration. to examine the relationship between macrophages and regeneration further the monocyte chemokine ccl was overexpressed in drg neurons in intact animals by viral infection using an aav containing the ccl coding sequence. the resulting overexpression of ccl led to the accumulation of macrophages in drgs even though no injury had taken place and subsequently to an increase in the intrinsic growth capacity of the sensory neurons. examination of changes in gene expression in the drgs in these animals revealed increased expression of the cytokine leukemia inhibitory factor and an increase in its downstream signaling pathway that involves the phosphorylation and nuclear translocation of stat . strikingly, pharmacological blockade of stat activation inhibited the increase in the neurons' growth capacity produced by the virus. these results reveal unexpected interactions between immune cells and neurons facilitating nerve degeneration and regeneration and could lead to therapies to improve regeneration after injury or in disease. lin j , , qiao k , , huang j , , zhao cb , , lu jh , . institute of neurology, fudan university, shanghai, china; department of neurology, huashan hospital, fudan university, shanghai, china. we used terminal latency index (tli) as a tool in differentiation between poems syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). comparison of median and ulnar nerve conduction studies including motor conduction velocity (mcv), distal motor latency (dml) and terminal latency index (tli) were studied in poems patients, matched cidp patients and normal controls. in this cohort, the average age at evaluation was . ± . years old in poems group and that of cidp patients was . ± . years old. except the ulnar terminal latency index in cidp group, poems and cidp patients demonstrated prolonged distal latencies, low conduction velocities and increased terminal latency indexes compared with the normal group. reduced conduction velocities and higher terminal latency indexes in poems group than in cidp group was found. increased tli was demonstrated in . %(median nerve) and . %(ulnar nerve) poems and that in cidp patients was . %(median nerve) and . %(ulnar nerve). decreased tli was found in . %(median) and . %(ulnar) cidp patients and none in poems. temporal dispersion (td) and conduction block (cb) were more often seen in cidp patients with increased tli than that in poems. compared with cidp and poems showed greater slowing of the intermediate nerve segments and relatively more uniform demyelination. about % cidp demonstrated more distal conduction slowing and more td and cb especially in those with increased tli. terminal latency index combined with td and cb may be helpful in differentiating poems from cidp. lin y , sung j , chang t , jowy t . department of neurology, taipei municipal wanfang hospital, taipei, taiwan. the purpose of our study is to exam whether electrophysiology changes could be detected in prediabetes patients and to discover the possible mechanism of nerve injury in prediabetes stage. we analysis and compare the nerve excitability test data between prediabetic patients and age-matched normal control subjects. prediabetes is defined by american diabetes association (ada) as one of the three following: hba c . % to . %, fasting glucose mg/dl to mg/dl, and hour oral glucose tolerance test to mg/dl. patients with radiculopathy, myelopathy, entrapment neuropathy such as carpel tunnel syndrome, and polyneuropathy were excluded. the strength-duration time constant (sdtc) and superexcitability showed significant difference (p< . ) between two groups. we also find increased threshold electrotonus in depolarization (ted) and reduced relative refractory period (rrp) and refractoriness in . msec. these early changes in prediabetic patient are similar in nerve excitability feature of diabetic patients. however, the above changes are not found in motor axonal excitability test. our data supports that nerve excitability test may be a useful, non-invasive, and less time dependent tool to detect peripheral nerve injury in prediabetic stage. the sensory axons are more vulnerable than motor axons. superexcitability is the most sensitive parameter in prediabetes. transthyretin-related familial amyloid polyneuropathy (ttr-fap) is an autosomal dominant disorder caused by mutations of ttr gene and is associated with variable penetrance. ttr-fap is rare, except for endemic areas. this is a retrospective study of ttr-fap patients diagnosed at our center between - . we identified four families with different ttr mutations. in one family with v a mutation, nine family members over four generations diagnosed with ttr-fap was followed since . in the other families the index cases with different mutations were identified between - . affected family members with v a mutation developed severe progressive polyneuropathy with cachexia, with onset of the disease between ages and . three patients presented with marked visual symptoms (one patient underwent vitrectomy). nine patients died to years after disease onset. two patients (sisters) underwent liver transplantation -one died after years of disease at age , second is years old and wheelchair-bound as her symptoms continue to progress. the ttr mutations diagnosed in the index cases of three other families are: d v, f l and v m. they all presented with similar clinical picture of late-onset ttr-fap with predominant progressive axonal sensory, motor and autonomic polyneuropathy. all three index cases were men, the onset of symptoms was between - years with numbness and paresthesia in the feet followed by weakness and autonomic dysfunction. all had excessive weight loss resulting in cachexia and were diagnosed with cardiomyopathy. no patient suffered from visual symptoms. all three patients progressed to stage ii of ttr-fap -walking with assistance. time to diagnosis was . - years. due to advanced stages of their disease these patients were not suitable for therapy with tafamidis or the liver transplantation. the ttr d v (p.d v) was confirmed as a de novo mutation, which is uncommon in ttr-fap. in the remaining families carriers of ttr mutations were identified and are followed up. pedigree analysis of the family with f l mutation revealed affected members who with high probability died from ttr-fap. our study suggests that patients with ttr-fap in poland exhibits clinical and genetic heterogeneity. liu x , fan d . department of neurology, peking university third hospital, beijing, china. objective: to identify the gene mutation of chinese charcot-marie-tooth pedigrees and investigate the correlation among the clinical manifestation, electrophysiology and mechanism of different genotype. methods: we included pedigrees with cmt enrolled in our hospital from january, to december . we recorded clinical features, cmtns and electrophysiological data at diagnosis. the patients underwent mutation analysis of pmp , cx , mpz, mfn , hspb , hspb using mlpa, dhplc and sanger gene sequencing. results: we found pmp duplication pedigrees ( . %), cx pedigrees ( . %), mfn pedigrees ( . %), mpz pedigrees ( . %) conclusions: in chinese han population, the proportion of pmp duplication is relatively low, the majority of clinical manifestation is classical cmt. axonal cmt can show isolated lower extremity injury, with central nervous system involvement. hmn may be an underestimated clinical types, the identification should be done with caution in differential diagnosis. liu y , liu b , sebastian b , wozniak km , wu y , slusher b , polydefkis m . johns hopkins school of medicine, baltimore, usa. chemotherapy-induced peripheral neuropathy (cipn) is a common dose-limiting toxicity in the treatment of many cancers. most cipn studies preferentially focus on sensory fiber loss and dysfunction. here, we compared the structural and functional recovery of autonomic fibers in sweat glands (sweat gland nerve fiber density, sgnfd) and sensory fibers (intra-epidermal fiber density, ienfd) in mouse footpads after exposure to a maximum tolerated dose (mtd) of several common chemotherapy agents. additionally, we assessed footpad sweat production as a functional correlate to sgnfd reductions. female balb-c mice ( -animals/group) were treated with a mtd of four anti-tubulin drugs: paclitaxel (pca, mg/kg), ixabepilone (ixa, mg/kg), eribuline (erib, . mg/kg), vinoelbine (vino, mg/kg), or corresponding placebo given intravenously, mwf for two weeks. recovery was assessed at -hours, , , , , and weeks following the last dose. footpads were processed to visualize epidermal nerve fibers using pgp . and autonomic nerve fibers with tyrosine hydroxylate and pgp . . ixabepilone-treated mice experienced significant reductions in sgnfd at hrs, while ienfd nadir occurred at a later time point, -weeks. the recovery to baseline levels occurred more quickly for ienfd ( -weeks) than sgnfd ( -weeks). in contrast, vinorelbine and eribuline treated mice experienced a maximum deficit in sgnfd and ienfd at hrs and sgnfd recovery was slower ( -weeks) compared to ienfd ( -weeks). pca-treated animals showed more severe ienfd and sgnfd deficits compared to the other agents with both ienfd and sgnfd not recovering completely until -months. reductions in th-sgnfd were comparable or more pronounced to decreases in pgp . -sgnfd for all agents and timepoints. pca-treated animals demonstrated reductions in footpad sweat droplet number thereby providing a functional correlate. together, these data indicate that in mouse models of cipn, autonomic nerve fibers are affected more severely than sensory nerve fibers, and also recover more slowly than intraepidermal nerve fibers. autonomic dysfunction may be an important and under-appreciated consequence of chemotherapy exposure. the pi -kinase vps (pik c ) synthesizes phosphatidylinositol -phosphate (pi p), a lipid critical for both endosomal membrane traffic and macroautophagy. human genetics have implicated pi p dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating charcot-marie-tooth (cmt) peripheral neuropathy. here, we investigated the role of vps , and pi p, in mouse schwann cells by selectively deleting vps in this cell type. vps -schwann cell knockout (vps scko ) mice show severe hypomyelination in peripheral nerves. vps −/− schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. upon reaching the promyelinating stage, vps −/− schwann cells are significantly impaired in the elaboration of myelin. nerves from vps scko mice contain elevated levels of the lc and p proteins, indicating impaired autophagy. however, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in vps scko mice is caused by impaired autophagy. endosomal membrane traffic is also disturbed in vps −/− schwann cells. we investigated the activation of the erbb / receptor tyrosine kinases in vps scko nerves, as these proteins, which play essential roles in schwann cell myelination, are known to traffic though endosomes. in vps scko nerves, erbb was hyperphosphorylated on a tyrosine known to be phosphorylated in response to nrg exposure. the overall level of erbb was also decreased during myelination. our findings suggest that the loss of vps alters the trafficking of erbb / through endosomes. abnormal erbb / signaling may contribute to the hypomyelination observed in vps scko mice. lombardi r , devaux j , cortese a , dacci p , benedetti l , demichelis c , lauria g . irccs foundation "carlo besta" neurological institute, milan, italy; aix-marseille université, marseille, france; irccs c. mondino national neurological institute, pavia, italy; university of genoa and irccs aou san martino-ist, genoa, italy. the recent identification of igg anti-neurofascin (nfascin) antibodies in a group of patients has widened the spectrum of presentation for chronic inflammatory demyelinating polyradiculoneuropathy (cidp). these patients can be distinguished by disabling tremor, poor response to intravenous immunoglobulin and distal and sensory disturbances. cell-adhesion molecule nfasc and cntn are expressed on the paranodal junction (pnj) of nodes of ranvier, and play key roles on sodium channel clustering and glia-axon interactions. quantification of unmyelinated intraepidermal nerve fibers (ienf) is a useful parameter employed in small nerve fiber pathology diagnosis. in addition the immunohistochemistry evaluation of dermal nerve fibers allows to examine morphological changes of myelin sheath and ranvier nodes structure. we performed immunofluorescent colocalization studies using antibodies to visualize axons (protein-gene-product . , neurofilament, tubulin), sheath of myelin (myelin-basic-protein) and specifically nodal/paranodal/juxtaparanodal structures (pannfascin, nfascin , nfascin , caspr, cntn , potassium and sodium channels) in skin tissues from seronegative and seropositive cidp patients. we analyzed axon and myelin sheath damage, abnormal nodal-paranodal-juxtaparanodal architecture and morphometric parameter as internodal length of ranvier nodes. our results on skin biopsies from three igg nfasc -positive cidp patients revealed complete loss of nfasc staining at the paranodes, asymmetrical paranodes and widening of the nodes of dermal myelinated nerve fibers. one igg cntn -positive cidp patient showed abnormal nodal/paranodal immunostaining with different features as compared with igg nfasc -positive patients suggesting specific changes. however, such alterations were not found in four seronegative cidp patients. our data support the hypothesis that examining specific axonal and myelin markers could provide diagnostic and prognostic clues on nodo-paranodopathies. the goal of this study is attempt a possible correlation between the presence of serum autoantibodies and structural changes in nodal/paranodal regions of dermal nerve fibers in cidp patients. knowledge of autoantibodies expression in the peripheral myelinated nerves of cidp patients could serve for stratifying patients and potentially guiding personalized treatments. lunati a , lerat j , dzugan h , , rego m , magdelaine c , , bieth e , calvas p , cintas p , gilbert-dussardier b , goizet c , journel h , magy l , toutain a , urtizberea j , sturtz f , , lia as , . charcot-marie-tooth disease is one of the most frequent inherited peripheral neuropathies ( / ). so far, mutations in more than genes have been identified causing either the demyelinating form (type ) or the axonal form (type ). duplication of pmp gene is the most frequent cause of autosomal dominant demyelinating form. autosomal recessive demyelinating form is often due to sh tc gene mutations. patients suffer then from early severe neuropathy starting in the first decade. scoliosis and deafness are often observed. we analysed patients suffering from peripheral neuropathy, by multiplex-ligation-dependant-probe-amplification (mlpa), followed by targeted next-generation-sequencing (ngs) using a -gene custom panel designed for the diagnosis of charcot-marie-tooth and associated neuropathies. mutations of interest were verified by sanger sequencing. diagnosis was positive for patients. as expected, the most frequent mutation was the pmp duplication detected in patients. deletion of pmp was observed in patients and pathogenic point mutations were detected in patients. sh tc gene appeared to be the most frequently mutated with nine patients diagnosed. associated with known mutations, four new mutations have been identified: two nonsense mutations and two missense mutations. all these patients presented deafness and/or scoliosis. sh tc appears to be an important gene involved in charcot-marie-tooth disease, often associated with deafness and /or scoliosis. it is important to pay attention to these associated symptoms in charcot-marie-tooth patients in order to guide their diagnosis and to improve their medical care. lupo v , , frasquet m , , sánchez-monteagudo a , , barreiro m , alberti ma , casasnovas c , quintáns b , , , camacho a , domínguez c , sedano mj , pelayo al , pardo j , sobrino t , sobrido mj , , , sevilla t , , espinós c , . mme (membrane metalloendopeptidase) mutations, inherited in an autosomal recessive fashion, have been recently identified in japanese probands (higuchi et al. ) . thus, mme has been included in the list of cmt genes as a new autosomal recessive axonal form, cmt t (mim ), and moreover, it is considered a strong candidate for the genetic diagnosis of unsolved late-onset cmt cases. in fact, few months later auer-grumbach et al. ( ) reported european probands with autosomal dominant late-onset cmt and mutations in mme. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy using an updated version of our custom gene panel, which includes mme. in this study, we report probands with cmt , intermediate cmt or dhmn/cmt and homozygous or compound heterozygous mutations in mme, and proband with cmt and a heterozygous mutation in mme. we have identified different type of mutations: novel splice donor variant, frameshift, nonsense and missense mutations. the two nonsense changes, p.trp * and p.arg *, and the splice donor variant c. + g>a, were detected in homozygous or compound heterozygous state in patients, while the frameshift mutation, p.pro leufs* , was detected in homozygous or heterozygous in the remaining patients. strikingly, the two nonsense and the frameshift mutations had been previously reported as causative for autosomal-dominant cmt t (auer-grumbach et al. ) . out of three missense mutations, one is novel (p.his tyr), and two are reported in control databases (p.asn lys and p.arg trp). this study shows that the autosomal recessive cmt t is common in spanish population, and moreover, it suggests that screening of mme using gene panel testing could help to improve diagnosis of unclarified inherited peripheral neuropathies cases. funds: isciii (pi / , pi / ); fundació per amor a l' art. inherited peripheral neuropathies (ipns) encompass a group of disorders highly heterogeneous, clinically and genetically. charcot-marie-tooth (cmt) disease is closely related to distal hereditary motor neuropathy (dhmn) or distal spinal muscular atrophy (dsma), and some patients show additional signs associated with amyotrophic lateral sclerosis (als). targeted gene panel and exome sequencing are considered to be powerful and cost-effective tools for diagnosis of these disorders. we have investigated a clinical series of patients diagnosed of motor or sensory-motor peripheral neuropathy: families were investigated by exome sequencing and, cases were tested using different updated versions of a gene panel . each version comprises , or ipn genes, respectively and it shows a high coverage performance: percentage of analyzable target base with > coverage was , %. both exome and gene panel capture libraries were based on sureselect capture technologies (agilent technologies), and sequencing was performed in miseq or hiseq illumina equipment. we have identified novel genes and novel mutations in known genes, broadening the phenotypical spectrum associated with ipns. exome sequencing has allowed us to identify causative gene in % of familiar or sporadic cases: morc , aars, bscl , kif a, gars, egr , fig , dnajb , drp , ighmbp , dao, sod , fig . gene panel testing has been mostly performed in sporadic cases, and it has allowed us to identify either disease-causing or candidate mutations in % of cases: kif a and bicd were the most common genes mutated. update gene panels neuro and neuro have revealed novel mutations in genes recently associated to cmt and cmt disease: mme and pmp , respectively. in sum, both strategies have helped us to achieve a more accurate clinical and genetic reclassification of these disorders, an impossible challenge using conventional sequencing methods. our study expands the clinical phenotype previously associated to known ipn causing-gene, and emphasizes that gene panels should be considered as a first diagnosis method for unclarified ipn patients. funds: isciii (pi / , pi / , pi / ); fundació per amor a l' art. magy l , mathis s , goizet s , tazir m , vallat j-m . department and laboratory of neurology, national reference center for rare peripheral neuropathies, chu limoges, france; department of neurology, chu bordeaux, france; department of medical genetics, chu bordeaux, france; department of neurology, chu algiers, algeria. charcot-marie-tooth (cmt) disease is a hereditary neuropathy with a relatively homogeneous phenotype but is genetically heterogeneous. moreover, nerve conduction studies distinguish different forms, adding another level of complexity. the current classification of cmt being difficult to understand for physicians, scientists and patients, we presented and published a proposal for updating this classification, based on inheritance, nerve conduction findings and gene/mutation involved. inputs from colleagues prompted us to conduct a survey in order to try to reach some consensus about our proposals. we conducted an internet survey between october and december . the link to complete the survey was sent several times by email with an introduction to more than people. participants were contacted through the emailing list from the last cmt meeting in venice (september ) and additional physicians and scientists who are involved in cmt care and research were contacted as well. one hundred seven people from various countries (mainly france, italy and the usa) answered the survey. most ( %) of the participants were between and years of age, % being physicians and % being scientists. the vast majority ( %) considered the proposal constituted an improvement over the historical classification whereas % wanted to keep the old one. about the order of information, % of participants thought the mode of inheritance should come first, whereas % felt the phenotype should be placed at the beginning. ninety-one percent of people thought cmt should be kept as a generic name for hereditary sensory and motor neuropathy. for pure sensory neuropathy, % favoured hsn over hsan although % thought the opposite and % of participants felt dhmn should be kept for distal motor neuropathy. about nerve conduction findings, % of participants thought the intermediate phenotype has to be kept and % favoured our proposal to replace " " by "de" (for demyelinating) and " " by "ax" (for axonal). finally, % of responders thought that genetic information should be included in the classification of cmt. overall, our proposal of a new classification received a very good appreciation from physicians and scientists implicated in the care of patients with hereditary neuropathy. mallik r , hubsch a , gaida a , barnes d . csl behring, kop, usa; csl behring, bern, switzerland; csl behring, ottawa, canada. the risk of hemolytic events (hes) with intravenous immunoglobulin (ivig) therapy appears to be linked to the isoagglutinin (anti-a and anti-b) level of the specific ivig product. using published anti-a and anti-b titers for seven ivig products and corresponding he rates reported to the eudravigilance database, we developed a mathematical model to predict the risk of he to patients receiving ivig products of given anti-a and anti-b levels. modeling was performed separately for the risk to patients with blood groups a, b, ab and o and the overall population risk was estimated assuming a blood group distribution of % a, % b, % ab and % o. applying the prediction model, we calculated the he risk for an ivig product produced via a chromatographic process (privigen ® , csl behring) a) without any isoagglutinin reduction measures ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) with an anti-a donor screening program eliminating approximately % of donors with high anti-a titers ( ) ( ) ( ) , c) incorporating an anti-a/anti-b specific immunoaffinity chromatography (iac, igisolo tm ) step in the manufacturing process (since ) and d) with both measures (b and c) combined; as well as for an ivig product produced with a cohn-like cold ethanol fractionation process (carimune ® nf/sandoglobulin ® , csl behring). isoagglutinin titers in ivig products, measured by european pharmacopoeia direct assay, were provided by dr c bellac, swissmedic, bern, switzerland. the predicted risk was highest with the chromatographically purified ivig without isoagglutinin reduction ( . cases expected per kg ivig used). anti-a donor screening reduced the predicted risk to . cases/ kg. a greater risk reduction was predicted with the iac isoagglutinin reduction step ( . cases/ kg). the combination of both methods produced little benefit ( . cases/ kg) versus iac alone. the predicted hemolytic risk with ivig produced by cohn-like ethanol fractionation was low ( . cases/ kg). an observational cohort study to confirm these hemolytic risk reductions is in progress. at present, the observed hemolytic risk for anti-a donor screening appears consistent with the prediction calculated by the model; results for iac isoagglutinin reduction are expected in . the cmt infant scale (cmtinfs) is an outcome measure of functional ability for young infants and children aged < years. cmtinfs aligns with the cmt pediatric scale and cmt neuropathy score to measure disease severity across the lifespan. to measure gross motor and fine motor function, cmtinfs comprises of two subscales: gross motor (e.g. head control, crawling, walking, jumping and hopping) and fine motor function items (e.g. grasping, reaching, tearing paper and buttoning). overall and subscale-specific function is expressed as a z-score based on normative reference values (positive z-scores indicate poorer function). a total of controls aged - months (mean age , sd m) have been assessed across australia (n= ), thailand (n= ) and usa (n= ). total cmtinfs z-scores did not differ significantly between sites (australia vs thailand) (p= . ) or gender (p= . ). data collection is ongoing and infants aged < years are eligible for inclusion. to date, infants ( % male) aged - months (mean age , sd m) with a range of cmt subtypes ( cmt a, cmt d, cmt c, cmt x and unidentified gene) have been assessed with cmtinfs. mean total z-score for infants with cmt ( . , sd . , range: − . - . ) was significantly higher than controls ( . , sd . , range: − . - . , t=− . , p= . ). differences between affected infants and controls were larger in infants older than months. infants with cmt a (cmtinfs z-score . , sd . ) and cmt c (z-score . ) were less affected than cmtx (z-score . ) and cmt d (z-score . ). the gross motor function subscale differed significantly between cmt cases and controls ( . , sd . vs . , sd . ; p=. ) and a significant difference was also observed for the fine motor function subscale ( . , sd . vs . , sd . ; p= . ). reliability, factor and rasch analysis of the cmt-infs is underway to assess validity. initial results support the sensitivity of cmtinfs in distinguishing between infants with and without cmt. preliminary analyses also suggest the scale is sensitive to genetic subtype. with increased power, cmtinfs promises to become a useful outcome measure of disease severity and function in infants with cmt. manso c , querol l , mekaouche m , illa i , devaux j . aix-marseille université, marseille, france; universitat autónoma de barcelona, barcelona, spain. contactin- , contactin-associated-protein- (caspr ), and neurofascin- (nfasc ) are essential for the formation of paranodal axoglial junctions. igg autoantibodies to contactin- , caspr , and nfasc are associated with subsets of patients with chronic inflammatory demyelinating polyradiculoneuropathy (cidp) presenting with common clinical features. anti-contactin- igg autoantibodies have been shown to be pathogenic and to affect the paranodal axoglial junctions in vivo and in vitro. by contrast, the pathogenic effect of anti-nfasc igg have not been demonstrated. here, we purified anti-nfasc igg from cidp patients' plasma and investigated their effects after passive transfer. to determine whether these antibodies can pass the paranodal barrier, we performed intraneural injections of anti-nfasc igg autoantibody. by contrast to anti-contactin- igg , anti-nfasc did not penetrate the paranodal regions after intraneural injections, but bound to the surface of the schwann cell. to perform chronic exposure, lewis rats were implanted with intrathecal catheter and anti-nfasc igg were administrated in a daily manner during three weeks. igg to nfasc , but not control igg , induced progressive clinical deteriorations characterized by gait ataxia and hindlimb paraparesis. these deteriorations were associated with nerve activity loss in motor spinal nerves and with a selective loss of the paranodal specialization characterized by the disappearance of the caspr /contactin- /nfasc complex at paranodes. the passive transfer of anti-nfasc igg thus seem to induce similar pathogenic effects as the anti-contactin- igg . however, the pathogenic mechanisms leading to paranode disappearance appear different. our findings indicate that igg directed against nfasc are pathogenic and further show that these antibodies are reliable biomarkers of a specific subset of cidp patients. better fit the needs of the consortium and improve patient experience. these enhancements include creating mobile friendly webpages, updating enrollment form content, access to a customized dashboard, and the ability of registrants to explore their data in comparison to other registrants. we will review these enhancements in depth, and demonstrate their impact on the growth and development of the rdcrn inc contact registry. martinez c , hubsch a , watson dj , shebl a , wallenhorst c , simon tl . institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl bering ag, bern, switzerland; csl behring llc, king of prussia, usa; csl bering gmbh, marburg, germany. hemolytic anemia (ha) is a complication of intravenous immunoglobulin (ivig) treatment, particularly in patients receiving high dose ivig for immune modulation, such as guillain-barré syndrome or chronic inflammatory demyelinating polyneuropathy. the primary mechanism for the increased risk is believed to be passive acquisition of anti-blood group a and b antibodies (isoagglutinins) from the ivig product. to reduce the quantity of isoagglutinins, an anti-a donor screening was implemented for the ivig privigen ® from - and donors with high titers were excluded from contribution to pooled plasma. anti-a donor screening was replaced since with an immunoaffinity chromatography step, which decreases isoagglutinins to a greater extent, but no data are available to test its clinical effectiveness. to test the effectiveness of the donor screening, two cohorts of patients treated with privigen ® before and after start of donor screening were identified from a hospital-based administrative database of us hospitals with in-and outpatient discharge diagnoses, procedures, drug utilization and laboratory tests between / and / (period ) and between / and / (period ). privigen ® dose per kg body weight was estimated from the daily quantity administered and age-and sex-specific us population body weight estimates. ha within days of privigen ® use was assessed from manual records review and the incidence rate of ha in the two periods compared. incidence rate ratios (irr) of ha were adjusted for sex, age, treatment setting, indication and dose per kg body weight using period as reference. the incidence rate of ha was . / , person-days ( % confidence interval: . - . ) in period ( has in , person-days) and . ( . - . ) in period ( has in , person-days). the adjusted irr was . ( . - . ). significantly less ha risk was found with high dose (≥ . g/kg body weight) privigen ® , irr . ( . - . , p= . ). we conclude that anti-a donor screening and exclusion of donors with high anti-a titers from plasma pools is associated with a decreased risk of ha with ivig. matsumoto a . department of neurology, keijinkai jozanki hospital, sapporo, japan. subacute myelo-optico neuropathy (smon) is the intoxication of clinoquinol with main clinical symptoms of paresthesia and spasticity of legs. these symptoms have been considered to be elicited by the disturbance of spinal cord and peripheral nerve as the intoxication of clinoquinol. however, as to the patients with smon who have been still living after the onset of disease, the examination of nerve conduction velocities are in normal ranges and the clinical symptoms of peripheral neuropathy are not observed now. in order to investigate whether the peripheral neuropathy were observed in the early stage of smon, we investigated the longtidunal changes of electrophysiological results in patients who could examine the nerve conduction studies from early stage of smon until the present time. as to the disturbance of pyramidal tract functions (myelopathy) in smon patients, the central motor conduction times were calculated by transmagnetic stimulation of motor cortex, cervical roots and lumbar roots. the peripheral nerve conduction velocities of sensory nerve were examined with the sural nerves. as the results, in patients with smon who could examine the electrophysiological examination from the early stages of smon until to years later, the central motor conduction times of leg muscles from motor cortex to lumbar roots were prolonged in the smon patients compared to the normal cases. these results suggest the presence of disturbances of conduction velocities of spinal cord. conduction velocities of sensory nerve velicities (sncvs) showed the delayed sncvs of sural nerves( - m/sec) at the first examination from the onset of - years. however from to years later, sncvs of these cases were covered to - m/sec. from these electrophysilogical examinations, it was suggested that the presenting main symptoms of smon were the disturbance of myelopathy, and the disturbance of peripheral nerve function had been recovered after onset of smon being elapsed a long time, natural killer (nk) cells are part of our innate immune system with regulatory and effector functions. they comprise the first line of defence in the recognition and destruction of virus-infected and pathologically altered cells. different studies suggest that the treatment with intravenous immunoglobulins (ivig) has an immunomodulatory effect on nk cells. ivig is a first-line treatment for various autoimmune diseases in particular in chronic inflammatory demyelinating polyneuropathy (cidp). the lack of a predictive marker for ivig responsiveness in cidp avoids the early preservation of non-responding patients. to better understand the effect of ivig in patients with cidp, we tested whether ivig treatment altered the nk cell status. additionally, we analysed if the alteration in the populations may serve as a surrogate marker in predicting the outcome of ivig treatment. using semi-quantitative pcr and flow cytometry in the peripheral blood of patients with cidp, we analysed the effects of ivig on the nk cell population before treatment initiation and h after first dose and correlated the changes with the reponsiveness to ivig. ivig administrations induced a reduction in the expression of several typical nk cell genes. interestingly, this ivig-induced reduction of nk cells was reversible four weeks after the ivig treatment. flow cytometry data revealed that ivig reduced the cytotoxic cd dim nk cell population, while regulatory cd bright nk cells remained almost unaffected or were even increased. interestingly, we found that the observed effects on nk cells almost exclusively occurred in cidp patients who responded to ivig therapy. correlation between the changes in the nk cell population and treatment efficiency suggests a crucial role for nk cells in the immunomodulatory mechanism of ivig. further studies are warranted to investigate whether the differences in the nk cell status of patients with cidp represent a reliable surrogate marker in predicting the outcome of ivig therapy. mccray b , sullivan j , woolums b , aisenberg w , lloyd t , sumner c . johns hopkins university, baltimore, usa. mutations in transient receptor potential vanilloid (trpv ), a calcium-permeable non-selective ion channel, cause charcot-marie-tooth disease type c (cmt c). trpv is unique in that it represents the only membrane-expressed ion channel in cmt and thus a potential therapeutic target. previous work has suggested that trpv mutations lead to gain of channel function and toxicity in cultured cells. neuropathy-causing mutations of trpv largely cluster in the cytosolic ankyrin repeat domain (ard) that is known to mediate protein-protein interactions, suggesting that pathogenesis may be related to disruption of such interactions. in order to identify trpv -interacting proteins, we performed two unbiased proteomics screens and identified multiple cytoskeletal-modifying proteins including syndapin- , a neuronal protein known to promote axonal outgrowth by influencing the actin cytoskeleton. in cultured cells, we have shown that trpv and syndapin- co-localize to highly dynamic actin-rich cellular processes and together stimulate robust neurite extension, but this facilitation of neuritogenesis is impaired by disease-causing mutations in trpv . we have also shown that over-expression of syndapin reduces trpv -mediated calcium influx in cultured cells and rescues toxicity of mutant trpv . in addition, syndapin over-expression suppresses mutant trpv phenotypes in a drosophila model of trpv -related neuropathy. further, we have demonstrated that treatment of drosophila with a specific trpv channel antagonist ameliorates trpv mutant toxicity. together, our data highlight the importance of trpv interaction with cytoskeletal proteins such as syndapin- in the pathogenesis of cmt c. specifically, our results suggest that mutations in trpv disrupt the normal role of trpv in regulation of cytoskeletal dynamics and that interactions with the cytoskeleton reciprocally modulate trpv channel function and influence toxicity of mutant trpv . mcgonigal r , yao d , barrie ja , crawford c , willison hj . university of glasgow, glasgow, uk. guillain-barré syndrome (gbs) is in part mediated by anti-gm ganglioside antibodies induced by preceding infections. anti-gm antibodies target plasma membrane gm that is extensively distributed in both glial and axonal membranes, particularly at the node of ranvier. antibodies deposited at this site in models of gbs are associated with complement deposition, conduction block, structural disruption of ion channels and macrophage infiltration. the wide distribution of the gm ganglioside target leads to unwanted complexity in ascribing pathological outcomes to injury of cell-specific membranes, in particular unravelling the consequence of paranodal schwann cell membrane injury on axonal function, and vice versa. to overcome this impasse, we have generated transgenic mice through glycosyltransferase manipulation that express gm exclusively in neurons or glia, thus allowing us to very specifically target and injure axonal or glial membranes with a single anti-gm ganglioside antibody. through this route we can create mouse models of both the axonal and demyelinating forms of gbs, induced by a single anti-gm antibody, thus creating otherwise highly comparable conditions. here, we show anti-gm antibody binding is restricted to the nodal axolemma in galnact −/− -tg(neuronal) mice and conversely to paranodal loops in galnact −/− -tg(glial) mice. when anti-gm antibody and a source of complement is added to a nerve-muscle ex vivo injury paradigm, there is a loss of axonal integrity (i.e. loss of neurofilament immunolabeling) when the neuronal membrane is targeted in galnact −/− -tg(neuronal). conversely, axonal integrity is maintained when the paranodal membranes are decorated by antibody and complement products ex vivo in galnact −/− -tg(glial) mice. in a passive immunisation model in vivo, galnact −/− -tg(neuronal) mice acutely develop weakness, respiratory dysfunction, associated complement deposition, and degenerative pathology in distal axons. in contrast, galnact −/− -tg(glial) mice have significantly fewer abnormalities under the same acute conditions. these data indicate the high vulnerability of axonal membranes to acute injury and underline the importance of developing specific axonal protection strategies. in summary, targeting the nodal axolemmal or glial membranes allows us to study associated nodal pathology, and determine the downstream consequences on function and axon fate, currently a major area in gbs clinical research. memon a , madani s , schultz l , grover k , arcila-londono x , sripathi n , ahmad bk . neuromuscular division, department of neurology, henry ford hospital, detroit, michigan, usa. objective: to differentiate sensory electrophysiology, tli and treatment response in patients with paraproteinemic cidp. background: low tli has been reported as a useful electrophysiological marker for mag-cidp. to our knowledge comparison of sensory electrophysiology and tli of paraproteinemic cidp subgroups have not been previously reported. methods: retrospective review(january -december ) of patients with cidp fulfilling electrophysiological criteria(aan ad hoc subcommittee and albers and colleagues).cidp patients with diabetes(n= ) were excluded. patients were divided into idiopathic (n= ) and paraproteinemic cidp(n= ). paraproteinemic cidp sub-groups: mag( ), non-mag( ) and igg( ) were compared to idiopathic cidp( ). these groups were compared for demographics, history of cancer, csf protein, sensory conductions, tli measurements and response to treatment using chi-square tests for binary and categorical variables and t-tests for continuous measures. results: there was a higher proportion of females in idiopathic-cidp compared to non-mag-cidp ( % vs %). idiopathic group having a higher proportion of patients on monotherapy( % vs %) and combination therapy( % vs %) compared to non-mag. higher mean csf protein compared to mag-cidp(p= . ) was seen in the idiopathic. the difference between idiopathic and igg-cidp was significant for overall rx response(p= . ) and rx response in patients with follow-up(p= . ). for both variables, patients in the idiopathic group had a higher proportion of patients on combination therapy and lower proportion of no treatment offered compared to patients in the igg-cidp. % of non-mag-cidp patients had a history of cancer vs % of mag-cidp. none of the other differences were significant. there were no group differences in sensory electrophysiology and tli. conclusions: sensory electrophysiology and tli may have no value in differentiating paraproteinemic cidp. csf protein is higher in idiopathic cidp compared to mag-cidp. idiopathic-cidp has a higher proportion of females compared to non-mag-cidp and a higher proportion of patients on combination therapy compared to igg-cidp. cancer screening should be considered in patients with non-mag-cidp. memon a , madani s , ahmad bk , schultz l , grover , arcila-londono x , sripathi n . department of neurology, henry ford hospital, detroit, michigan, usa. introduction: sensory electrophysiology and terminal latency index (tli) differences have been described in various cidp sub-groups. objective: evaluate electrophysiology, tli and treatment response in idiopathic and diabetic cidp. methods: retrospective review of patients with cidp who underwent electrodiagnostic evaluation (january -december . patients fulfilled electrophysiological criteria described by ad hoc subcommittee of american academy of neurology (aan) and albers et al. we excluded patients( ) with acute inflammatory demyelinating neuropathy, hereditary sensorimotor neuropathy, vasculitis and polyneuropathy with paraproteinemia. patients were divided into idiopathic( ) and diabetic( ) groups. these groups were compared for age, sex, history of cancer, csf protein, response to treatment, sensory response abnormalities and tli measurements using chi-square tests for binary and categorical variables and t-tests for continuous measures. all testing was at the alpha= . level. results: group differences for age, sex, history of cancer, csf protein and treatment response were not significant. comparing tli values in measurable responses, the difference between the two groups for tibial tli was significant (p= . ), with idiopathic group having a lower mean as compared to the diabetic. tli values differences for median, ulnar and peroneal nerves were not significant. the difference in abnormal rates of sensory responses was significant for the sural nerve with the idiopathic group having a lower rate compared to the diabetic group ( % vs %, p< . ). no differences were noted for the ulnar, median and radial nerves. tibial tli and sural sensory responses have some value in differentiating the two groups. larger prospective studies are needed to confirm our findings. ivig is an important treatment option for cidp. although recommended by treatment guidelines, little is known about the consequences of temporary ivig withdrawal to assess ongoing immunoglobulin need. path is a randomized, double-blind trial of the subcutaneous immunoglobulin (scig) igpro (hizentra ® , csl behring) in cidp. before scig randomization, subjects underwent periods of ivig withdrawal ( weeks or until pre-determined indications of clinical deterioration) and ivig restabilization (igpro ; privigen ® , csl behring). subjects not showing deterioration during withdrawal period were withdrawn from study. to proceed to scig randomization, subjects had to achieve "cidp stability" (no relevant change in incat score at last two restabilization visits and at least the same total score as at screening). subjects entered the ivig withdrawal period. ( %) of these qualified for igpro restabilization; subjects ( %) were not ivig dependent, and ( %) were withdrawn for other reasons. one subject withdrew consent before igpro dosing. at the end of the igpro restabilization period, cidp stability was achieved in % of subjects ( did not reach stability, were withdrawn for other reasons). post-study follow-up information was available for / subjects who did not reach stability: ( %) had improved to baseline clinical status and had not, meaning at least % of the subjects improved to their pre-study status. during the restabilization period, / subjects ( %) improved in at least one of the predefined outcome measures. on average, subjects improved by . points in incat total score, . points in i-rods centile score, kpa in mean grip strength (dominant hand), and . points in mrc sum score. improvement occurred with a median of days after the first igpro dose in one or more efficacy outcome measures and in % of cases after the third igpro maintenance infusion. headache and nasopharyngitis were the most frequently reported adverse events (aes) during restabilization. aes deemed causally related were mostly mild or moderate. no unexpected aes or laboratory or vital sign findings associated with igpro occurred during the study. in summary, igpro reversed neuromuscular disability and improved activity/participation after previous clinical deterioration during an ivig withdrawal period. pmp duplication is the most frequent cause of charcot-marie-tooth disease (cmt). since it discovery, more than genes have been identified to be potentially responsible for cmt disease. however only single nucleotide variations (snvs) or small indels have been described. this could be due to the new sequencing strategy (ngs), especially ngs by amplicon sequencing, for whose few convenient tools are available and easily usable to detect cnvs responsible for inherited disease. to overcome this problem, we designed "cov'cop", a user-friendly tool able to detect cnvs among amplicons sequencing data. using the run's coverage file provided by the sequencer, "cov'cop" simultaneously analyzes all the patients of the run using a two-stages algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. we validated our method on several datasets, including those of our targeted ngs panel screening genes known to be involved in cmt and close pathologies. cov'cop detected easily pmp duplication and deletion in our patients, confirmed by mlpa. in addition, cov'cop permitted the detection of new cnvs different from the pmp duplication, in cmt patients. we confirmed these cnvs by quantitative pcr and cgh array. we present here one of these cnvs: the duplication of aars gene detected in cmt patients and we discuss the pathogenicity of this new cnv. additional cnvs responsible for cmt disease are probably still to be discovered and we believe that cov'cop will help molecular geneticists to rapidly identify them. poems (polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscraisia and vascular endotherial (vegf) overproduction. although number of therapeutic interventions for plasma cell dysorders have been applied to poems syndrome, there have been no randomized clinical trials. this phase / double blind, randomized, placebo comtrolled trial was performed to investigate the safety and efficacy of thalidomide for patients with poems syndrome who are not eligible for stem-cell transplantation. the primary endpoint was the reduction rate of serum vegf concentrations at weeks in intention to treat analysis. additional outcomes of long-term extension study included progression-free survival. twenty-five poems patients were randomly assigned to either thalidomide plus dexamethasone or placebo plus dexamethasone from nov , , to july , . one patient in the placebo group was excluded from analyses because of a protocol violation. the adjusted mean serum vegf reduction rate at weeks was . (sd, . ) in the thalidomide group compared with − . ( . ) in the placebo group (p= . ). the kaplan-meier rate of progression-free survival at months was . in the thalidomide group, as compared with . in the placebo group (hr, . ; % ci, . to . ). in the randomized study period, mild sinus bradycardia was more frequent in the thalidomide group than in the placebo group ( % vs %; p= . ). thalidomide suppresses serum vegf concentrations and lengthened pregression-free survival in poems patients who are ineligible for stem cell transplantation. although thalidomide treatment has a risk of bradycardia, the benefits would exceed the risk. this study is registered with the umin clinical trials registry, umin . montes-chinea ni , coutts m , vidal c , courel s , rebelo a , abreu l , zuchner s , saporta, ma , . department of neurology, university of miami, miami, usa; department of human genetics, university of miami, miami, usa. autosomal dominant mutations in synaptotagmin- (syt ), a synaptic vesicle protein that functions as a calcium sensor for neurotransmission, have been previously linked to presynaptic neuromuscular junction (nmj) dysfunction and motor neuropathy in two families. both pathogenic mutations (asp ala and pro leu) were located in the c b domain of syt , which is essential for neurotransmitter release at the nmjs. we report a family with a new missense mutation in the c b domain of syt and a similar phenotype characterized by a slowly progressive, predominantly motor neuropathy and evidence of presynaptic nmj dysfunction on nerve conduction studies. the index case is a year-old woman with gradually progressive weakness of her extremities. she had normal developmental milestones, but was found to have bilateral high arched feet and hammertoes and occasional falls around the age of . she gradually developed progressive leg weakness, worsening bilateral hand cramping, weak handgrip, and only mild paresthesias on distal extremities. family history is remarkable for similar symptoms reported by her maternal grandfather, two maternal uncles, her mother and a younger sister. her neurological exam revealed inability to walk on heels or toes, significant distal lower extremity weakness and absent ankle deep tendon reflexes. cranial nerve examination and coordination were normal and there were only non-specific sensory changes in the lower extremities. emg/ncs revealed normal sensory responses throughout; however, motor nerve evaluation demonstrated globally reduced amplitudes with a > % increment after brief isometric contraction. further electrophysiological evaluation with slow ( hz) repetitive nerve stimulation of the right ulnar motor nerve revealed a % decremental response in amplitude and a > % increase in amplitude immediately after a one-minute period of sustained muscle contraction, which rapidly extinguished after one minute. voltage-gated calcium channel (vgcc) antibodies and a chest ct were normal. targeted sanger sequencing revealed an ile lys mutation in syt , which is located in the c b domain and is predicted to impair protein function. syt -related neuropathy is a rare disease, but should be suspected in patients presenting with a combination of pre-synaptic nmj dysfunction (resembling lambert-eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history. charcot-marie-tooth (cmt) disease affects roughly in , individuals and is described as an inherited peripheral neuropathy primarily affecting distal muscles. limited studies detail patient-reported impact of muscle weakness on functional activities. this anonymous survey was developed with input from clinical experts and patient interviews and aimed to better understand the prevalence and impact of various cmt clinical manifestations on patients' lives. the survey was administered online to the hereditary neuropathy foundation's (hnf) patient contact database and is ongoing through june . here we present preliminary data on patient characteristics and disease impact for cmt patients collected february - , . respondents were mostly female ( %) and mostly from the us ( %). median age (range) at symptom onset was years ( - years), at diagnosis was years ( - years), and at present was years ( - years). the sample was representative of all cmt types (cmt , , , ,and x). the most common physical and clinical manifestations of cmt were problems with balance ( %), ankle weakness/foot drop ( %), loss of feeling or abnormal sensation in the lower leg/foot ( %), and hand muscle weakness ( %). maintaining balance, walking long distances, and climbing up and down stairs were key challenges associated with ankle weakness/foot drop. foot drop was considered by % to be the primary factor contributing to falls, which averaged . falls to ground per month. of those with foot drop, % had bilateral weakness. a majority of respondents ( %) used some form of assistive device for mobility, including ankle-foot orthotics/below-the-knee leg braces ( %), canes/walking sticks ( %), and custom foot orthotics/inserts ( %). the most common drug therapy included pain and anti-inflammatory medications ( %). foot surgery was the most common surgical procedure received ( %) and toe surgery was the most common surgery considered ( %). key symptoms that affected quality-of-life "very much" included problems with balance ( %), ankle weakness (foot drop) ( %), and fatigue ( %). these data suggest a high prevalence of lower leg muscle weakness; therefore, therapies aimed at improving ankle weakness and the resulting foot drop and imbalance may be beneficial to patients' daily functioning and quality of life. morano m , , gambarotta g , ronchi g , , cillino m , fornasari be , , fregnan f , , tos p , cordova a , moschella f , geuna s , , raimondo s , . department of clinical and biological sciences, university of turin, orbassano (to), italy; neuroscience institute of the "cavalieri ottolenghi" foundation (nico), university of turin, orbassano (to), italy; plastic and reconstructive surgery. department of surgical, oncological and oral sciences, university of palermo, palermo, italy; hand microsurgery and surgery, gaetano pini hospital, milan, italy. nerve fiber regeneration and complete functional recovery after peripheral nerve injury do not always occur and can be influenced by many factors including patient age, gender, lesion site, injury severity, size of the gap between damaged nerve stumps and time interval that elapses before performing surgical repair. the poor outcome occurring after a long delay can be due to loss of the neuron ability to regenerate, loss of the schwann cell ability to support regeneration and, of course, progressive muscle atrophy. the aim of this study was to investigate the nerve regeneration after delayed repair and to study the degenerative processes of the denervated distal nerve stump and denervated muscle. in particular, the analyses were focused on the role of nrg/erbb system, that is expressed both in nerve and in muscle tissue, during degenerating and regenerating processes. functional recovery analysis performed after nerve repair showed that only the group repaired immediately and not the groups repaired with a delay of or months, recovered partially. nevertheless, morphological analyses demonstrated that, despite the delay, the nerve fibers are still able to regenerate, even if they are fewer and smaller than the immediate repaired group. moreover, the analysis of the nrg /erbb system showed a significant decrease of soluble nrg in both degenerating and delayed-repaired nerves. the poor outcome after delayed nerve regeneration might be explained by schwann cell impairment and the consequent ineffective support for nerve regeneration. as regards denervated muscle analysis, results showed that erbb receptors expression is related to the innervated state of the muscle, with an upregulation of erbb clearly associated with denervation state. interestingly, nrg isoforms are differently regulated depending on the type of nerve injury. future experiments will be needed to address the in vivo efficacy of different isoforms of nrg both in injured nerve and denervated muscle. we planned a multicenter, prospective, randomized, single blind, controlled study to evaluate the efficacy and safety of an innovative rehabilitation protocol based on the use of treadmill training in a cmt a population. the protocol required that subjects were blindly randomized into two treatment groups, spe (three months of respiratory, proprioceptive and stretching exercises) or trespe (the same treatment plus aerobic training at the treadmill). subjects were evaluated at baseline (t ), after three month of treatment (t ) and further three months of follow up free of therapy (t ). the full assessment included: -mwt (primary outcome measure), -mwt, walk- , short physical performance battery (sppb); lower limbs dynamometric strength evaluation; berg balance scale (bbs); cmt neuropathy score; medical outcomes study short form (sf ). a total of subjects (mean age of . ± . years) were recruited. at t we found a significant improvement in both groups in the -mwt (p< . ), -mwt (p< . ), bbs (p< . ) and sppb (p< . ), while at t only the -mwt was still significantly improved (p< . ) in the spe group. no significant differences between groups were observed for any of the outcome measures. performances on walk did not significantly change during follow-up (p= . ). concerning the sf , we did not observe consistent changes during follow-up or consistent differences comparing the two treatments. in conclusion, this multicenter, prospective, randomized, single blind, controlled study shows that the combination of respiratory, proprioceptive and stretching exercises has a positive impact on the performance of cmt patients, especially regarding walking tests. the aerobic exercise at the treadmill, is well tolerated but apparently does not add any further improvement to the conventional treatment. we speculate that the relatively low clinical severity of the patients, due to the selection criteria, may have prevented a positive effect of treadmill exercise. müller m , dohrn m , romanzetti s , reetz k , gess b . university rwth aachen, department of neurology, aachen, germany. muscle mri is increasingly used in neuromuscular patients to detect changes in muscle volume, muscle fat infiltration and edema. muscle mri are mostly analyzed by qualitative means as quantitative analysis is time-consuming and not well established. here, we developed a novel method for semi-automated segmentation of muscle mri data sets. based on axial t -weighted dixon mri stacks, muscle volumes were quantified by an adapted water-shed algorithm. muscle volumes of thighs and calves were determined separately and the ratio of thigh/calf was calculated. myopathy, neuropathy patients and healthy controls were included in the study. muscle volumes determined by semi-automated segmentation were very similar to manually segmented data sets, differences being < %. this was the case for patients as well as healthy controls. the time-saving effect of automated segmentation was very strong ( vs min. per patient). muscle volumes of the thigh and also of the calf of myopathy patients showed a highly significant difference (p< . ) compared to healthy subjects. in neuropathy patients there was a just significant difference (p< . ) of muscle volumes compared to healthy patients that did not sustain in bonferroni's multiple comparison test. the ratio of thigh/calf muscle volume was significantly different comparing patients with myopathy and neuropathy (p< . ). subgroup analyses of different groups of myopathy patients showed highly significant differences (p< . ) in myositis, limb-girdle-muscular dystrophy and metabolic myopathy, compared to healthy patients, but no significant differences in-between these groups. taken together, the data shows that automated segmentation of muscle mri allows for exact and fast quantification of muscle volumes in neuromuscular patients. higher patient numbers are necessary to test differences between specific disease groups. further studies should also address the possible use as a marker of disease progress for clinical studies or therapy monitoring. murakami t , nishimura h , nagai t , hemmi s , kutoku y , sunada y . department of neurology, kawasaki medical school, kurashiki, japan; department of pathology, kawasaki medical school, kurashiki, japan. familial amyloidotic polyneuropathy (fap) is an autosomal dominant hereditary systemic amyloidosis caused by mutation of transthyretin (ttr) gene, and usually shows sensory dominant polyneuropathy and autonomic neuropathy at the initial stage. the pathogenesis of neuropathy is not well understood, and explained by several mechanisms, including such as mechanical compression, vessel occlusion, ttr toxicity and schwann cell dysfunction. we describe a sporadic patient with late-onset fap due to ttr e k. she noticed dysesthesia first in the foot at age . the symptoms were slowly progressive, and abnormal sensations were extended up to the both upper arms and the both knees at age . distal muscle weakness and atrophy was also observed in the extremities. she noticed difficulties in walking and frequent diarrhea. echocardiogram revealed diffuse left ventricular hypertrophy, suggesting cardiac amyloidosis. amyloid deposits were not detected in the endoneurim or perineurium of the sural nerve years after the onset of the disease, but a marked loss of myelinaed and unmyelinated nerve fibers was observed in it. ttr-derived amyloid deposits were confirmed in the peroneous brevis muscle, salivary gland and heart tissue. dna analysis revealed the heterozygote mutation, p.e k (e k)/c. g>a, of ttr gene, and she was diagnosis as fap. these findings suggest that the proximal parts of peripheral nervous system might be strongly involved by ttr aggregates or amyloid fibrils. blood-nerve barrier in the distal part of peripheral nerves could be preserved until later in the patient. several biopsy sites other than nerve may be helpful and necessary for diagnosis of ttr amyloidosis in mild or late-onset fap as our case. amyloidgenic element (cae) encoded by the ' utr. this study also identified a de novo c. _ dup frameshift mutation predicting p.lys glnfs* in nefh from a cmt family with atypical clinical symptom of proximal dominant weakness. this mutation is located near the previously reported frameshift mutations, suggesting a mutational hot spot. these relatively frequent deletion/duplication events with this resign might be caused by the putative hairpin structure. patient's lower limb mri revealed a marked hyperintense signal changes in the hip muscles than those in the thigh or lower leg muscles. this study also observed an anticipation pattern of earlier onset ( yrs old for mother to yrs old for daughter) and more severe symptoms in later generation. therefore, this study suggests that the stop loss and translational elongations by the ' utr of the nefh mutations may be relatively a frequent genetic cause of axonal peripheral neuropathy with the specific characteristics of proximal dominant weakness and an anticipation pattern. neil j , haghi ashtiani b , choumet v , musset l , léger jm . department of immunology, pitié-salpêtrière hospital (ap-hp), paris, france; department of neurology, national referral center for rare neuromuscular diseases, pitié-salpêtrière hospital (ap-hp), paris, france; institut pasteur, emerging diseases epidemiology unit, paris, france. the myelin-associated glycoprotein (mag) is a transmembrane glycoprotein localized in periaxonal shwann cells and oligodendroglial membranes of myelin sheaths. mag contains a carbohydrate epitope (hnk- ) that is a target antigen in autoimmune peripheral neuropathy associated with monoclonal igm gammopathy. in these neuropathies, numerous studies report the absence of correlation between the titers of anti-mag antibodies and the disease course. anti-mag titers and igm level at diagnosis are not always associated with disease severity and there is not good correlation between pre-and post-treatment anti-mag titers in patients who respond clinically to immunomodulators. mag belongs to siglec- a family and the linkage of sialic acid to the underlying sugars is an important determinant of siglec binding. mag shows high affinity for alpha- , -linked sialic acid ( , -sa).moreover, human monoclonal igm possesses heavy chain glycosylation sites at asn , , , and with sialylated olidgosaccharides and high-mannose type oligosaccharides. igm may bind to mag via these glycan epitopes as an alternative and additional route of antigen binding other than through the fab v regions. this mag-glycans igm interaction may be clinically neutral but could lead to an overvaluation of the biological results. in this study, we analyzed sera from patients with igm reactivity against mag: of them had an anti-mag neuropathy with various degrees of severity, and the last one had igm monoclonal gammopathy, strong serum anti-mag reactivity but no neurological disease. igm were extracted and purified from these sera by affinity chromatography. for each batch, an aliquot was digested by jack bean alpha-mannosidase and anti-mag reactivity was performed by elisa and indirect immunofluorescence (iif), before and after demannosylation. these extracts, tested in an iso quantitative way with regard to the original serum, showed a decrease of activity (elisa) and intensity (iif) after demannosylation. furthermore, elisa anti-mag was carried out in sera from patients with igm monoclonal gammopathy without neurological impairment: of them ( . %) showed a significant biological response. taking into account the fact that anti-mag antibodies are pathogenic (in animals models), these results support the hypothesis of neutral intermolecular interactions between igm and mag. ng cjb , ng jph , tay lb, t , umapathi . yong loo lin school of medicine, national university of singapore, singapore; lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. patients with postural orthostatic tachycardia syndrome (pots) have marked tachycardia with no associated decrease in pbp. we asked if the reflex tachycardia, which corresponds to postural dizziness in these patients, could be a result of a decrease in casp. two male patients, and years of age, with clinical features typical of pots went through a complete battery of autonomic screening tests. the sympathetic and parasympathetic responses were normal other than a heart rate increase of and beats per minute, respectively, on standing. there was no significant decrease or increase in pbp on standing for and minutes. casp was measured non-invasively by a device bpro r that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure waveform from which a numerical casp value is derived. the casp measurements for both patients did not decrease on standing for and minutes. our preliminary observation suggests that the basis of tachycardia in pots patients may not be a decrease in central blood pressure. we are proceeding to systematically study more pots patients to corroborate the above observation. we are also trying to compare the difference between pbp and casp in pots and age, gender-matched normal controls. the major limitation of the study is the model-based mathematical derivation, rather than direct measurement, of casp. we are proceeding to systematically stud y more pots patients to corroborate the above findings. ng jph , ng cjb , t umapathi . lee kong chian school of medicine, nanyang technological university, singapore; yong loo lin school of medicine, national university of singapore, singapore; national neuroscience institute, singapore. recent developments have validated non-invasive means of measuring central arterial blood pressure (casp) and have shown that casp and peripheral blood pressure (pbp) are unidentical entities. orthostatic hypotension (oh) is a prominent component of autonomic dysfunction (ad), the consequent hypoperfusion of vital organs responsible for considerable morbidity and mortality. these structures are exposed to casp rather than pbp. we sought to understand the relationship of casp to peripheral blood pressure (pbp) in patients with autonomic dysfunction exposed to orthostatic stress. we reviewed autonomic function tests of patients tested at our laboratory over a -year period. the patients were divided into cohorts: ( ) no ad and no oh ( ) no ad, with oh ( ) mixed ad ( ) parasympathetic dysfunction ( ) sympathetic dysfunction. casp was measured non-invasively by a device, bpro r , that imputes the measured radial waveform onto the brachial blood pressure to generate a pressure-wave form from which a numerical casp value is derived. the difference and ratio of casp to pbp was recorded at rest and after minutes of standing. out of patients had complete data and a definitive final diagnosis. cohorts - had , , , , patients respectively. mean casp-pbp difference in cohorts - were − . , − . , − . , − . , − . respectively at minutes, and − . , − . , − . , − . , − . respectively at minutes. mean casp/pbp ratio in cohorts - were . , . , . , . , . , . respectively at minutes, and . , . , . , . , . respectively at minutes. there was no significant difference in the response of casp and pbp to orthostatic stress across abnormal cohorts to and in comparison with the "normal" cohort (p= . to . ). there was also no relationship to symptoms, namely postural dizziness. in conclusion, autonomic dysfunction does not seem to affect the casp-pbp relationship as measured by non-invasive means. the absence of true normal controls, the exclusion of significant number of patients because of incomplete data and the model-based mathematical derivation rather than direct measurement of casp are limitations that we aim to address in follow-on studies. niu jw , guan hz , cui ly , guan yz , liu ms . the department of neurology, peking union medical college hospital, chinese academy of medical sciences, beijing, china. we aimed to explore the correlation between afterdischarges in motor nerve conduction studies and clinical motor hyperexcitability in patients with voltage-gated potassium channels (vgkc) antibodies. six patients with positive serum antibodies to contactin-associated protein-like (caspr ) or/and leucine-rich glioma-inactivated protein (lgi ) were recruited, including with autoimmune encephalitis, and with cramp-fasciculation syndrome. electromyography (emg), nerve conduction studies (ncs) and f waves were performed, and afterdischarges were assessed. one patient was followed up. five patients had clinical evidence of peripheral motor nerve hyperexcitability (myokymia or cramp), and four of them had abnormal spontaneous firing in concentric needle electromyography. prolonged afterdischarges following normal m waves were present in all six patients, including the two patients who had no emg evidence of peripheral nerve hyperexcitability (pnh). in the patient who was followed up, afterdischarges disappeared after treatment with intravenous immunoglobulin (ivig). afterdischarges in motor nerve conduction study might be more sensitive than needle electromyography for detecting peripheral motor nerve hyperexcitability in patients with vgkc antibodies, and could disappear gradually in accordance with clinical improvement and reduction of antibodies. in our research, multiple sites measurement of cross sectional areas (csa) by ultrasound was performed to differentiate charcot-marie-tooth type a (cmt a) and chronic inflammatory demyelinating polyradiculoneuropathy (cidp). twenty-eight patients with cidp, patients with cmt a, and healthy controls (hc) were recruited prospectively. consecutive ultrasonography scanning was performed from wrist to axilla on median and ulnar nerves. csas were measured at predetermined sites of each nerve. cmt a had significantly larger csas at all sites of median and ulnar nerves (all p< . ). in cmt a, csas increased gradually and homogeneously from distal to proximal along the nerve, except potential entrapment sites. cidp displayed three different morphological patterns, including mild enlargement in patients, prominent segmental enlargement in , and slight enlargement in one, among which different treatment responses were observed. all patients with mild nerve enlargement treated with intravenous immunoglobulin (ivig) were responsive ( / ), while less than half of those with prominent segmental enlargement ( / ) were responsive (p< . ). the patterns of csa enlargement were different in cmt a and cidp patients. consecutive scan along the nerve and multiple sites measurement by ultrasound could supply more detailed morphological feature of the nerve and help to differentiate cidp from cmt a. guillain-barre syndrome (gbs) mainly consists of acute inflammatory demyelinating polyradiculoneuropathy (aidp) and acute motor axonal neuropathy (aman). in aman, conduction block (cb) could be reversible or followed by axonal degeneration. we aimed to identify the correlation between existence of cb and the functional outcome for patients with gbs. gbs patients were prospectively recruited for serial electrophysiological tests and disability evaluation. all patients received treatment of intravenous immunoglobulin (ivig), and their disabilities were evaluated on the hughes functional grading scale before and month after treatment. patients were classified into aidp, aman, equivocal or normal according to electrodiagnostic criteria described by rajabally et al. aman patients who had follow-up nerve conduction studies were further classified into three groups. group was typical aman without conduction block, group had reversible conduction block, group had conduction block and subsequential axon degeneration. electrophysiological study results showed aidp, aman, equivocal and normal. probable or definite conduction block was observed in aidp patients and aman patients. aman with cb had higher reduction of hughes grade at one month ( . ± . vs . ± . ,p= . ), and lower percentage of patients with slow recovery (unable to walk independently at six months) ( % vs %, p= . ) compared with aman without cb. there were no significant differences between aidp with cb and without cb, in the reduction of hughes grade at one month. among the aman patients who were followed up, were typical aman without cb (type ), had reversible cb (type ), had cb and subsequential axon degeneration (type ). hughes grades at nadir were similar, while patients with reversible cb (type ) had the largest hughes grade reduction at one month (type - . vs type - . vs type - . ). none of the patients with axon degeneration (type ) showed rapid recovery, while % of those with reversible cb (type ) had rapid recovery (improvement by two or more hughes grades within four weeks after onset). electrodiagnosis of aman with conduction block, especially reversible conduction block, might be a marker of good recovery. we report a follow-up study of nerve ultrasound in a patient with primary neurolymphomatosis. a -year-old female presented with -months history of asymmetric limb pain, paresthesia, and weakness. electrodiagnostic studies and spinal cord mri showed an axonal neuropathy involving cervical and lumbosacral root, brachial plexus and left median nerve. detection of malignant b lymphocytes by cytology and flow cytometry of cerebral spinal fluid confirmed the diagnosis of b-cell non-hodgkin lymphoma. nerve ultrasound showed dramatic enlargement of upper, middle and lower trunks of left brachial plexus (cross sectional area-csas mm , mm , mm respectively), middle trunk of right brachial plexus (csa mm ), and proximal part of left median nerve (csa - mm ). five months later, after five chemotherapy of rituximab and high-dose methotrexate, and intrathecal injection of cytosine arabinoside and dexamethasone, the patient had clinical improvement. nerve ultrasound also showed alleviation of nerve enlargement. the csas of upper, middle and lower trunks of left brachial plexus were mm , mm , mm respectively; the csa of middle trunk of right brachial plexus was mm ; the csa of proximal part of left median nerve was - mm . peripheral nerve ultrasound could help locate the distribution of nerve involvement, and reveals disease progression. nolano m , provitera v , stancanelli a , caporaso g , saltalamacchia am , borreca i , lullo f , califano f , lanzillo b , iodice r , manganelli f , barone p , santoro l . irccs "salvatore maugeri" foundation, institute of telese terme (bn), italy; "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; center for neurodegenerative diseases (cemand), department of medicine and surgery, neuroscience section, university of salerno, italy. a peripheral nerve involvement has been demonstrated in pd with the evidence of a small fiber pathology as possible intrinsic feature of the disease and a higher occurrence of large fiber neuropathy in patients longtime treated with l-dopa. however the role that disease itself and ldopa have on small and large fiber pathology in pd is still debated. we studied morphology and function of cutaneous innervation, in idiopathic pd patients ( male, aged . ± . ), including naïve and l-dopa treated subjects without electrophysiological signs of neuropathy, with the aim to assess and characterize small and large fiber involvement and the effect of l-dopa on it. all patients underwent a screening to rule out potentially neurotoxic conditions such as glucose intolerance, dysendocrinopathies, vitamin e, b and folic acid deficiency, hepatic or renal failure, hiv or connective tissue disorders. skin biopsies were obtained from thigh, leg and fingertip from the more affected side and bilaterally from thigh and leg in patients. samples were processed with indirect immunofluorescence technique using primary antibodies to mark different sensory and autonomic fiber populations. density of intrapapillary myelinated endings (ime), meissner's corpuscles (mc) and epidermal nerve fibers (enfs) was obtained as well as a semi-quantitative assessment of sudomotor, pilomotor and vasomotor innervation. further evaluation included sympathetic skin response, quantitative sensory testing and dynamic sweat test. morphological and functional findings were compared with data extracted from our age and sex stratified normative dataset. ienf, ime, mc densities were lower (p< . ) compared to controls in both naïve and l-dopa treated patients without differences between them except for mc density that was lower in l-dopa treated subjects ( . ± . vs . ± . /mm ). a loss of autonomic nerves was also found in both groups compared to controls. significant abnormalities (p< . ) of thermal sensory thresholds, tactile thresholds, mechanical pain perception and reduced sweating output were present and similar in both groups. our work confirms in pd an intrinsic peripheral nerve pathology involving both small and large fibers. small fiber pathology isn't affected by l-dopa treatment while sensory large fibers involvement, already present in naïve patients worsens with ldopa treatment. noto y , garg n , li t , timmins hc , park sb , shibuya k , kiernan mc . brain and mind centre, the university of sydney, sydney, australia. the diagnosis of amyotrophic lateral sclerosis (als), a progressive, fatal neurodegenerative disorder defined by combined upper and lower motor neuron involvement, remains clinically based. the purpose of this study was to determine the ultrasound appearance of peripheral nerves in als patients, and to investigate whether parameters such as distal/proximal ratios of nerve cross-sectional areas (csas) may effectively differentiate disease mimics from als. nerve ultrasound of the median, ulnar, and tibial nerves was performed in als patients compared to mimic patients ( patients with peripheral nerve hyperexcitability syndromes (pnhs) and patients with multifocal motor neuropathy (mmn)). comparison of nerve and the distal/proximal ratios was undertaken by ultrasound and compared across clinical and neurophysiological parameters.compared to normal controls, csa of the median nerve at the upper arm was decreased in als (p < . ). in comparison to als mimic disorders, csa at the proximal site of the median, ulnar and tibial nerve and the forearm/upper arm ratio of the median and ulnar nerves had diagnostic values. in addition to csa of the median, ulnar, and tibial nerves, the median and ulnar nerve forearm/upper ratios may provide a useful marker in for the diagnosis of als. approximately in , individuals are diagnosed with charcot marietooth (cmt) disease, making it the most common hereditary peripheral neuropathy. there is no documented cure for cmt, however, many of those affected, report difficulty with mobility, imbalance, and weakness of the feet and hands. in the general population, patients reporting difficulty walking, falls and/or fear of falling, and poor strength are often referred to physical (pt) and occupational therapists (ot) to skillfully address the impairments and help restore function and quality of life (qol). for patients diagnosed with cmt this is unfortunately not the norm, often leaving patients without any skilled guidance on managing their functional impairments. patients ( males) with a mean age of . years ( - ), went through a progressive and skilled pt intervention over the course of months. the program included: therapeutic activities and exercise, neuromuscular reeducation, and manual techniques to address the documented deficits. patients were progressed through the program based on the borg scale. each patient was assessed prior to and post the commencement of the program with the berg balance scale, minute walk test, timed up and go, sit to stand in seconds, foot self selected and fast gait speed, the lower extremity functional scale, activities balance confidence form, upper extremity functional index, oswestry, and the sf- qol measure. the patients were seen by the same skilled pt - a week for weeks. all but patients improved in all measures taken, indicating an improvement in function and overall quality of life. participants reported a total of falls in the months prior to the initiation of the study and only fall was reported during the month pt intervention. this study makes a strong case for the utilization of skilled pt to address deficits in patients with cmt. additionally, the utilization of objective, valid and reliable outcome measures in this population may help healthcare practitioners establish baseline function and response to change. a large randomized control trial is recommended to study the effects of a specific pt intervention on outcome measures in patients with cmt. ogata h , fujita a , yamasaki r , matsushita t , kira ji . department of neurology, neurological institute, graduate school of medical sciences, kyushu university, fukuoka, japan. clinical features of chronic inflammatory demyelinating polyneuropathy (cidp) patients with autoantibodies against neurofascin (nf) , one of the paranodal proteins, have been elucidated while the relation between anti-nf antibody levels and long-term clinical course in these patients still remains elusive. we retrospectively collected clinical, electrophysiological and immunological data of three anti-nf antibody-positive cidp patients. they were all males and their ages at onset were , , and years old. their clinical severity was evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale. anti-nf antibody levels were measured by flow cytometry using hek cell lines stably expressing human nf . after immunotherapies of various combinations, including intravenous immunoglobulin, plasmapheresis, corticosteroids and other immunosuppressants, were introduced, their clinical parameters were gradually improved. decreased or absent dtrs were normalized and grip strength was increased by more than kg. hughes functional scale scores were decreased by at least one point compared with those at nadir. ncs findings of all three patients also showed obvious amelioration. for example, their f wave latencies in the right ulnar nerve were improved from to ms, from to ms, and from to ms, respectively. anti-nf antibody levels after treatment were decreased in two patients whose pre-and posttreatment sera were available. when dose of oral prednisolone was being tapered, they experienced re-exacerbation of clinical parameters, especially dtrs and grip strength. their ncs findings and serum anti-nf antibody levels were also deteriorated. exacerbation of these laboratory data in one patient preceded his clinical fluctuation, which suggests that ncs and serum anti-nf antibody levels could be used as early disease activity markers. in this case series, not only clinical but also laboratory data support a notion that anti-nf antibody-positive cidp patients were reactive to combined immunotherapies including corticosteroids. even though various treatments were administered to them, efficacy of oral corticosteroids seemed to be dose-dependent. optimal disease activity markers and immunotherapies for long-term maintenance of remission in anti-nf antibody-positive cidp should be identified. evaluated by deep tendon reflexes (dtrs), grip strength and hughes functional scale, after starting immunotherapies, ohnmar o , kamyw , ng lfp , t umapathi . university of medicine , yangon, myanmar; singapore immunology network, a*star, singapore; national neuroscience institute, singapore. singapore's zika virus (zikv) outbreak started in late august . over a period of months, we studied patients enrolled into our institution's prospective guillain-barré syndrome (gbs) database for relationship to zikv infection. we also studied gbs controls that were seen before the established outbreak and non-gbs controls. the index cases tested negative for zikv pcr in blood and urine. we proceeded to test zikv igg, igm, dengue virus (denv) igg and igm, and neutralization assays against zikv and denv. one patient with anti-gq b igg positive miller fisher syndrome had detectable zikv igm and zikv igg. the serum showed low titre denv igm and denv igg. follow-up serum at about months showed increase in zikv igg. we believe this patient has zikv-gbs. one patient with acute motor sensory axonal neuropathy and another with acute inflammatory demyelinating polyneuropathy had high zikv igm but low denv igm and igg. another patient with mfs showed high levels of zikv and denv igm but low igg. the latter two patients had gbs before the zikv outbreak in singapore. we suspect these patients could have zikv-gbs, but are awaiting convalescent sera for confirmation. two patients seen during the outbreak had detectable levels of zikv igg but serial testing showed a decline after a period of - months. the initial and follow-up sera showed raised denv igm and igg levels in one and raised igg levels in the other. in addition, both had stronger neutralizing capacity against denv than zikv suggesting that the initially detectable zikv igg levels was due to cross reactivity with previous denv infection. four patients, gbs control and non-gbs controls also showed serological response consistent with previous exposure to denv. one normal control showed nil exposure to both viruses. in summary, using various overlapping serological methods we diagnosed definite and suspect zikv gbs cases. our findings highlight ) insensitivity of blood and urine pcr to diagnose zikv-gbs ) the problems of interpreting zikv serology from cross-reaction with denv ) serial serology increases diagnostic accuracy. okar sv , ergunay k , bekircan-kurt ce , , erdem-ozdamar s , , tan e , . department of neurology, hacettepe university, ankara, turkey; virology unit, department of medical microbiology, hacettepe university, ankara, turkey; neuromuscular disease research laboratory hacettepe university, ankara turkey. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated polyradiculoneuropathy. it is believed that acute inflammatory demyelinating poliradiculoneuropathy is caused by t-cell mediated autoimmune response targeting peripheral nerve myelin. molecular mimicry plays a role in the pathogenesis of some gbs cases. this mechanism has been well demonstrated in the acute motor axonal neuropathy (aman) variant, in which autoantibodies to camphylobacter jejuni share epitopes with peripheral nerve gangliosides. this molecular mimicry mechanism can be attributed to some cases with atypical triggers such as zika virus or west nile virus infections. moreover there are accumulating clinical data for vector borne viral infections triggering gbs. we evaluated vector-borne viral infections in our gbs and aman patients. eight patients with gbs, two patients with aman and as a control group, seven patients with normal pressure hydrocephalus were included. gbs and aman was diagnosed with clinical, electroneuromyograpic and cerebrospinal fluid (csf) findings. cerebrospinal fluid serum and urine samples were examined for vector borne viral infections via generic flavivirus and phlebovirus pcr. we also documented our patients prognostic scores such as modified erasmus gbs outcome score (megos) and modified erasmus gbs respiratory insufficiency score (megris). the mean age of the patients was , ( - years) , six of them were female. all csf, serum and urine samples of our patients and control patients were negative for flavivirus and phlebovirus families. the preliminary results of our study in this our small cohort did not show any correlation between the vector-borne viral infections and gbs. further studies with broad number of patients are needed for more suggestive results. neuromyotonia (nmt) consists of spontaneous motor unit activity that reflects increased peripheral nerve excitability, leading to involuntary, persistent muscle activity, visible as muscle twitching at rest, with generalized, easily provoked cramps. since the first electrophysiological (emg) description of nmt by denny-brown and foley ( ) , there has been discussion about the origin of the abnormal electrical activity recorded in needle emg studies. we studied two patients. patient 's nmt is aggravated by cold, and he has an associated non-progressive mild polyneuropathy with demyelinating features. he is now -year-old and has been followed in our centre for years. he is negative for anti-vgkc antibodies. firstly, he was treated with carbamazepine and phenylhydantoin with poor response, but he has shown major improvement on intravenous immunoglobulin (ivig) during the last years. patient is a year-old man with nmt, followed for year, with high titters of anti-vgkc antibodies ( pmol/l; normal< ). he improved on ivig during the last months. screening for neoplasia was unremarkable in both patients (negative anti-neuronal antibodies, in particular anti-hu, anti-yo and anti-ri antibodies; normal computerized tomography scan of the chest and abdomen). in addition to routine studies, we tested synchronicity to spontaneous discharges in different motor units in simultaneous recordings made with two needle electrodes in the first dorsal interosseus muscle. time-locked fasciculations in these double recordings would represent abnormal ectopic activity initiated in a nerve trunk with ephaptic stimulation of a nearby axon. in patient , this research protocol was applied once, years after regular ivig treatment. patient was investigated before and year after ivig. both patients improved after ivig, mirrored by a striking decrease in the amount of spontaneous activity on emg. moreover, our technique did not detect synchronous spontaneous activity (time-locked fasciculations) on the second assessment, although this was predominant before treatment in patient . in nmt, abnormal discharges originate both in distal axonal branches and in more proximal segments. it appears that ivig is more effective in blocking antibody activity in proximal axonal segments, perhaps related to factors such as blood-nerve barrier, temperature or differing ion channel distributions. celiac disease (cd) is a chronic, multisystem and immune-mediated disorder characterized by small-bowel sensitivity to dietary gluten in genetically predisposed individuals. neurological manifestations may occur in about % of patients, including peripheral nerve involvement. recent growing evidence strongly suggests that peripheral neuropathy in cd may be autoimmune and associated with anti-ganglioside antibodies. a -year-old woman presented with slowly progressive weakness of her right hand and fingers extensors. medical and family history were unremarkable. on examination, muscle strength (medical research council-mrc) was scored for right wrist and finger extensors, for right abductor pollicis longus and for right ankle dorsiflexion. right triceps brachii and brachioradialis reflexes were weak, but normal elsewhere. the remainder of the neurological examination was normal. neuroaxis magnetic resonance was unremarkable, specifically with no gadolinium-enhancing lesions. motor and sensory nerve conduction studies were normal. no conduction block or abnormal temporal dispersion was found. needle electromyography showed severe neurogenic changes with abnormal spontaneous activity in right radial-innervated muscles, and chronic neurogenic changes in homolateral tibialis anterior, peroneus longus and extensor digitorum brevis. ganglioside antibody testing was positive to igg anti-gm antibody, but negative to anti-gm and other anti-ganglioside antibodies. additional blood tests were unremarkable, in particular cryoglobulin testing was negative. intravenous immunoglobulin improved weakness, as right extensors of the wrist and fingers scored (mrc). a monthly treatment was initiated, which after months was changed to every weeks to preserve function. a diagnosis of mama was established. later, cd was diagnosed in her daughter due to chronic diarrhoea. our patient underwent anti-tissue transglutaminase antibodies determination and small-bowel biopsy after years disease' duration, and a diagnosis of cd was made. gluten-free diet was started, but her neurological picture did not change after six months. in our case the presence of igg anti-gm antibody may support a causal link between mama and biopsy-confirmed cd, and the lack of response to gluten-free diet may be explained by chronic axonal injury induced by memory t-cells. this case broadens our knowledge about neurological manifestations in cd, raising a probable association with purely axonal multifocal motor neuropathies and anti-ganglioside antibodies. Õunpuu s , , pogemiller k , acsadi g , pierz k , , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa. orthopaedic surgical intervention of the foot and ankle is performed in persons with charcot-marie-tooth (cmt) for improving foot pain, ankle instability, orthosis fitting/comfort issues and shoe wear. the impact of these surgeries on ankle function during gait is not known. therefore, the goal of this study was to measure gait changes in ankle function following surgical intervention by means of computerized motion analysis. fourteen patients with cmt ( ± years pre and ± years post-operative) with bilateral orthopaedic surgery were included. all patients had a plantar fascia release plus some combination of other soft-tissue (posterior tibialis and tendo-achilles lengthenings; extensor hallucis longus, peroneus longus and anterior tibialis transfers) and bony procedures (metatarsal and cuboid osteotomies). all patients completed two gait analyses (pre and on average . years post-surgery) during barefoot walking using a standardized d motion analysis protocol. the changes in ankle kinematics and kinetics and temporal-spatial parameters were analyzed in reference to a control group of patients with cmt without intervening surgeries with . years between gait analyses as well as normal reference data. the surgical group showed a significant increase in height between the pre and post-operative analyses and no changes in walking velocity (pre: ± , post: ± , normal: ± cm/sec). similarly, the ankle kinematics and kinetics showed no changes as a result of surgery. however, there was a trend for increased peak ankle dorsiflexion (pre: ± , post: ± , normal: ± degrees). as in the surgical group, the control group showed an increase in height but no simultaneous increase in walking velocity (test : ± , test : ± cm/sec). the control group also showed no changes in ankle kinematics and kinetics suggesting that the impact of the disease is, for the most part, not noticeable over . years. the results show that surgical intervention that primarily addresses foot alignment does not negatively impact ankle kinematics and kinetics during gait. the comparison with the control group supports this finding. however, there are some other findings such as the increase in peak dorsiflexion during stance in some patients that will need to be examined further in larger cohorts. Õunpuu s , , pogemiller k , pierz k , , , acsadi g , . center for motion analysis, connecticut children's medical center, farmington, ct, usa; school of medicine, university of connecticut, farmington, ct, usa; division of orthopaedics, connecticut children's medical center, farmington, ct, usa; division of neurology, connecticut children's medical center, farmington, ct, usa. ankle-foot-orthoses (afos) are commonly prescribed for patients with charcot-marie-tooth (cmt) to improve gait and reduce ankle instability and falling. there are no studies that examine bracing outcomes using objective evaluations. the purpose of this study was to examine the impact of various afo designs on gait parameters in children and adolescents with cmt. we predicted that the afos would improve excessive and delayed peak dorsiflexion in stance and equinus in swing. fifteen patients (mean age ± years) were analyzed barefoot and with their prescribed afos by means of a standardized motion analysis protocol. a full clinical examination was also completed including strength and passive range of motion measures. the afos included solid, hinged and posterior leaf spring (tapered, less supportive) designs. sagittal plane ankle kinematics and kinetics and temporal-spatial parameters were analyzed in comparison to normal controls. walking velocity improved from ± to ± cm/sec demonstrating the functional benefits of afos. ankle plantar flexion angle at initial contact improved from − ± to − ± degrees demonstrating improvement in drop foot in swing and at initial contact reducing the risk of tripping. however, the degree of peak ankle dorsiflexion in stance remained the same and excessive at ± degrees barefoot and ± degrees with afos suggesting that in many patients the afo design was not sufficiently supportive. peak ankle plantar flexor moment showed improvement from . ± . to . ± . nm/kg highlighting the improved base of support provided by the afos that compensate for ankle plantar flexor weakness. however, peak power generation was reduced from . ± . to . ± . w/kg indicating that some of the available ankle strength was impeded with the afos. the results of this study suggest that orthoses can provide improved gait outcomes which will improve overall function. however, there are individual differences in patient impairment (strength, range of motion and bony deformity) and associated gait presentation that need to be accounted for in afo design. afos do not always function as intended due to the complex interaction between patient impairment, orthosis stiffness and orthosis design. motion analysis can assist in identifying the specific afo needs for an individual with cmt. painous c , lópez-pérez ma , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, barcelona, spain; hospital san pedro, logroño, la rioja, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is an autoimmune neuropathy with a heterogeneous clinical spectrum. specific autoantibodies define different clinical phenotypes. cidp with nf antibodies constitutes a specific cidp subset in which a high incidence of postural and intention tremor has been described and that responds poorly to immunoglobulins. we report a patient with an anti-nf positive cidp that presented head, voice and limb tremor that improved with immunotherapy. a -year-old man with unremarkable medical history, presented at the age of with progressive distal weakness and paraesthesia. numbness, gait ataxia and action tremor involving voice, head and limbs appeared sequentially. the emg showed features of acquired demyelination fulfilling cidp diagnostic criteria. a first course of intravenous immunoglobulins was ineffective. the weakness, ataxia and tremor worsened significantly, needing two walking aids first and becoming wheelchair bound later on. he received six plasmapheresis cycles that were also ineffective and oral corticosteroids ( mg/kg) were started with mild improvement. after corticosteroid tapering the patient developed a severe relapse and was referred to our centre. five plasma exchange cycles followed by rituximab ( mg/m , doses) were added to the corticosteroids. three months later the weakness, ataxia and tremor, including the head and voice tremor, started to improve significantly. six months later the patient presented a significant improvement and was able to walk unaided. the anti-nf antibody titres fell from / pre-rituximab to be undetectable. limb tremor is known to occur in patients with inflammatory neuropathies and, specifically in anti-nf + cidp patients but, to our knowledge, this is the first report of a cidp patient presenting with head and voice tremor ever reported. the improvement of tremor with immunotherapy strongly suggest that anti-nf autoantibodies are involved in its pathogenesis, expanding the phenotype of anti-nf specific clinical features. palaima p , peeters k , l. pelayo-negro a , garcía a , gallardo e , garcía-barredo r , de vriendt e , infante j , berciano j , jordanova a . vib and university of antwerp, center for molecular neurology, molecular neurogenomics group, antwerp, belgium; university hospital "marqués de valdecilla", santander, spain. charcot-marie-tooth disease type g is an autosomal dominant and slowly progressing inherited neuropathy which was first described over years ago. it has been attributed to a single spanish family consisting of individuals with affected members spanning four generation. initially, the genetic defect was linked to a . mb region in q -q . . however, extensive sequence and structural variant analyses using whole genome sequences (wgs) of three affected individuals did not reveal any known or novel genetic causes within the region. over the last decade, serial clinical re-evaluation of the entire pedigree was performed leading to changes to the affection status of seven individuals redefining the disease-linked region to chr q . -q . (z max = . at theta = ). additional family members were then submitted for whole exome sequencing. this has led to the identification of a novel missense variant in the e ubiquitin-protein ligase lrsam (p.cys tyr) that was previously not covered by wgs. the variants co-segregated with disease and were absent from controls. other mutations that are known to disrupt the ring domain of lrsam have been previously reported to cause both autosomal dominant and recessive cmt type p (cmt p). unlike prior reports, we demonstrated that the mutation does not influence overall protein levels of lrsam , nor of its ubiquitylation target tsg in patient derived lymphoblasts. transcriptomics analysis identified significant upregulation of another e ubiquitin-protein ligase (nedd l) and of a key regulator of axonal degeneration (tnfrsf ). notably, magnetic resonance imaging of lower-limb musculature systematically showed fatty atrophy in both clinical and subclinical mutation carriers emphasizing its use for the identification of mildly affected members. our findings demonstrate that the isolated genetic entity cmt g is caused by a missense mutation in lrsam and should be reclassified as cmt p. moreover, we reveal novel molecular players associated with lrsam dysfunction, and highlight pathways and therapeutic targets shared with amyotrophic lateral sclerosis and alzheimer disease. damage to peripheral nerves is a prerequisite for neuropathic pain. however, it remains unclear why some neuropathy patients have pain, but others with identical nerve conduction, qst and ienfd do not. we propose to examine the distribution of trpv and its co-localization with cgrp and sp in epidermal nerve fibers in patients with and without neuropathic pain. we aimed to define the expression pattern of trpv in normal healthy subjects first, and study the co-localization of trpv with cgrp and sp in normal controls and patients with painful neuropathy. skin biopsies from neuropathy patients and normal subjects were utilized. anti-trpv antibody generated in our university with support from ninds-funded dept. of neuroscience monoclonal core (ns ) was used. combined immunohistochemistry were performed to identify co-expression of trpv with cgrp, sp and pgp . . the distribution of trpv in controls revealed a proximal to distal gradient similar to that observed for ienf labeled by pgp . . trpv staining was more intense in nerve terminals in the epidermis. combined immunostaining revealed that % of pgp . labeled fibers in the epidermis were trpv +, while % of cgrp+ fibers trpv +. patients with pain had a higher density of trpv + fibers compared to that of patients with numbness. a greater proportion of cgrp+ fibers ( %) in the painful patients were trpv +. expression of trpv in controls exhibits a distal to proximal gradient. trpv expression and co-localization with cgrp were altered in neuropathic pain patients, suggesting that this receptor plays an important role in pathological states. activation of the nop-receptor (nop-r) by its endogenous ligand nociceptin/orphanin fq or non-peptide agonists modulates nociception and analgesia in neuropathic pain models. the wide distribution of nop-r and its endogenous ligand represents an attractive treatment target. since pain is the most prominent feature in pc, we defined expression of the nop-r in plantar skin biopsies and assessed whether alterations exist in pc-affected vs pc-unaffected and anatomically matched control skin. skin biopsies from k a pc and control subjects were immunohistochemically stained for nop-r. combined immunostaining for nop-r with pgp . , neurofilament h (nfh) and cgrp was used to define nop receptor expression in the epidermis and upper dermis. robust nop-r was detected in epidermal keratinocytes and a subset of pgp . + fibers in both epidermis and dermis. staining was inhibited through pre-incubation with a nop-r blocking peptide and western blot analysis using homogenized human skin tissue demonstrated a band at ∼ kd consistent with nop-r molecular weight. nop-r expression occurred in dermal nfh+ a beta-fibers in all groups though no cgrp+ fibers co-expressed nop-r. pc-affected skin had slightly lower nop-r expression than in pc-unaffected skin and a similar pattern in anatomically matched locations from healthy control subjects was observed. nop-r is expressed in human plantar skin epidermal keratinocytes as well as a subset of epidermal and dermal nerve fibers. these fibers are pgp . +, cgrp-and many co-express nfh. nop receptor is a viable pharmaceutical analgesic target in pc patients irrespective of its slight down-regulation as compared to pc-unaffected skin. this work was supported by grünenthal gmbh. pareyson d , stojkovic t , leonard-louis s , reilly mm , laurà m , parman y , battaloglu e , tazir m , bellatache m , bonello-palot n , sacconi s , guimarães-costa r , attarian s , latour p , megarbane a , schenone a , ursino g , sabatelli m , luigetti m , santoro l , manganelli f , quattrone a , valentino p , murakami t , scherer ss , dankwa l , shy me , bacon cj , herrmann dn , pisciotta c , previtali s , bolino a . milan, italy; paris, france; london, united kingdom; istanbul, turkey; algiers, algeria; marseille, france; nice, france; lyon, france; genoa, italy; rome, italy; naples, italy; catanzaro, italy; kurashiki, japan; philadelphia, usa; iowa city, usa; rochester, usa. cmt b and b are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, myelin outfoldings, association with loss-of-function mutations in myotubularin-related protein- and − (mtmr , mtmr /sbf ), respectively, involved in the metabolism of ptdins p and ptsins( , )p phosphoinositides, key regulators of membrane trafficking. in a multicentre retrospective study to better characterise cmt b, we collected a minimal dataset of information including cmtes/cmtns on cmt b patients, cmt b ( unrelated families) and cmt b ( families). cmt b patients were younger and with earlier onset than cmt b : last visit was performed at a mean age of years (sd . ; range - ) for cmt b and years ( . ; - ) for cmt b ; disease onset occurred at a mean age of . years ( . ; - ) in cmt b as compared to . years ( . ; - ) in cmt b ; delay in motor milestones occurred in / cmt b and / cmt b subjects. eleven cmt b patients became chair-bound, whereas all cmt b subjects but one are still ambulant, although with afos for patients and requiring unilateral support in two cases. both disease types are characterised by vocal cord involvement ( / cmt b and / cmt b ); respiratory involvement was seen almost exclusively in cmt b patients (n= , four requiring non-invasive ventilation and one tracheostomy, as compared to one cmt b patient on niv at age ); two cmt b subjects and an affected cmt b relative not included in the present study died of respiratory complications. glaucoma (n= ) and buphthalmos (n= ) occurred only in cmt b . cmtes/ cmtns scores were higher in cmt b patients in spite of their younger age, indicating more severe disease: cmt b = cmtes mean . (n= ; sd . ; range - / ), cmtns mean . (n= ; sd . ; range - / ,; cmt b = cmtes mean (n= ; sd . ; range - / ), cmtns mean (n= ; sd . ; range - / ). our data confirm that cmt b is more severe than cmt b . interestingly, mtmr interacts with mtmr . mtmr is a catalytically active phosphatase, whereas mtmr is a catalytically inactive protein, known to increase mtmr enzymatic activity. thus, in cmt b nerves a residual enzymatic activity of mtmr may result in a less severe clinical phenotype as compared to cmt b . the charcot-marie-tooth disease (cmt) national registry is fully operative (https:www.registronmd.it) with collection of clinical/genetic information (minimal dataset) and reporting of clinical scales; cmt patients have registered thus far and have chosen one of the reference centers; information collected during the ad hoc visit have been entered in the registry for of them. registered patients have the chance to participate to a study that requires filling online self-reported questionnaires related to five important issues: disease course and complications during pregnancy; use, efficacy and tolerability of orthotics and assistive devices; outcome of surgery for skeletal deformities; safety of anesthesia; occurrence of sleep disorders (including evaluation of fatigue, anxiety and depression). by february , patients and relatives/friends (as healthy controls) have filled the questionnaires. we are performing a first explorative analysis of results, but data collection on all questionnaires will be prolonged until novemebr , , to obtain a larger sample. pregnancy: / cmt women had at least one pregnancy; complications ranging from mild to severe occurred in / pregnancies vs / in controls. cmt worsened in pregnancies ( patients) with no recovery in instances. prenatal diagnosis was performed in / pregnancies. satisfaction related to surgical procedures for foot deformities, assessed with vas (score - ), was . ± . (n = ). repeat surgery was required in / instances. sleep: the epworth sleepiness scale questionnaire revealed abnormalities of sleep in / cmt patients ( %) and in / controls ( %). the vast majority of cmt subjects ( / ; mean . ± . ), but also of controls ( / ; . ± . ) were not good sleepers according to the pittsburgh sleep quality index (psqi) (range - , good sleep). fatigue: scores of modified-fatigue impact scale (range - , no fatigue) were higher for cmt (mean ± . ) than controls (mean . ± . ). hospital scale for anxiety and depression: / cmt subjects had mild-to-severe anxiety and / mild-to-severe depression as compared to / and / controls, respectively. data analysis on orthotics and anesthesia is ongoing. in conclusion, the first data analyses confirm that there are problems related to all the five domains explored, that will need to be specifically addressed in patients' care. supported by telethon-uildm grant gup . charcot-marie-tooth disease (cmt) is the most frequent motor and sensory peripheral neuropathy and is known to have the uniform demyelination. the traditional definition of conduction block is the reduction of negative peak of compound muscle action potential (cmap) of proximal segment relative to adjacent distal segment more than %. in this study, we tried to find the frequency of conduction block in cmt a patients and to investigate the differences of the clinical manifestation. we enrolled unrelated cmt a patients ( males and females) with pmp duplication and undertook nerve conduction studies from to . stimulation sites were wrist, elbow and axilla for median nerve study. eleven patients ( . % of all enrolled patients) had ncs features suggestive of conduction block. compared to cmt a patients without conduction block, functional disability scale was significantly higher in cmt a patients with conduction block (p < . ). however, onset age and disease duration were not different between cmt a patients with and without conduction block. in addition, conduction block was more frequently observed in distal segments than proximal segments. we suggest that the frequency of conduction block in cmt a patients is not low, and there is some heterogeneity of demyelination in cmt a patients. also, in cmt a patients, the conduction block might have relationship with clinical disability. park jg , park ms . yeungnam university college of medicine, daegu, korea. the subacute combined degeneration (scd) is diagnosed by a characteristic clinical manifestation, spinal mri, and decreased serum vitamin b levels. we report a case of scd with elevated homocysteine and methlymalonic acid level in the situation of spurious elevation of vitamin b concentration. an years old man presented with progressive gait disturbances and sensory disturbances. about a year ago, he felt both hands and feet paresthesia and gait disturbances. four months before the visit, nerve conduction tests were performed elsewhere due to symptoms. polyneuropathy was found. the laboratory test showed megaloblastic anemia. however, serum vitamin b concentration was increased to pg / ml (normal: - pg / ml). his symptoms progressed gradually. at presented, neurological examination showed spasticity of the lower limb without weakness, and vibration and position sensation were decreased in both lower limbs, and showed ataxic gait. spinal cord mri showed a lesion with a long t high signal intensity from c to t in the posterior column of the spinal cord. further laboratory test were added, then while folate was normal level but homocysteine at . umol/l (normal: - umol/l), and methylmalonic acid at . um / l (normal: - . um / l) were increased. finally, scd caused by vitamin b deficiency was diagnosed. in addition, gastric endoscopy was performed to find the cause of vitamin b deficiency and chronic atrophic gastritis was found. after cobalamine treatment for months, hemoglobin level was improved and his symptoms were all improving with a little gait disturbances. false-elevation of vitamin b level could lead to delayed diagnosis and cause irreversible changes in the nervous systems. one of the most commonly used methods to measure vitamin b concentrations is competitive-binding assay, which may result in normal levels even with vitamin b deficiency due to the effect of anti-intrinsic factor antibodies. when vitamin b deficiency is suspected, even if the vitamin b concentration is normal, additional tests of homocysteine and methylmalonic acid should be considered. parman y , durmus h , deymeer f , oflazer-serdaroglu p , battaloglu e . istanbul university, istanbul faculty of medicine, department of neurology, istanbul, turkey; bogazici university, istanbul, turkey. hereditary motor and sensory neuropathies, also called cmt neuropathies, are the most common group among the hereditary neuropathies. cmt subtypes are grouped by axonal, demyelinating or intermediate phenotype, or by autosomal-dominant (ad), autosomal-recessive (ar) or x-chromosomal inheritance. cmt- is considered axonal and characterized by distal muscle wasting, mild sensory loss and normal or near-normal nerve conduction velocities. mutations in more than genes are known to be able to cause autosomal dominant (ad) or recessive (ar) cmt- to date. the genetic heterogeneity in charcot-marie-tooth (cmt) is a challenge for genetic diagnostics. clinical clues and frequencies of mutations in cmt genes from large cohorts may help to develop strategies for efficient genetic testing. here, we present the clinical, electrophysiological and genetic features of patients from turkey, diagnosed as genetically confirmed cmt- in the department of neurology, istanbul faculty of medicine our clinic between and . genetic testing was performed for gdap by dna sequencing and samples from patients were exome sequenced (wes). twenty-one male and ten female patients from unrelated families were investigated. segregation was ar in eight, ad in six families and two were isolated cases. intraand interfamilial variability in the age of onset with a range of - (mean . ± . years) and disease progression rate were striking. slowly progressive weakness and atrophy of distal muscles in the feet and/or hands were the most common presenting symptoms. mfn was found in four unrelated ad kinship and in unrelated ar kinships and was most common gene mutated among whole cohort. gdap was the most commonly mutated gene among ar families ( / ). wes revealed further mutations in aars, nefl, kif b and hspb , however, the causative mutation could not be identified in known cmt genes in about % of patients. our data indicates the marked intra-and inter-familiar phenotype variability in cmt- as previously described in literature. many more genes causing arcmt- remain to be discovered. pasnoor m , veerapaneni k , murphy r , statland jm , kimple d , hamasaki a , glenn md , herbelin l , barohn rj , jawdat o , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. electrodiagnostic (edx) studies involving electromyography/nerve conduction study (emg/ncs) are useful for diagnosis of neuromuscular disorders. although these are safe procedures, emg and ncs often evoke anticipated pain and anxiety. there are no published research studies to support the notion that providing detailed information to patients prior to these procedures would reduce procedural pain and ameliorate anxiety levels afterwards. the objective of this study was to perform a prospective survey to assess anticipated pain and anxiety in patients presenting for edx studies, design a detailed patient education form, and assess the change in perceived pain and anxiety pre and post procedure in the standard of care (soc) education versus the printed detailed education instrument group. after completing a brief pain/anxiety questionnaire, patients were randomly assigned to either soc verbal education group or to read the detailed education form. another brief questionnaire was completed post procedure. seventy-eight patients were enrolled at the time of abstract submission, in intervention and in soc groups. mean age of patients enrolled was ± years. pain was anticipated by % patients as a visual analog scale (vas) score mean of . ± . with no significant difference between both groups (p= . ). post-procedural pain was reported in % of patients with mean pain score of . ± . and no significant difference between the two groups (p= . ). anxiety pre-procedure was reported by % patients with a mean likert-like score of . ± . score in all cases (p= . between both groups). post-procedural anxiety frequency was reduced to % with mean level of . ± . (p= . between both groups). most patients reported pain with both emg and ncs ( %). we found that edx testing evokes moderate pain in most and in some cases moderate anxiety level. the detailed education intervention did not attenuate the frequency or severity of pain. post-procedural anxiety was expectedly reduced in severity and frequency in both groups with a suggestion that anxiety severity is lesser in the detailed education group but numbers were small. further studies with more detailed questionnaire and perhaps web-based education in larger population may be useful to reduce pain/anxiety in patients presenting for edx studies. pasnoor m , roach c , barohn rj , statland j , jawdat o , dick a , glenn m , dimachkie mm . department of neurology, neuromuscular division, the university of kansas medical center, kansas city, ks, usa. diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (cidp) have been designed for use in clinical trials. there is limited data on the comparative diagnostic utility of these criteria in the clinic setting and it is unknown if some of these criteria are better for predicting treatment response. the objective of this study is to compare the sensitivity of various diagnostic criteria and review treatment response. after obtaining local irb approval, we performed a retrospective chart review of cidp patients seen at the university of kansas medical center between and . we abstracted the clinical, electrodiagnostic, and treatment information. we determined the frequency of patients fulfilling each of the following criteria: efns , aan, saperstein and incat. we defined treatment response based on treating physician's impression of change, patient-reported functional improvement or one point grade change in the mrc grade. fifty-three cidp patients charts were reviewed, were excluded due to missing data. mean age was . , and ( %) were female. elevated csf protein was seen in / ( %). twenty-eight ( %) fulfilled efns definite criteria, ( . %) efns probable, ( . %) aan, ( . %) incat, and ( . %) the saperstein criteria. treatment response in patients who fulfilled efns definite/probable criteria included / to ivig, / to iv or oral corticosteroids, / to mycophenolate mofetil; among patients who fulfilled aan criteria, these were respectively / , / and / ; among those meeting incat criteria / , / and / ; and for saperstein criteria it was / , / and / . half to two-thirds of patients responded to plex based on different criteria. while all cidp criteria can similarly predict ivig treatment response, the efns criteria are most sensitive for the clinical diagnosis of cidp. pellegatta m , canevazzi p , forese mg , podini p , quattrini a , taveggia c . division of neuroscience and inspe, axo-glia interaction unit, san raffaele scientific institute, milan, italy; division of neuroscience and inspe, experimental neuropathology unit, san raffaele scientific institute, milan, italy. axonal nrg type iii is an essential instructive signal for pns myelination, since its expression determines whether axons are myelinated and also the thickness of the myelin sheath. previous studies have shown that secretases' cleavage controls nrg type iii activity at post-transcriptional level. specifically, the beta-secretase bace- cleavage of nrg type iii promotes myelination, whereas the alpha-secretase tace inhibits pns myelination by inactivating nrg type iii. after a damage, the axon located distally to the site of injury degenerates and loses the myelin sheath, following a process termed as wallerian degeneration. unlike the cns, axons in the pns can regenerate and regrow. several studies have shown that regeneration is favored by the trans-differentiation of schwann cells, which start to create a permissive environment for axonal regrowth. in addition, it has been shown that nrg regulates the early stages of the dedifferentiation process and is essential for creating a permissive environment to regeneration. similarly, the beta secretase bace- promotes pns regeneration and remyelination. in the present study, we analyzed the role of the alpha-secretase tace during wallerian degeneration. our data indicate that tace is upregulated in sciatic nerves after injury, distally to the site of damage. since tace is expressed in schwann cells and axons, we analyzed the role of both glial and neuronal tace during degeneration and regeneration processes. thus, we performed crush injury in sciatic nerves of two different transgenic lines carrying a conditional deletion of tace either in schwann cells (p -cre//tace flox/flox ) or in neurons (chat-cre//tace flox/flox ). the analyses of the different phases of the wallerian degeneration in these animals suggest a role of glial tace during the early stage of the process. in fact, we observed an increased number of myelinated axons only in p -cre//tace flox/flox mice, while we did not detect substantial differences in mutant chatcre//tace flox/flox mice. these results suggest that even during regeneration tace has an inhibitory role, as deletion of tace in schwann cell likely induces the activation of a neuro-protective program that we are currently investigating. péréon y , nizon m , küry s , besnard t , quinquis d , boisseau p , magot a , mussini jp , mayrargue e , barbarot s , bézieau s , isidor b . reference centre for neuromuscular disorders; dept. of genetics; dept. of paediatric surgery; dept. of dermatology, university hospital, nantes, france. the hereditary sensory and autonomic neuropathies (hsan) constitute a clinically and genetically heterogeneous group. hsan are associated with sensory dysfunction, altered pain and temperature perception with varying degrees of autonomic dysfunction, and abnormal small fibers neurodevelopment. more than ten genes have been described so far in the hsan group. original classification encompassed four entities, but additional subgroups continue to be described. hereditary sensory neuropathies (hsan) type ii are characterized by autosomal recessive inheritance, onset at birth and self-mutilating behavior. until now, one homozygous frameshift variant, c.[ _ del], p.(lys phefs* ), was reported in arl ip (adprribosylation-like factor -interacting protein ) in a consanguineous family. patients presented with spastic paraplegia, diffuse sensory and motor polyneuropathy and acromutilation. the disorder was classified as autosomal recessive spastic paraplegia spg . here, we described a new patient with congenital insensitivity to pain, sensory neuropathy, self-mutilation, and spastic paraplegia. whole exome sequencing showed a homozygous frameshift variant c. [ _ del], p.(lys phefs* ) in arl ip . the protein harbours reticulon-like short hairpin transmembrane domains and has a role in endoplasmic reticulum shaping. the variant causes an additional c-terminus hydrophobic domain which could alter its function. arl ip interacts with atlastin- responsible for spg a and hsan type d. this report highlights the role of arl ip in pathophysiology of insensitivity to pain and spastic paraplegia. péréon y , nguyen a-l , leclair-visonneau l , fayet g , nguyen j-m , magot a . laboratoire d'explorations fonctionnelles, hôtel-dieu, nantes, france; département de statistiques médicales, hôtel-dieu, nantes, france. carpal tunnel syndrome (cts) is responsible for sensory and/or motor symptoms that may be increased by wrist flexion or extension (phalen sign). previous studies have shown that short duration (e.g. min or less) flexion or extension was not responsible for significant changes of median nerve conduction parameters. the objective is the study was to determine how prolonged extension (reverse phalen's test) affects median and ulnar nerve conduction parameters in normal subjects. after providing informed consent, normal subjects ( females, age rank - years) underwent motor and sensory testing of both median and ulnar nerves from the dominant side. edx testing was performed using standard techniques with wrist in neutral position, then during passive wrist extension ( ∘ ) for minutes, with sequential recording of both motor action potential (cmap) and sensory action potential (snap) latencies and amplitudes every minutes after extension onset. ulnar nerve conduction parameters remained unchanged in all the subjects. regarding median nerve, groups of subjects could be individualized: subjects with no changes; subjects with only significant decrease of snap amplitude (− %); subjects with both snap amplitude and velocity decrease; subjects with both cmap and snap significant changes. when present, changes did not appear before min, with snap decrease being the earliest observed abnormality. prolonged wrist extension was responsible for median nerve edx parameter modifications in / normal subjects. sensory fibers were firstly affected, corresponding to the chronology clinically encountered in cts. based upon these results, the same protocol will be used in patients presenting with mild clinical cts, in order to try and identify an additional edx technique being useful in the diagnosis of cts. one could also wonder whether the subjects with the most important changes would be more prone to present cts in the future. perez-siles g , , drew a , , ellis m , kidambi m , takata r i , speck-martins c e , hagerman k a , siskind c e , day j w , ginzberg m , nicholson g , , , kennerson m l , , . northcott neuroscience laboratory, anzac research institute, sydney, australia; sydney medical school, university of sydney, sydney, australia; molecular medicine laboratory, concord repatriation general hospital, sydney, australia; sarah network rehabilitation hospitals, brasilia, df, brazil; department of neurology, stanford health care, stanford, ca, usa; pediatric neuromuscular unit, wolfson medical center, holon, israel. mutations in the atp a gene cause of x-linked hereditary distal motor neuropathy (dhmnx). to date, missense mutations (t i and p s) in this copper transporting atpase are the only confirmed mutations causing dhmnx in two independent families. next generation sequencing (ngs), including whole exome and whole genome sequencing, is increasing the rapid detection of variants in genes known to cause disease, however the absence and size of additional families, make it challenging to determine the pathogenic or benign status of variants identified. we have recently identified new variants in atp a in patients with progressive peripheral neuropathy, suggesting further genetic heterogeneity of dhmnx. two of these new variants, pe v and pm v, are located at highly conserved amino acids within domains of atp a (a-domain and p-domain, respectively) that are critical for the catalytic cycle of the copper transporter. we have also identified a third variant (c. - a>g) located bases upstream exon that is predicted to abolish a conserved branch-site, a consensus intronic sequence necessary for the processing of immature rna during splicing. our recent investigations using both patient fibroblasts and an atp a conditional knock in mouse model for dhmnx expressing atp a t i , the orthologue of the human atp a t i mutation, showed reduced atp a protein levels and defective retrograde trafficking of atp a, as pathological hallmarks of dhmnx atp a causative mutations. to elucidate if the newly identified pe v, pm v and c. - a>g atp a variants are pathogenic, we have systematically assessed patient fibroblasts harbouring these mutations for altered parameters previously found in the pathogenic t i atp a mutation. we predict that functional characterisation of atp a using patient fibroblasts harbouring newly identified variants will provide the evidence to ascertain whether these variants are disease causing. establishing a systematic functional readout for atp a variants will improve accuracy of genetic counselling and patient management of dhmnx in those cases where genetic evidence is limited. this study represents a complementary and necessary approach to the use of ngs, for validating the pathogenic status of variants identified and for expanding the genetic heterogeneity of dhmnx. month of birth (mob) have been defined as a risk factor for more than diseases. for instance, susceptibility for multiple sclerosis (ms) seems to be associated with being born in may, while lower risk of ms is observed in those born in november. there are no studies that assessed potential association between mob and a risk for guillain-barré syndrome (gbs). we sought to determine if the risk of gbs is associated with the mob. study included gbs patients diagnosed in serbia, montenegro and republic of srpska in the period from january , until december , . mob of patients and of general population were compared. patients with gbs had tendency to be born in october ( % increase compared to general population, p= . ) and were less likely to be born in june ( % decrease, p= . ). when consider specific seasons, gbs patients were more likely to be born in winter months ( % increase, p< . ). no associations were found between month/season of birth and disease severity. results of this pilot study showed that gbs patients are more likely to be born in cold months, and less likely to be born in june. this might be explained by higher exposure to different pathogens during pregnancy in cold months. in accordance with this, it is well known that early exposure to infective agents reduces risk of allergic and autoimmune diseases. further studies are needed to test our findings in different cohorts and in regions with different climate. these results may shed new light on the disease pathogenesis. majority of patients with guillain-barré syndrome (gbs) tend to have a successful recovery, but in number of them significant long-term consequences may negatively affect their quality of life. aim of this research was to analyze the outcome of the disease one year after the acute episode of gbs. among patients diagnosed with gbs in seven tertiary centers in serbia, montenegro and republic of srpska during , subjects were retested after one year ( % males, mean age ± years). functional disability of patients was estimated based on the gbs disability scale (gds). severe form of the disease (gds score - ) was registered in % of patients at admission, % at nadir, and % on discharge. after one year follow-up period, % of patients had no symptoms of gbs, % had mild symptoms, % was able to walk but not to run, % needed unilateral support during walking, while % was wheelchair bound or bedridden. lethal outcome was registered in three patients during acute phase of gbs and in four more during one-year follow-up. paresthesias were present in % of gbs patients, musculoskeletal pain in %, and fatigue in % one year after acute phase of the disease. factors associated with the worse functional outcome (gds grade above ) after one year were: age above , preceding respiratory infection, and worse gds on discharge. one year after the onset of the disease, significant number of our gbs patients had neurological impairments including sensory symptoms, pain, fatigue and muscle weakness which may significantly affect patients' everyday functioning. phetteplace je , saade d , bacon c , shy me . university of iowa hospitals and clinics, iowa city, ia, usa. charcot-marie-tooth (cmt) disease, also known as hereditary motor and sensory neuropathy, is a heritable peripheral neuropathy caused by genetic mutations in many different genes. cmt type a is the most common form of cmt. individuals affected with cmt a commonly have distal muscle atrophy, foot deformities, abnormal gait, and reduced nerve conduction velocities caused by demyelination of the peripheral nerves. cmt a is known to be caused by a duplication of the pmp gene at p . . the proposed disease mechanism of cmt a is believed to be increased gene dosage of the pmp gene, although the exact function of pmp is not known. there have been previous reports in the literature of individuals who carry a homozygous duplication of pmp , therefore having four copies of the gene. however, many of these reports have focused on adult patients who symptoms may not vary significantly from individuals who harbor only a duplication of pmp . a month old male presented to the university of iowa hospital due to a family history of cmt a and concern for delay in independent ambulation. he was known to have both a maternal and paternal family history of cmt a, although the families were believed to be unrelated. on exam he was unable to walk independently, but could take a few steps when his hands were held. per report he began crawling and scooting at months of age. he was not found to have any structural abnormalities or atrophy in his upper or lower extremities. electrodiagnostic testing revealed a demyelinating neuropathy. only one nerve was tested, as the patient was unable to tolerate additional shocks. the peroneal nerve was found to have a nerve conduction velocity of m/s. based on the clinical presentation, electrodiagnostic examination and family history deletion and duplication testing for pmp was performed. this testing revealed a homozygous duplication of pmp , which has been previously reported to cause cmt a. this case supports the gene dosage disease mechanism, as our patient appears to be more severely affected than a typical infant or toddler affected with cmt a. piscosquito g , saveri p , provitera v , stancanelli a , ciano c , magri s , taroni mpz mutations are associated with the demyelinating early-onset charcot-marie-tooth (cmt) type b and with the axonal later onset cmt i/cmt j. paresthesias/dysesthesias and pain have been already reported in some cmt types and also in cmt i/j. we report patients ( from families, sporadic cases,) carrying the mpz c. dela mutation (p.asp thrfster ). this is predicted to be a loss-of-function mutation, leading to p haploinsufficiency. most of the subjects have genetic ancestry from puglia region (southern italy). two patients carried the mutation in homozygosity, showing a severe phenotype: onset at birth, severe scoliosis, proximal weakness, distal limb plegia, no autonomous walking, cmt examination score (cmtes) - / . all the other heterozygous patients had mild adult-onset (mean age , range - ) neuropathy, walked without aids or ankle-foot orthoses, had cmtes < / . five showed mild foot deformities, / had foot and / hand weakness (mrc never less than / ), / sensory loss in lower limbs (ll). paresthesias/dysesthesias (mostly tingling and burning in type) localized in hands and ll were reported by / subjects, neuropathic pain by / . in the homozygous patients, all cmaps and saps were absent. in all other subjects, motor conduction velocities (cv) were > m/s (range - m/s) and sensory cv > m/s (range - m/s). cmaps as well as saps were normal or moderately reduced in amplitude, indicating only slight axonal loss. we confirm that heterozygosity is associated with very mild cmt, whereas homozygosity causes a very severe neuropathy. our data suggest that paresthesias and neuropathic pain are very common and should be considered as part of the phenotype. although the clinical picture suggests a small fiber neuropathy, no such evidence is provided in the current literature. in the attempt to further investigate this field, we found in a -year-old male patient of our series that all thresholds were increased at quantitative sensory testing, and several abnormalities of small nerve fibers were present in skin biopsy. apparently, p aneuplody causes neuropathy and small fiber involvement through a mechanism different from other mpz missense mutations. poitras t , chandrasekhar a , singh v , martinez j , zochodne dw . neuroscience mental health institute, and division of neurology, department of medicine, university of alberta, edmonton, alberta, canada. a microarray mrna analysis of dorsal root ganglia examined in long-term diabetic mice yielded an unexpected upregulation of a unique set of proteins known as major urinary proteins (mups & ). originally, this family of barrel shaped proteins was described as including mediators of pheromone secretion in the urine of rodents, apparently unconnected to other functions. we identified its unanticipated expression in sensory neurons of adult mice and rats and explored its potential impact on the properties of these neurons. analysis of rat and mouse sensory neurons showed widespread and pan-neuronal cytosolic mup expression, not only in the cell body, but also in distal sensory projections located in the dermis of the footpad. to assess the role that mup might offer in the growth behaviour of adult neurons, we examined sirna-induced knockdown of mup on pre-injured dissociated drg primary cultures. mup knockdown in both species showed a significant increase in neurite outgrowth following mup sirna treatment when compared to a scrambled sequence control. mice treated with streptozotocin (stz) model features of human type diabetic polyneuropathy, including decreases in multi-fiber motor and sensory measurements, and changes in thermal sensation in the extremities. to explore the potential role of mup upregulation specifically in diabetes, we examined the impact of non-viral intranasal injection of mup sirna on indices of neuropathy in chronic type diabetic mice. following treatment, mice treated with mup sirna showed improvement in both motor and sensory nerve conduction velocities compared to scrambled controls. taken together, this data suggests that mup plays a growth-suppressive role in both diabetic and non-diabetic neurons and may also contribute to the electrophysiological and behavioral abnormalities associated with diabetic polyneuropathy. the repurposing of mups in sensory neurons identifies an interesting role for a supposed pheromone protein. [supported by cihr, cda and adi] enhancement of their outgrowth properties (singh et al, ) . here we asked whether similar activation, using low dose capsaicin, might similarly ramp up the regenerative activity of sensory neurons. using rat adult primary sensory neurons, we identified a dose dependent relationship between capsaicin exposure and their outgrowth with enhancement at lower doses and expected neurotoxicity at higher doses. doses that enhanced outgrowth were nonetheless associated with rises in intraneuronal calcium. we next tested whether a similar impact of trpv activation in vivo might exist. we used a high sciatic crush injury mouse model to assess the impact of capsaicin at low ( um) or high ( mm) doses applied directly at the site of axon outgrowth after injury. by days following the single application of capsaicin, low doses of capsaicin showed more rapid recovery of thermal, but not mechanical sensation, whereas high dose capsaicin showed no significant difference compared to vehicle treated mice. in parallel with the neurotoxicity observed in vitro, the high dose capsaicin resulted in a decrease in sensory nerve conduction velocity, while the low dose showed no significant difference when compared to the vehicle treated animals. there was no impact on motor axon recovery. taken together, subtoxic trpv activation with intraneuronal calcium mobilization, as applied to regenerating sensory axons, enhances their outgrowth after injury, and improves behavioural recovery similar to that following extracellular electrical stimulation. [supported by cihr] hereditary transthyretin (hattr) amyloidosis is a rapidly progressive, life-threatening disease caused by ttr gene variants, resulting in ttr misfolding and amyloid deposition in multiple organs, including peripheral nerves. hattr amyloid polyneuropathy has prominent small fiber involvement characterized by neuropathic pain and dysautonomia. measurement of intraepidermal and sweat gland nerve fiber density (ienfd, sgnfd) in skin biopsies has been useful in identifying small fiber abnormalities in hattr. we report the effect of patisiran, an investigational rnai therapeutic for the treatment of hattr, over a -month period on ienfd, sgnfd and dermal amyloid content from the phase open-label extension (ole) study. skin biopsies (distal leg, distal thigh) were obtained every months for up to years within the phase ole study (patisiran . mg/kg iv, q w, nct ) and analyzed in a blinded manner by a central laboratory. all analyses are exploratory; nominal p-values are reported without adjustment for multiple testing. twenty-seven patients enrolled; preliminary data indicated patisiran was generally well tolerated; resulted in sustained mean serum ttr lowering of ∼ % for > months and a mean . -point decrease in mnis+ (n= ). twenty-four patients underwent serial skin punch biopsies. at baseline, mean ienfd was . and . fibers/mm, while mean sgnfd was . and . m/mm for distal thigh and distal leg, respectively. amyloid was detected in ∼ % of skin biopsies, mean baseline dermal amyloid content . % and . % for distal thigh and distal leg, respectively, which inversely correlated with ienfd and sgnfd. overall, ienfd was stable throughout the -year treatment. sgnfd increased over time relative to baseline, reaching statistical significance at , , and months for distal thigh (p< . , all time points) and at months for distal leg (p= . ). dermal amyloid content decreased over time and reached statistical significance at , and months for distal thigh (p< . , all time points) and at , , , and months for distal leg (p< . , all time points). in summary, improvements in sgnfd and dermal amyloid content observed over a -year period suggest that these parameters have the potential to serve as biomarkers of response to patisiran treatment. polydefkis m , neuhaus s , doherty l , ebenezer gj . department of neurology, johns hopkins university, baltimore, md, usa. misdiagnosis and delayed identification of an etiology for sensory neuropathies hampers adequate management and delays initiation of therapy. we present several cases that illustrate variable presentations of amyloid neuropathy (an) and an complicated by amyloid arthropathy. a -year-old former college athlete developed painless difficulty walking. he was unable to keep up with family members during routine outings and subsequently was unable to navigate uneven ground during a hike, something he had previously excelled at. balance decreased, making bicycling difficult. neurological evaluation -months after symptom onset led to a diagnosis of peripheral neuropathy. a reversible neuropathy panel was normal though a c was elevated ( . %). over several months, his strength/sensation deteriorated despite diet improvement, losing lbs and a c improvement ( . %). upon referral, emg revealed a sensorimotor neuropathy with markedly reduced/absent sural, peroneal and tibial motor amplitudes. there was mild orthostasis. nis was . skin biopsies revealed severe ienfd, sgnfd reductions and positive congo red staining, confirmed with birefringence. a sural nerve biopsy confirmed amyloid deposits. genetic testing revealed the ttr-fap variant leu his. there was no history or suspicion for a family history of amyloid. a -year-old male with a history of controlled diabetes x years developed abrupt onset of rls months after a lymphoma diagnosis. treatment with rituximab led to radiographic resolution of the lymphoma though the patient developed progressive peripheral neuropathy confirmed by ncv/emg. new-onset early satiety, mild orthostasis and ed emerged. skin biopsies demonstrated a lichenoid pattern of amyloid deposition consistent with aa-amyloid. a -year old woman with leu his ttr-fap since developed progressive leg weakness, reduced ability to walk and low back pain. emg demonstrated progression of her sensorimotor neuropathy and superimposed multilevel radiculopathy. spine mri demonstrated severe lumbar stenosis requiring surgical decompression. postoperatively, she regained strength, walking more easily. congo red staining of vertebral bone demonstrated widespread positive staining with birefringence consistent with amyloid arthropathy. these cases illustrate to wide sprectrum of amyloid neuropathy presentation, an example of amyloid arthropathy-induced spinal stenosis and the utility of skin biopsy to diagnose and manage amyloid neuropathy. the differentiation between hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (cidp) in children is especially significant because of completely different treatment possibilities and prognosis in these conditions. the aim of the study was to compare electrophysiological abnormalities in a group of children and young adults with demyelinating neuropathy of chronic or subacute onset. retrospective analysis of clinical and nerve conduction study (ncs) data included children and young adults with charcot-marie-tooth neuropathy x type (cmtx ), children with charcot-marie-tooth neuropathy type a (cmt a) and children with cidp. in our study / cmtx had both axonal and demyelinating changes in ncs study. the aan and efns electrophysiological cidp criteria were fulfilled in / cmtx , / cmt a and / cidp patients. additionally patients with cmtx are classified with efns criteria as "probable/possible cidp". a distal compound muscle action potential (dcmap) was > ms in all cmt a and / cidp patients but none with cmtx . abnormal median/normal sural snap (amns) parameter was observed in // cidp and / ctmx patients but not any cmt a patient while abnormal sural/normal median snap (asnm) parameter was found in / patients with cidp. a difference between conduction velocities (cv) of two corresponding nerves > m/s was seen in / cmtx and / cidp patients but no one with ctm a. one patient with cmtx had especially conspicuous difference between nerve conduction in lower limbs, with axonal neuropathy and demyelinating features, and upper limbs with no changes. one -years-old patient with genetically confirmed cmtx had no abnormalities in ncs. electrophysiological data of cmtx , cmt a and cidp indicate diffuse demyelination, however in cmtx axonal changes coexist, seen in / patients, and asymmetrical abnormalities between corresponding upper and lower limb were observed in / patients. our study has showed that duration of dcmap is useful not only in diagnosing an inflammatory neuropathy but also in differentiating cmtx from cidp. however presence of the not homogenous abnormalities in ncs in patients with cmtx may mimic inflammatory neuropathy. prada v , mori l , francini l , accogli s , ursino g , gemelli c , schizzi s , grandis m , bellone e , mandich p , schenone a . department of neurosciences, rehabilitation, ophthalmology, genetics and maternal and child health, university of genoa, genoa, italy. the overwork weakness (ow) problem in cmt disease has been debated for long time. it has been reported that the non-dominant hand (ndh) of patients with cmt disease is stronger than the dominant hand (dh) as a result of ow and some authors verified this hypothesis using mrc on different muscles (van pomeren, ). more recently, piscosquito et al. ( ) found that the ow phenomenon does not exist using a dynamometer and the hole peg test, a dexterity test. we recruited cmt patients and healthy controls. we evaluated the strength of the -pinch and of the hand-grip with a dynamometer, the opposition ability with the thumb opposition test (tot) and applied an instrumental testing of hand function using the sensor engineered glove test (segt), which previously demonstrated its sensitiveness to measure severity of hands dysfunction in cmt patients. tot was significantly higher in the ndh compared to dh (ndh= , ± , ; dh= , ± , , p< . ) in cmt patients, instead there was no difference in dh and ndh in healthy controls. in the evaluation of -pinch and of the hand-grip, ndh performed slightly but not significantly better than the dh ( -pinch: dh= , ± , n; ndh= , ± , n; ± , n; ndh= , ± , n, p:ns) . in normal controls we confirmed the % rule (noguchi & demura, ). finally, segt results were similar between the ndh and dh in cmt patients but in normal controls there was a better performance in the dh. in conclusion, this study supports the existence of the overwork weakness in cmt, evident in every measurements. dexterity and overall ability of the hands impaired in the dh, probably because of compensating movements, compared with the healthy controls in the weaker hand. finally, our results support the importance of avoiding supramaximal exercises and educating the cmt patients to prevent incorrect movements, which may overload the hand muscles. prasad k , ohnmar o , teng a , cheng yj , umapathi t . national neuroscience institute, singapore; yangon general hospital, yangon, myanmar; yong loo lin school of medicine, national university of singapore, singapore. antecedent infections that have been linked to guillain-barré syndrome (gbs) include campylobacter jejuni, haemophilus influenza, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and influenza a virus. in south-east asia, the burden of mosquito-borne flavivirus infections is considerable. even in a small country like singapore, with a population of only . million and in spite of excellent sanitation and public health measures, dengue virus (denv) infections can exceed , a year. both symptomatic and asymptomatic denv infections are associated with gbs. zika virus (zikv) infections were detected in singapore in late august . doctors working in south-east asia anecdotally report that gbs cases increase after rainy season when mosquitos breed. we are currently planning studies to estimate the hitherto unknown burden of gbs associated with denv, zikv and other mosquito-borne viruses in this region. as a preliminary step, we reviewed the gbs cases seen at our institution in for evidence of denv and zikv. three out of the tested cases were positive for denv serology. one of these was also positive for denv pcr. all these patients had symptomatic dengue. two had acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) subtype of gbs. none the patients tested were positive for zikv pcr in serum and urine. one patient, with miller fisher syndrome (mfs), had serological evidence of recent zikv. he did not have clinical zikv symptoms. in mfs cases, the initial detectable zikv igg was later proven to be due to cross reactivity with previous denv infection. in cases ( amsan, aidp, mfs) the initial zikv igm was raised. we await analysis of convalescent sera to decide if they indeed had zikv or the results were related to previous and co-infection with denv. our preliminary findings indicate that flavivirus infections may account for at least some of the gbs cases in singapore. the lack of symptoms in some cases and the interactions between zikv and denv antibodies make accurate diagnoses challenging. prior r , , benoy v , , vanden berghe p , van den bosch l , . ku leuven-university of leuven, department of neurosciences, experimental neurology and leuven research institute for neuroscience and disease (lind), leuven, belgium; vib-center for brain & disease research, laboratory of neurobiology, leuven, belgium; laboratory for enteric neuroscience, translational research center for gastrointestinal disorders, k.u. leuven, leuven, belgium. charcot-marie-tooth disease (cmt) is the most common inherited neurodegenerative disorder of the peripheral nervous system. it is divided into two main subtypes, a demyelinating subtype (cmt type ) and an axonal form (cmt type ). cmt is a length-dependent disease, in that it effects the longest neurons in the body, thus the muscles in the peripheral regions are affected first and foremost. moreover, the majority of cmt patients share a classical phenotype with shared pathological hallmarks, such as distal muscular atrophy, reduction in nerve conductions, etc. but also molecular pathological hallmarks, like the breakdown in the transport of organelles and vesicles in neurons in a process called axonal transport. currently, there is no cure or effective treatment available to cmt patients. histone deacetylase (hdac ) has been shown to be a key regulator in axonal transport. moreover, inhibiting hdac has been demonstrated to stabilize microtubules, which act as molecular tracks for motor proteins and facilitates axonal transport of cargos. our labs current focus is on cmt type a, the main cause of cmt, which is caused by a duplication of a segment of chromosome p . containing the gene encoding peripheral myelin protein (pmp ). pmp is mainly expressed by schwann cells, the cells that myelinate neurons in the peripheral nervous system. in the work presented, the commercial mouse schwann cell-line, sw cells, was transfected to overexpress pmp using a lentivirus vector system to mimic that of cmt a patient schwann cells. the overexpression of pmp was confirmed using immunofluorescence and western blot techniques. these transfected sw schwann cells were then co-cultured with primary mouse dorsal root ganglion neurons (drgs) isolated from adult mice. these co-cultures where then analysed after weeks in vitro for axonal transport. the investigated groups were: a co-culture of drgs + sw cells, a co-culture of drgs + sw cells transfected with pmp , and a co-culture of drgs + a sw cell-line transfected with green fluorescent protein using a lentivirus vector system, and a monoculture of drgs. furthermore, this work demonstrates that the overexpression of pmp in schwann cells can impair axonal transport in co-cultured drgs in comparison to the other co-culture groups. moreover, these axonal transport defects were able to be rescued by the treatment of a hdac inhibitor, tubstatin a. to conclude, selective hdac inhibitors have been shown to be a beneficial treatment for a number of cmt subtypes in preclinical studies in our lab and offer as a viable treatment for a currently, untreatable debilitating disease. provitera v , caporaso g , stancanelli a , piscosquito g , di caprio g , saltalamacchia am , santoro l , nolano m . "maugeri" clinical and scientific institutes irccs, institute of telese terme (bn), italy; department of neurosciences, reproductive and odontostomatological sciences, university 'federico ii' of naples, naples, italy. the observation of rate and patterns of cutaneous nerves regrowth after mechanical or pharmacological distal axonotmesis, has proven to be an excellent tool to study human nerve regeneration in normal and pathological conditions. most of the observations, however, are referred to small fibers, possibly due to the availability of excellent models of reversible distal cutaneous small fiber axonopathy. no such model is available to study the regeneration of myelinated fibers in human. we studied regrowth of cutaneous large fibers in fingertip of patients with carpal tunnel syndrome after surgical decompression. we recruited patients (m/f / ; age . ± . years) with carpal tunnel syndrome candidate to surgery. patients underwent clinical and electrophysiological evaluation, quantitative evaluation of discriminative threshold at rd fingertip. in the same site, patients also underwent mm punch skin biopsy. skin sections were stained by immunohistochemical techniques and cutaneous innervation was analyzed by confocal microscopy. meissner corpuscles and their myelinated afferences were assessed following previously published procedures. twelve months after surgery, patients repeated functional evaluation and underwent a second skin biopsy two mm apart from the first one. mean density of mcs/mm was . ± . at time and . ± . at follow-up, mean density of myelinated endings was . ± . at time and . ± . at follow-up. however, not in all patients regeneration occurred. based on the variation of mc density between time and follow-up we were able to identify patients in which active regeneration had occurred. in this subgroup mean density of mcs/mm was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . in the same group, mean density of myelinated endings was . ± . at time and . ± . at follow-up (p< . ) with a mean delta of + . . we describe morphological patterns associated to nerve regrowth in the biopsies of patients in which nerve regeneration occurs. we propose an in vivo model to study regeneration of large myelinated fiber endings in human skin. in addition to the count of nerve endings, the identification of patterns associated to nerve regeneration can increase the diagnostic yield of skin biopsy. the most common type of charcot-marie-tooth disease is caused by a duplication of pmp , leading to dysmyelination, axonal loss and progressive muscle weakness (cmt a). currently, no approved treatment for cmt a patients is available. among others, preclinical therapeutic approaches aim to correct the pmp overexpression in order to ameliorate axonal loss and muscle weakness. we previously reported that the novel polytherapeutic drug pxt , a low-dose combination of baclofen, naltrexone and sorbitol, improved dysmyelination and axonal loss after long-term application in adult pmp transgenic rats, a known animal model of cmt a. interestingly, after short-term application in young cmt a rats we observed a long-lasting prevention of muscle weakness as well but without obvious effects on dysmyelination and axonal loss. improved distal motor latencies in the electrophysiological recordings and a shift in the axonal diameter distribution raised the hypothesis that therapy may improve the neuromuscular endplate pathology as another therapeutic target for cmt a. here, we report a preclinical trial in adult cmt a rats treated from postnatal week for consecutive weeks as the most effective regimen in order to facilitate a deeper understanding of the mode-of-action of pxt . long-term therapy effects were confirmed by an ameliorated behavioral phenotype, improved distal motor latencies and also nerve conduction velocity in the electrophysiological recordings. histological analysis revealed an increased number of neuromuscular endplates, which were lowered to wildtype level after pxt treatment. further investigations include detailed analysis of peripheral nerve myelin thickness, internodal and nodal length, terminal axonal sprouting at the neuromuscular endplate and muscle fiber subtyping in treated cmt a rats. charcot-marie-tooth disease (cmt) is a rare hereditary neuropathy for which novel treatments are being developed and urgently needed. only few clinical trials and natural history studies have been performed. evaluation of intervention efficacy is hampered by slow progression and lack of sensitive outcome measures. the interindividual disease variability is high and prognostic disease measures are not established for cmt patients. previously, we have identified skin-derived disease and progression biomarkers in a large european and us-based cmt a cohort. within the german charcot-marie-tooth disease network (cmt-net, www.cmt-net.de) we aim to establish easier accessible biomarkers in blood of cmt a patients. blood analysis is less invasive and can be repeatedly performed during clinical trials and routine patient care. we have started clinically assessing approximately young, adolescent and adult cmt a patients at clinical centres (münster, münchen, göttingen) including a large set of clinical outcome measures (cmtnsv , onls, mwt, mwt, walk , hpt, sf- , fss, psqi, ess, bdi-ii). we will sample blood and skin tissue once a year at , and months for an observational period of years. tissue samples will be analyzed on the transcriptional, translational and epigenetic level. we envision that the diagnostic and progression biomarkers may be used to measure therapeutic effects within clinical trials and later in clinical routine monitoring. this is the first trial testing "circulating" biomarkers from blood in a prospective observational trial in cmt a patients. igm peripheral neuropathy is a slowly progressive and heterogeneous disease with symptoms ranging from mild foot numbness and minor imbalance to severe neuropathic pain and sensory and motor dysfunction. international consensus regarding assessment and treatment of patients with igm peripheral neuropathy is lacking. this might be caused by the repeated use of suboptimal outcome measures, the small trial sizes with low numbers of treated patients, the indolent disease course needing a longer follow-up period to capture relevant changes, or the possibility that administered treatments were not aggressive enough. the imagine study is an international, multi-centre, prospective, observational cohort study, which will result in a unique collection of a large number of prospectively collected and highly standardized clinical data, and a biobank from a large population of well-defined patients with igm peripheral neuropathy. the main objective is to describe in detail the variation in clinical subtypes, clinical disease course, past and current practice of treatment, antibody titres, and clinical picture at the various levels of assessing outcome. patients being at least years, and fulfilling the international criteria for igm peripheral neuropathy are eligible. exclusion is primarily based on concomitant diseases influencing peripheral nerve function. several study parameters measuring weakness, sensation, activity and participation, ataxia, pain, and quality of life are of interest. in february an enmc kick of meeting is organized, bringing together an igm peripheral neuropathy study group. the meeting will focus on the development of a core set of outcome measures to be used in future studies in igm peripheral neuropathy. the imagine study started in the netherlands in september , and to date patients were included. the imagine study starts in the united kingdom, italy, and france in the beginning of . centres recruiting at least patients with igm peripheral neuropathy are eligible to participate in the study. during the pns global recommendations from the enmc meeting, as well as the first results of the imagine study, will be presented. puget s , paolantonacci p , burlot l lfb biomédicaments, les ulis, france; lfb biotechnologies, les ulis, france. in dysimmune neuropathies patients (mmn, cidp, gbs), ivig have become the gold standard due to their efficacy and tolerability. acute renal impairment, a rare but serious adverse event, can be induced by all ivig. its incidence is not determined precisely for each ivig. it mostly occurs in patients at risk, with pre-existing conditions (prior renal insufficiency, diabetes, mellitus, arterial hypertension, elderly > years old, dehydratation, hypervolemia, sepsis, paraproteinemia or concomitant use of other nephrotoxic drugs) and treated by immunomodulatory doses. as the number of these patients increases over the years, the choice of ivig in association with the precautionary measures will be decisive to reduce or avoid the occurrence of this adverse event. from s to s, the nephrotoxicity of ivig has been explained by tubular toxicity related to osmotic nephrosis. sucrose, a stabilizer of some ivig, has been implicated as one of the causes of this. even if several new ivig without sucrose are used, renal impairment cases have been described and studies have highlighted that sucrose is not the only ivig stabilizer associated with tubular damage. however, in a recent study, renal impairment cases ( / ) have occurred in cidp elderly patients treated by free-sucrose ivig. the occurrence of renal failure seemed to be low (only / ) for patients treated with sucrose-ivig in association with precautionary measures (hydratation, adaptation of dosage according body mass index, rate infusion). otherwise, since s, cases of renal impairment secondary to hemolysis have been increased with some ivig but would be rare for full ethanolic fractionated products as they have been identified to generate very low occurrence of hemolysis. the occurrence of renal impairment related to ivig seems to be multifactorial, need to find out all causes (mechanisms of different stabilizers, hemolysis, and concentration of ivig). registries would be helpful for physicians to define the incidence, relative risk of ivig-related renal impairment and the right ivig in association with precautionary measures in these patients at risk. the published ultrasound (us) studies on chronic inflammatory demyelinating polyradiculoneuropathy (cidp) report diffusely increased cross-sectional area (csa) of nerves. these data have not been correlated with histological patterns. to date, no further information about us ultrastructural nerve modification has been provided because of limited frequency range ( - mhz) of the us probe. the aim of this preliminary study is to evaluate the correlation between histological findings from sural nerve biopsies and us patterns found with uhfus ( mhz) from sural nerve at the ankle. four patients with cidp underwent uhfus nerve evaluation of clinically affected sural nerve before undergoing a sural nerve biopsy. us findings included: cross-sectional area of the nerve, connective tissue depth and changes in echogenicity of fascicles. those data were then compared to the histological findings obtained in transversal and longitudinal sections. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened and in two of the four patients hyperechogenicity of fascicles was observed. also, thanks to the ultra-high-resolution of the probe, a direct correlation of the histological and us images was possible, so as a direct measurement of internal microstructure of the nerve. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of sural nerve can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue, as confirmed by the histological study. in particular, we have observed a hyperechogenicity of fascicles in most severe patients; in those cases, histology confirmed an increase of endoneurial depth. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed and will be presented during the meeting. puma a , cambieri c , panicucci e , desnuelle c , raffaelli c , sacconi s . high-resolution ultrasound (hfus, mhz) of peripheral nerves is a valuable non-invasive, painless complement to neurophysiology, especially in the workup of cidp. nevertheless, the current spatial resolution of echographic images doesn't allow a detailed study of the nerves. ultra-high frequency ultrasound (uhfus, mhz) is a new tool with a - times higher spatial resolution than traditional hfus. the aim of this preliminary study is to evaluate sensory and motor nerves structural characteristics found with uhfus in patients with definite cidp. seven patients with cidp underwent uhfus nerve evaluation of median, ulnar, peroneal and sural nerves, bilaterally. us findings included: cross-sectional area of the nerve, connective tissue depth, nerve vascularization and changes in echogenicity of fascicles. patients also underwent electroneurography (eng) and plexus mri. in all patients, uhfus nerve changes were as following: csa was increased; connective tissue was thickened. two of the seven patients presented an epineural hypervascularization, observed at the doppler evaluation; none of them was treated by iv immunoglobulines. echographic changes were present even in the absence of mri abnormalities (root hypertrophy). eng characteristics correlated with the us patterns. uhfus may be of adjunctive diagnostic value in cidp assessment. more detailed images of nerves can be obtained compared with high-frequency us, allowing a better evaluation of the internal structure of the nerve. the increased csa observed in all our subjects seems to relate to an enlargement of connective tissue. in particular, uhfus allows a more detailed study of nerves, demonstrating that the structural abnormalities hit connective tissue, while -conversely to previous studies -fascicles anatomy seems to be spared. we did not show nerve vascularization except in non-treated patients. we speculate that those findings may explain a preliminary involvement of connective tissue in the pathogenesis of the cidp; our findings of nerve enlargement may be tool to monitor disease activity in cidp, and better understand disease pathogenesis. further studies are needed to confirm these findings and additional data are being processed. purcell l , , wojciechowski e , , gibbons p , , jamil k , , menezes, m , , burns j , . sydney children's hospitals network (randwick and westmead), new south wales, australia; university of sydney, new south wales, australia; universiti kebangsaan, kuala lumpur, malaysia. - % of children with cmt are reported to have hip dysplasia. we aimed to investigate the relationship between radiological hip dysplasia indicators and walking pattern in children with cmt. thirty children ( female; . ± . yrs; ± . cm; ± . kg cmt a, cmtx , cmtx , cmt f, cmt a) underwent d gait analysis ( dga), and had an anterior-posterior pelvis radiograph within months of assessment. radiographs were reviewed by two orthopaedic surgeons, and the reliability of measures of dominant limb hip health via radiograph was assessed. of the measures, measures had an intraclass correlation coefficient > . between raters, and the more experienced surgeon's measures were used for further analysis. the measures of acetabular index (ai), centre edge angle (cea), neck shaft angle (nsa), medial joint space, head width, lateral uncoverage, migration percentage and triradiate were used to investigate correlations with kinematic and kinetic dga variables of the pelvis and dominant limb hip, knee and ankle in planes, temporal spatial parameters and gait profile score. dga data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model. gait data were compared to typically developing children ( female; . ± . yrs, ± . cm, ± . kg). five of affected children had a migration percentage > ∘ , representing % of our sample. maximum hip abductor moment in terminal stance was significantly lower than normative reference values, and was moderately correlated with of the radiographic measures (ai r=− . , p= . ; nsa r=− . , p= . and medial joint space r=− . , p= . ). walking speed (normalised to leg length) was correlated with medial joint space (r= . , p= . ) head width (r=− . , p= . ) and triradiate (r=− . , p= . the ganglionopathies are a unique group of disorders with a varied etiology which includes autoimmune disorders, paraneoplastic syndromes and toxin induced causes. it has been observed that despite extensive investigations the cause of a sensory neuronopathy is often idiopathic in approximately - % cases. electrodiagnostic criteria to diagnose a possible ganglionopathy requires at least one sensory action potential absent or three sensory action potentials < % of the lower limit of normal in the upper limbs plus less than two nerves with abnormal motor nerve conduction study in the lower limbs. ulnar sensory-motor amplitude ratio values (usmar) lower than . is useful in differentiating ganglionopathy from axonal length-dependent neuropathy. we performed a retrospective analysis of patients who were either admitted or evaluated in the outpatient department in department of neurology, nizam's institute of medical sciences, with a possible ganglionopathy as per the diagnostic criteria proposed by camdessanchéet al. consecutive patients who sought treatment during a year period with a clinical pattern of sensory neuronopathy were analysed retrospectively. we reviewed the electrodiagnostic studies of patients and calculated the ulnar sensory-motor amplitude ratio values in comparison with patients with a clinical and electrophysiological diagnosis of diabetic axonal sensorimotor neuropathy. the clinical profile of a ganglionopathy from the indian subcontinent comprised of a prominent sensory ataxia as the initial presenting clinical manifestation with a predilection towards involvement of large-fiber sensory modalities. electrophysiologically a pattern of absent snaps was characteristically encountered with a significant value of usmar less than . , enabling differentiation from axonal length-dependent neuropathy. rahman im , jahan it , nahar s , khalid mm , jahan i , hayat s , , , islam z . guillain-barré syndrome (gbs) is a post-infectious autoimmune polyradiculoneuropathy where immune response is triggered by molecular mimicry between glycolipid antigens expressed by an infective agent such as campylobacter jejuni (c. jejuni) and human peripheral nerve gangliosides. cd molecules are mhc-like structures specialized in capturing and presenting a variety of glycolipids to antigen-specific t lymphocytes. the objective of this study was to investigate the possible role of coding region polymorphisms of cd genes in the pathogenesis of gbs in bangladeshi population. single nucleotide polymorphisms (snps) of exon of cd a (* /* ) and cd e (* /* ) in well defined gbs patients and healthy controls were studied to delineate their effect in developing gbs. we genotyped exon of both cd a (cys/tryp) and cd e (gln/arg) genes through pcr-rflp. to validate these findings, direct sequencing of pcr product was performed for at least samples for each position. we found no differences in genotypes and allele frequencies of both genes in gbs patients compared to controls. however, compared to control individuals with cd a* /cd e* haplotype were . times more likely to develop gbs, whereas individuals with cd a* /cd e* haplotype had a reduced relative risk by . times. a positive association of cd a* /cd e* haplotype was observed only in axonal form of gbs ( . % vs. . %, p = < . ). haplotype cd a* /cd e* was prevalent among anti-gm antibody positive gbs patient compared to anti-gm antibody negative patients ( . % vs. . %) though it was not statistically significant. snps in cd a and cd e were not associated with antecedent c. jejuni infection, disease severity and disease outcome at months of follow up. in conclusion, cd polymorphisms are not a susceptibility or disease causing factor in gbs. conversely, increasing knowledge of this field may offer new dimension for the research to elucidate better answer for disease pathogenesis and also contribute to conduct high power meta-analysis. extensive genetic testing was performed on a unique patient, who received national media attention due to her severe cmt phenotype. the index patient was never able to walk independently, had difficulty breathing and swallowing and demonstrated aspiration by a barium swallow study. nerve conduction velocity testing revealed velocities of m/s in her upper extremity. she required continuous positive airway pressure (cpap) for an upper respiratory infection and developed bilateral vocal cord paralysis. we used the combined brief assessment of motor function (bamf) scale to evaluate her disability. her fine motor scale was a / , her upper extremity gross motor scale was a / and her lower extremity gross motor scale was / . unfortunately, she died at age in her sleep, presumably from respiratory arrest. the proband's father was asymptomatic, however, his neurological exam showed pes cavus bilaterally and mild atrophy of the first dorsal interossei muscles in his hands. his nerve conduction velocities were slowed to m/s in the upper extremities and his cmt neuropathy score (v ) was / . we performed whole exome and subsequently whole genome sequencing in the trio. comprehensive structural variant analysis for copy number variations, large deletions, and recombinations was completed by a combination of software tools. known cmt genes were excluded as the underlying cause and the only viable candidate gene remaining was sh tc . we showed expression of sh tc in peripheral nerve and schwann cells. sh tc is a paralogue to sh tc , which causes the recessively inherited dysmyelinating form cmt c. sanger sequencing confirmed the variants in our family, p.v m (c. g>a, chr : ) and p. a p (c. g>c, chr : ) , and showed the segregation of the heterozygous variations. we hypothesize that the heterozygous paternal allele had a minimal effect and let to a very mild or subclinical form of peripheral neuropathy. of interest, the recessive sh tc gene has also been shown to cause mild aberrant forms of peripheral nerve degeneration in carriers of heterozygous disease alleles. in summary, this paper will present genetic and molecular evidence from an extensive n= study proposing a novel cmt gene. charcot-marie-tooth (cmt) is an important cause of morbidity worldwide. it is a heterogeneous disease manifesting as progressive weakness, wasting and loss of feeling in a length-dependent pattern. there are an increasing number of cmt-related genes in the literature. more recently, recessive mutations in the hint gene have been reported as causative of predominant motor axonal neuropathy associated with neuromyotonic discharges on emg. one of the characteristics of autosomal recessive cmt is it varying frequency in different populations and ethnic groups. we sought to evaluate the frequency of mutations in the hint gene in a brazilian cohort with axonal hereditary neuropathy. all patients included in this study born within south east area of brazil. the group consists of consecutive patients with axonal neuropathy screened for recessive or sporadic axonal neuropathy in the neurogenetics laboratory of clinical hospital of ribeirão preto. direct sequencing of the coding region of hint gene was done. among patients screened, were suspected of having recessive axonal neuropathy without neuromyothonic discharges, and two have axonal neuropathy associated with neuromyothonic discharges on emg. we did not find any disease causing mutations among our patients. some previously reported studies reported a high frequency of mutations in the hint gene among recessive axonal neuropathies in some european countries. our results demonstrated that frequencies of mutation underlying genetic hereditary neuropathies are different between different ethnic groups and have implications for the organization of services management of cmt, for genetic counseling, and for gene flow in different world populations. funded by cnpq, fapesp, faepa, pronas (ministry of health). rodriguez-menendez v , ballarini e , malacrida a , , ceresa c , semperboni s , , meregalli c , cavaletti g , nicolini g . school of medicine and surgery, experimental neurology unit, university of milano-bicocca, monza, italy; phd program in neuroscience, university of milano-bicocca, monza, italy. bortezomib (btz) is a proteasome inhibitor widely used for multiple myeloma treatment. btz induced peripheral neuropathy (bipn) is the most frequent adverse effect. bipn in humans, is a dose dependent painful sensory neuropathy characterized by nerve axonopathy and a tendency to recover in the follow-up period after btz withdrawal. bipn affects principally dorsal root ganglia (drg) and different rodent models have shown alterations in sensory neurons, small unmyelinated axons, large myelinated axons, axonal mitochondria and schwann cells. in this work, we evaluated the effects of btz in vitro on drg neurons isolated from adult mice. our interest focused on dystonin, a protein able to interact with all the three components of cytoskeleton (microtubules, microfilaments and intermediate filaments) and able to bind map b (a mt-associated protein) through which can influence golgi apparatus morphology. there are different neuronal isoforms of dystonin and in particular dystonin-a is able to modulate tubulin acetylation and stability through interaction with map b. it is noteworthy to underline that map b expression in the mature nervous system is restricted to sensory neurons. western blot analysis demonstrated that a treatment with nm btz induces a statistically significant decrease in tubulin acetylation. this result does not go along with our previous study where we observed a tubulin polymerization increase after btz treatment. therefore, we have decided to focus our interest on soma organelle organization that is well known to be dependent from cytoskeleton structure. immunofluorescence images showed an altered distribution of acetylated tubulin in soma cytoplasm after hour treatment. moreover, confocal analysis of cis golgi (gm ) demonstrated that in btz treated neurons the normal golgi structure is lost showing a spot-like non perinuclear labeling distribution. additionally, dystonin distribution seemed comparable to that of cis golgi apparatus suggesting that btz could induce a change in dystonin localization which in turn affects golgi organization, probably through map b.these data suggest that the cytoskeleton alterations, induced by btz, could probably cause wrong maturation and trafficking alteration of golgi vesicles consequently impairing the correct sensorial function. romão tt , aleixo bfl , wedemann d , herculano fgn , prado h , cal h , pupe c , bittar c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. acute motor and sensory axonal neuropathy (amsan) is a clinical variant of guillain-barré syndrome (gbs) which has rarely been reported in association with human immunodeficiency virus (hiv) and neurosyphilis. we describe a case of a -year-old woman who started with muscle weakness in the left leg followed by weakness in the right leg and in upper limbs in one month. motor symptoms were followed by urinary and fecal incontinence. no preceding symptoms, as diarrhea or fever were referred. neurological examination showed global areflexia with impairment of vibratory and tactile modalities, as well as loss of proprioception. pinprick sensation was moderately compromised. no impairment of higher mental functions was observed. other causes of neuropathies were ruled out. cerebrospinal fluid (csf) examination showed cell/mm , increased protein level, normal glucose level and negative vdrl test. blood analysis showed hiv and positive serology tests, vdrl : and positive t. pallidum hemagglutination assay. igm cytomegalovirus antibody was negative, cd was in normal range and hiv load was undetectable. cranial and spinal cord magnetic resonance imaging revealed extensive involvement of the posterior spinal cord tracts which supports the diagnosis of neurosyphilis. nerve conduction studies showed a significant reduction in compound motor action potentials amplitudes, particularly at lower limbs, and absent sural nerve responses. electromyography revealed positive waves and fibrillation at lower limbs. treatment with methylprednisolone, human intravenous immunoglobulin and ceftriaxone has been settled, resulting in improvement of upper limbs paresis and sphincters dysfunction. antiretroviral therapy was initiated. this amsan case is associated with central nervous system involvement, encompassing an encephalomyeloradiculopathyrelated to hiv/t. pallidum co-infection. c.jejuni is the main etiological agent associated with amsan, but there are some reports of hiv/cmv co-infected patients who presented this gbs variant. facing a patient with amsan, mainly when cns involvement is present, it is of crucial importance to undergo investigation of an ongoing infectious process, such as hiv and syphilis, considering the impact of early treatment on prognosis. roodbol j , , yiu e , doets a , , de wit mcy , monges ms , van nes s , catsman-berrevoets ce , van den berg b , , jacobs bc , and on behalf of the igos-kids consortium. only a few prospective cohort studies so far have investigated the clinical presentation and course of guillain-barré syndrome (gbs) in children using age-adjusted outcome scores. in we started a study based on the igos protocol but adjusted for children with gbs (igos-kids). (pediatric)neurologists from argentina, australia and the netherlands joined forces to determine the clinical and biological determinants of disease progression and recovery in gbs in children from different geographical areas. for each age category specific pain scales are used including the comfort score for children < years old, pain faces revised age - years old and numerical rating scale in children ≥ years old and outcome measures. a new strength score was developed: gbs kids score. this score will be used and validated in addition to the mrc-sum score. the onls, r-ods and fss are also be used in igos-kids. age-dependent quality of life questionnaires were added to the igos-kids protocol, namely the pedsql and the pedsql multidimensional fatigue scale. these scales are available for children ≥ years old. an activity score validated for neuromuscular disorders for children ≥ years old was also added. currently there are children included in igos-kids, eight children from australia and five from the netherlands. additional clinicians and researchers interested in gbs in children are most welcome to participate in igos-kids. prognostic models have been developed to predict the highly variable clinical course of guillain-barré syndrome (gbs) in adults. the clinical course of gbs is equally variable in children, but the current prognostic models have not been validated in children. in this study, we aimed to identify in children with gbs the characteristics at hospital admission that predict the clinical severity and outcome. the study was conducted in two patient cohorts from europe: one (largely) german cohort of children and one dutch cohort of children. clinical information was obtained regarding preceding infection, first symptoms, neurological deficits at admission and nadir, results of additional investigations (cerebrospinal fluid and nerve conduction studies). clinical severity, course and outcome was defined by the gbs disability score, especially the ability to walk unaided, at a follow-up of month, months, months and months after onset of symptoms. univariate and multivariate regression analyses were performed initially on the two separate cohorts. combined the cohorts consisted of children (age median years, range - years) including boys ( %). the median duration between onset of symptoms and hospital admission was days (range ). pain at admission was remarkably frequent and present in ( %) children. in the combined cohorts, children ( . %) developed respiratory failure and one child died. multivariate regression analysis showed that in both cohorts strong predictors of respiratory failure available at hospital admission were cranial nerve involvement, a higher gbs disability score and a shorter period in days between onset of symptoms and admission. this information was used to develop and validate a prognostic model for children with gbs that will be presented at the conference. in conclusion, based on the analysis of two independent cohorts of patients the predictors of respiratory failure and clinical recovery were identified and validated. similar factors were identified for adult patients, although the prognosis in children in general is better than in adults. this information will be used to develop a simple prognostic model for current clinical practice to predict the chance of respiratory failure and outcome in children with gbs. the negative trials of vitamin c in cmt a have highlighted the lack of sensitive outcome measures in charcot-marie-tooth disease (cmt). neurofilaments are abundant neuronal cytoskeletal proteins and their concentration in blood is likely to reflect axonal breakdown. we therefore examined plasma neurofilament light chain (nfl) concentration as a potential biomarker in cmt. blood samples were collected from patients with cmt and age matched healthy controls over a -year period. disease severity was measured using the weighted cmt examination and neuropathy scores. plasma nfl concentration was determined using an in house developed simoa assay based on the nfl antibodies from the nf-l light elisa kit (umandiagnostics). plasma nfl concentrations were significantly higher in cmt patients (median: . pg/ml; iqr: . - . ) than in controls (median: . pg/ml; iqr . - . , p< . ) and correlated with disease severity as estimated by the weighted cmt examination (n= , r= . , p< . ) and neuropathy scores (n= , r= . , p= . ) . concentrations were also significantly higher when subdividing cmt patients by genetic subtype; cmt a (n= , p< . ), sptlc (n= , p< . ) and gjb (n= , p= . ) or into demyelinating, cmt (n= , p< . ) or axonal, cmt (n= , p< . ) forms compared to healthy controls. there was no significant difference in the plasma nfl concentration after year in patients with cmt (n= , mean difference − . pg/ml, % confidence interval − . - . ) or healthy volunteers (n= , mean difference − . pg/ml, % confidence interval . - . ) which is unsurprising as cmt is a slowly progressive disease in which the rates of axonal degeneration are likely to be constant and elevated. in summary, we have shown that plasma nfl levels are significantly raised in patients with cmt and that they correlate with disease severity. this is of relevance not only for the field of cmt but for peripheral neuropathies in general, and suggests that plasma nfl holds promise as a biomarker of peripheral neuropathy in both routine practice and clinical trials. neurolymphomatosis (infiltration of the peripheral nervous system by lymphoma cells) is a rare and usually devastating condition belonging to the spectrum of lymphoma-associated neuropathies. cerebrospinal fluid examination with cytologic examination, flow cytometry and clonality testing by pcr may show malignant cells especially when nerve root involvement is prominent. however, nerve biopsy remains a useful tool to confirm the presence of malignant cells invading the nerve. neuropathological features are important and pcr-based immunoglobulin or t-cell receptor clonality testing on nerve fragments may add notable value for the diagnosis of neurolymphomatosis, although this has not been systematically investigated. we retrospectively studied clinicopathological data and clonality results of nerve biopsy samples in patients with nl from centres, performed between and . among patients with nl, we found % of b-cell lymphoma and one t-cell lymphoma. nl was the first manifestation in , % of patients. the main clinical pattern was symmetrical progressive sensorimotor polyneuropathy in % of patients and pain was a prominent feature in %. clonality testing showed a monoclonal rearrangement in ( %) cases, oligoclonal rearrangement and no amplification in ( %). the main histological pattern was perivascular infiltration of predominant b-cells in the epineurium, without signs of vasculitis. the extent of axon loss was highly variable between patients. various chemotherapeutic regimens were used and the median overall survival was months. only one case showed a monoclonal pattern among control nerve samples from patients with other types of neuropathy including vasculitis and cidp. overall, we found that clonality testing on nerve biopsy specimens may provide decisive information on the presence of neurolymphomatosis, with a sensitivity of . %, a specificity of . %, a positive predictive value of . % and a negative predictive value of . %. we confirm the utility of nerve biopsy for the diagnosis of nl and show the great yield of pcr-based clonality testing to assess the malignant feature of peripheral nervous system lymphoid infiltrates. despite an accurate diagnosis, neurolymphomatosis still remains a devastating disease with an overall poor prognosis. in the last years, cumulative data have shown that patients with amyotrophic lateral sclerosis (als) and mouse models of the disease present loss of small epidermal and dermal nerve fibers and sensory dysfunctions, in addition to the classical motor symptoms. our objective is to characterize this small fiber neuropathy and to clarify if axonal loss involves all sort of fibers equally, or if there is some specificity. for this purpose, we performed an immunohistochemical characterization of total intraepidermal nerve endings (protein gene product; pgp . ), peptidergic (calcitonin gene-related peptide; cgrp) and nonpeptidergical nerve epidermal endings (isolectin b ; ib ) of the sod g a mouse at different stages: presymptomatic stage ( weeks), disease onset ( weeks) and in symptomatic stage ( weeks). the sympathetic sweat gland innervation was immunolabeled for vasoactive intestinal peptide (vip) from very early stage ( weeks) to disease onset ( weeks). the results showed a marked reduction of the intraepidermal nerve fibers already in the presymptomatic stage compared to the wildtype littermates (p< . ). this axonal loss affected more markedly the nonpeptidergic axons from the disease onset stage ( % axonal loss, p< . ), whereas no significant differences were found in the cgrp positive fibers ( . % axonal loss). a reduction of the sympathetic innervation of the sweat glands was also found from the disease onset stage ( % axonal loss, p< . ). in summary, we have found that nonpeptidergic and sympathetic innervation of the skin are predominantly affected in the sod g a mouse model. these findings suggest that this specificity could be used as an accessible biomarker for the disease. hereditary and inflammatory neuropathies are peripheral nerve disorders of different pathophysiology whose identification is crucial for therapeutic approach. diagnosis of cmt is easy when there is a family history, disease course is slowly progressive, neurophysiological findings are homogeneously abnormal. cases of cmt patients with inflammatory-like phenotypes leading to a misdiagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (cidp) have been described. the presence of "red flag signs", such as slowly progressive decline, mild sensory symptoms, minimal electrophysiological progression and no response to therapy should prompt re-evaluation of the diagnosis of cidp. the introduction of nerve neuroimaging has contributed to the diagnostic work-up. we describe cmt patients ( men, women, mean age . ± . yrs, mean neuropathy duration . ± . yrs) with genetically confirmed cmt, who were initially diagnosed with cidp. neurophysiology showed demyelinating features in patients, the remaining patients had axonal features. csf analysis showed albumin-cytological dissociation in patients, oligoclonal igg bands were present in patient. nerve ultrasound in patients with demyelinating neuropathy showed diffuse increased cross-sectional area. mr-neurography in one patient confirmed diffuse nerve hypertrophy and increased signal intensity, supporting the hypothesis of a overlap syndrome. all patients were treated with immunomodulatory therapies. among patients with demyelinating features, patients underwent ivig for months, without benefit; the remaining were treated with steroids, showing temporary improvement. the patients with axonal neuropathy complaining of a progressive history, underwent ex adjuvantibus plasma exchange and ivig, without benefit. given the lack of benefit from therapies, screening for hereditary neuropathies was performed. among patients with demyelinating neuropathy, had cmt a, cmt b, cmt d. among the patients with axonal neuropathy, one was diagnosed with cmt k. in patients an overlap syndrome was present. several clinical and laboratory features (csf protein elevation), might contribute to the misdiagnosis of cidp in cmt patients. refractoriness to immune-modulatory treatment should rise the suspicion of a hereditary neuropathy. an overlap cmt/cidp syndrome may be considered, in front of acute/subacute deterioration and/or proximal muscles involvement. two patient with an overlap syndrome showed benefit after either steroids or ivig. ruiz m , tripodi sm , campagnolo m , zara g , salvalaggio a , ruggero s , toffanin e , anglani m , briani c . neurology, department of neuroscience, university of padua, padua, italy; neuroradiology, padua hospital, padua, italy. facial diplegia is a rare regional subtype of guillain-barrè syndrome characterized by rapidly progressive bilateral facial palsy in absence of other cranial neuropathies, ataxia or limb weakness. the diagnosis is based on history, clinical examination, laboratory data. the outcome of this variant appears to be better than that of classical gbs. although about % of gbs patients clinically exhibit facial nerve involvement, there are only few reports that demonstrate mri gadolinium facial nerves enhancement. a -year-old previously healthy man sought neurological advice for the acute onset of right perioral paresthesias and hyposthenia at the extensor hallucis longus bilaterally. in the previous month, during a trip to china and japan, he had suffered from gastroenteritis. at neurological examination he presented with bilateral extensor hallucis longus hyposthenia. ankle jerk reflexes were absent. one week later, he developed bilateral facial palsy. neurophysiology disclosed absence of activity in the facial nerves innervated muscles bilaterally and signs of mild partial denervation at right l metamer. csf analysis showed increased protein levels ( mg%) and . leucocytes/ l. serological tests for infectious agents were negative and serum levels of angiotensin converting enzyme were normal. antibodies to monosialoganglioside gm of igg isotype were positive ( / ). t brain mri showed, after gadolinium administration, marked bilateral enhancement of the facial nerves in their extra-and intra-canalicular segments. t lumbosacral mri scan ruled out the presence of disc diseases as well as signal modifications within conus medullaris and cauda equine nerve roots. the patient was treated with iv immunoglobulins ( . g/kg/die for days) with benefit on facial weakness. at follow-up examination months later, the patient presented a further improvement of the facial diplegia and neurophysiology disclosed a partial recovery of activity in the facial nerves, with persistent axonopatic damage. bilateral extensor hallucis longus hyposthenia persisted. in conclusion, we report on a regional subtype of guillain-barrè syndrome with the curious association of facial diplegia and bilateral extensor hallucis longus hyposthenia, and igg anti-ganglioside antibodies, that are not commonly described in facial diplegia. moreover, we provide t brain mri evidence of facial nerve involvement. rumora ae , tabbey ma , lograsso g , dolkowski j , haidar j , lentz si , feldman el . department of neurology, university of michigan, ann arbor, usa; department of internal medicine, division on metabolism, endocrinology and diabetes, university of michigan, ann arbor, usa. diabetic neuropathy (dn) is the most common complication of diabetes affecting up to % of diabetic patients. the pathogenesis of dn in type diabetes is directly related to the metabolic syndrome associated with the western diet composed of elevated levels of long chain saturated fatty acids (sfas) and low levels of medium chain sfas. long chain sfas are associated with insulin resistance and dyslipidemia while medium chain sfas have been associated with decreased lipid accumulation and improved mitochondrial function. since dn is primarily a disorder of the sensory dorsal root ganglion (drg) neurons, we sought to evaluate the impact of sfa hydrocarbon chain length on mitochondrial trafficking mechanisms that are critical for distributing atp throughout drg axon to provide energy for synaptic transmission. we hypothesize that sfas with longer hydrocarbon chains will impair mitochondrial trafficking whereas medium length sfas will not impact mitochondrial movement along the drg axon. in this study, we examined the impact of sfa hydrocarbon chain length on mitochondrial trafficking, directionality and velocity in primary mouse drg neurons. drg neurons were treated with increasing concentrations of long chain sfas, stearate and palmitate, or medium chain sfa, laurate, ranging from . to micromolar for twenty-four hours. drg neurons treated with long-chain sfas, palmitate and stearate, showed a significant decrease in the percentage of motile mitochondria whereas medium chain sfa, laurate, does not alter mitochondrial motility. we next assessed whether motile mitochondria in drg neurons treated with palmitate or stearate exhibited altered directionality or velocity of mitochondrial trafficking. palmitate and stearate treatments resulted in a trending decrease in the number of mitochondria trafficking in both anterograde and retrograde directions. furthermore, . to micromolar palmitate and stearate induced a significant decrease in mitochondrial velocity. laurate treatment, on the other hand, retained directionality and velocity of mitochondrial trafficking. these results suggest that hydrocarbon chain length of saturated fatty acids plays an important role in regulating mitochondrial trafficking mechanisms in dorsal root ganglion neurons. impaired mitochondrial trafficking in drg neurons exposed to elevated levels of long-chain sfas may play a critical role in the progression of dn. dyslipidemia is a critical factor that contributes to the development of diabetic neuropathy (dn). the progressive nerve damage associated with dn correlates with the dyslipidemic state characterized by elevated circulating levels of harmful saturated fatty acids (fas) and low levels of beneficial unsaturated fas. in dn, primary sensory dorsal root ganglion (drg) neurons exhibit energy dyshomeostasis and mitochondrial dysfunction, however, little is known about the differential impact of saturated and unsaturated fas on mitochondrial mechanisms in drg neurons. mitochondrial trafficking is an essential mechanism for transporting mitochondria throughout drg axons to provide cellular atp for neuronal function and neurotransmission. since mitochondrial trafficking is regulated by metabolic flux, we sought to determine whether saturated fa, palmitate, and unsaturated fa, oleate, have differential effects on mitochondrial trafficking in drg neurons. we evaluated mitochondrial trafficking patterns and the mitochondrial membrane potential (mmp) in primary drg neurons treated with physiologically relevant concentrations of palmitate, oleate, and combinations of both fas. primary drg neurons treated with . to micromolar palmitate induced a significant and dose-dependent reduction in the percentage of motile mitochondria. these palmitate treatments also induced a dose-dependent reduction in mitochondrial velocity but did not impact the directionality of mitochondrial movement. alternatively, . to micromolar oleate treatments did not impair the percent of motile mitochondria, the direction of mitochondrial movement, or mitochondrial velocity. since palmitate and oleate have differential effects on mitochondrial motility, we next assessed whether oleate could counter the inhibitory effect of palmitate on mitochondrial trafficking. surprisingly, oleate/palmitate mixtures at ratios of : or : prevented palmitate-induced impairment of mitochondrial trafficking. we assessed the impact of palmitate and oleate on mmp by staining palmitate and oleate-treated drg neurons with tetramethylrhodamine methyl ester. drg neurons treated with micromolar palmitate exhibited an increase in the percentage of depolarized mitochondria while mitochondria in oleate-treated drg neurons retained mmp. interestingly, drg neurons treated with : oleate/palmitate mixtures also maintained polarized mitochondria. these results suggest that saturated and unsaturated fas have a distinct impact on mitochondrial trafficking mechanisms in drg neurons and that equimolar ratios of oleate/palmitate can prevent impairment of mitochondrial trafficking. of g alpha- in m r overexpressed sensory neurons significantly reversed the m r-induced reduction in relative levels of total neurite outgrowth (mean±sd: . ± . (control) vs . ± . (g alpha- knockdown), p< . , n= neurons). further, treatment of m r overexpressing neurons with mt or pirenzepine sequestered g alpha- / proteins at the m r and significantly reversed the impaired cytoskeleton-mediated reduction in total neurite outgrowth (mean±sd: . ± . (control), ± (+ nm mt ), ± . (+ micromolar pirenzepine); n= , and neurons, respectively; for treated groups p value= . vs control by one-way anova). mt and pirenzepine also significantly restored mitochondrial trafficking and abundance of mitochondria in the distal neurites. our findings suggest a novel mechanism in which modulation of m r influences tubulin polymerization, mitochondrial distribution in nerve terminals and controls axonal outgrowth and regeneration. funded by cihr and nih. muscarinic receptors are a group of five g-protein coupled receptors (gpcrs) that are targeted by drugs for the treatment of several human pathophysiological conditions. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) antagonists of the muscarinic acetylcholine type receptor (m r) induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt et al., ) . one of the major cellular effectors activated by gpcrs is extracellular signal-regulated kinase (erk). the erk signaling cascade regulates a variety of cellular processes including growth and proliferation. both g protein and beta-arrestin mediated signaling pathways can lead to erk activation by phosphorylation through different kinases. activated erk in turn can phosphorylate about cellular substrates, thereby mediating diverse functions. in this study, we have analyzed beta-arrestin recruitment, as part of the receptor internalization process induced by agonist/antagonist binding. in addition, we studied phosphorylation of erk by mt and pirenzepine using isoelectric focusing with phospho-erk specific antibodies and a variety of cell-based assays including beta-arrestin and g protein (gnas/gnasl/gnaq/gna /gna /gna ) knockout cell lines. our study revealed that beta-arrestin is recruited to the m r upon mt and pirenzepine treatment. treatment with nm mt and micromolar pirenzepine significantly increased the dual phosphorylation of tyr and tyr residues of erk / in primary rat sensory neurons (p< . ) in comparison to muscarinic agonist carbachol ( micromolar) and general antagonist atropine ( micromolar). we have identified multiple distinct phosphorylation events on the m r by isoelectric focusing that are specific to mt and pirenzepine induction. further, we have shown that mt and pirenzepine-mediated erk phosphorylation is dependent on both g protein and beta-arrestin recruitment to m r. finally, we reveal that increased erk phosphorylation by mt and pirenzepine significantly (p= . and . , respectively) increased phosphorylation of camp responsive element binding protein (creb) at ser . these results show for the first time that antagonists of the m r can activate the erk signaling pathway and possibly drive phenotypic change in adult sensory neurons. funded by cihr # mop- . charcot-marie-tooth disease (cmt) is representative of inherited neuropathies affecting an estimated in people. the immense advances in gene discovery gained from next-generation sequencing (ngs) projects have revealed the extent of cmt's genetic heterogeneity, with over loci already identified. this knowledge is rapidly translated into clinical comprehensive gene testing panels, often containing over genes. such a large genomic space will invariantly yield variants of uncertain clinical significance (vus) in nearly any person tested. this rise of the number of vus creates major challenges for genetic counseling. in addition, less individual mutations in already known genes are being published as the academic merit is decreasing, and most such testing now happens in clinical laboratories. we propose to capture more of this data in the cmt field to gain a more complete collection of alleles in cmt genes, ideally in conjunction with detailed phenotypic data. this represents a rational approach to eventually reduce the number of vus. thus, we have created a unique, community-driven variant database for cmt researchers and clinicians. the inherited neuropathy variant browser provides simple, user-friendly access to currently reported cmt variation, including patient-level genotypic and phenotypic information. we have also designed an interactive rating system of genetic variation to assist the community with interpretation of vus. for the initial release, we have collected genetic variation, along with genotypic and phenotypic data when available, from published literature, clinical lab reports, and our in-house database. we highly encourage new submissions of not only observed pathogenic variation, but also variation of unknown significance. the goal is to provide a platform for the cmt community to store, share, and discuss genetic data in order to resolve variation of uncertain significance as a joint-effort. with active participation, we aim to provide the community with a more complete mutational spectrum in cmt genes to assist allelic interpretation and patient diagnosis. neurofilaments are strictly neuron-specific intermediate filaments crucial for maintaining axonal architecture. pathogenic mutations in nefl, which encodes the light chain of neurofilament, cause dominantly and recessively inherited charcot-marie-tooth neuropathy (cmt). the nefl-associated neuropathy can be either axonal (type e) or demyelinating (type f). most pathogenic nefl variants are dominantly inherited missense mutations, which are thought to cause disease by inducing nefl aggregation, leading to the disruption of axonal transport. however, investigation of disease mechanisms caused by the mutations has been complicated by the neuronal specificity of nefl. we identified a homozygous nefl variant c. c>t predicting a nonsense change p.arg * in a patient with early-onset cmt. to elucidate the disease mechanism, we used pluripotent stem cells, reprogrammed from patient's skin fibroblasts, to differentiate patient-specific spinal motor neurons. the motor neurons revealed a near complete loss of nefl mrna, and absence of nefl protein. our results establish that nefl is not essential for the development of human nervous system but its absence causes progressive axonal neuropathy. we currently profile the transcriptomic alterations of the motor neurons lacking nefl using single cell rna sequencing to identify compensatory pathways. friedreich's ataxia (frda) is the most common autosomic recessive ataxia, due to a trinucleotide expansion within the frataxin gene. within the wide phenotype, frda patients present also with an axonal neuropathy whose pathological mechanisms are not completely known. eight patients ( women, mean age . yrs, range - ) with genetically confirmed frda underwent neurophysiological and nerve ultrasound evaluation at four limbs bilaterally. echogenicity and cross-sectional area (csa) of median, ulnar, radial, peroneal, tibial, and sural nerves were recorded. mr neurography and diffusion tensor imaging (dti) analysis were performed in one patient; fractional anisotropy (fa), radial (rd) and axial (ad) diffusivity of median, radial and ulnar nerve were calculated at proximal, intermediate and distal sites. all patients presented with sensory axonal neuropathy. seven patients ( %) presented with increased csa of median and ulnar nerves at arm and axilla. mean median nerve csa at mid-upper arm was . mm (normal values < mm ), mean ulnar nerve csa at mid-upper arm was . mm (normal values < mm ). mean median nerve csa at axilla was . mm (normal values < mm ), mean ulnar nerve csa at axilla was . mm (normal values < mm ). mr neurography (performed in one patient) confirmed diffuse swelling and signal hyperintensity of median and ulnar nerves at the arm and dti analysis showed abnormal values of fa, ad and rd along the whole course of evaluated nerves thus suggesting a wide alteration of nerves structure. frda patients presented with an axonal neuropathy characterized, at ultrasound, by a nerve enlargement strictly limited to mid-upper limbs in all patients, findings that cannot be solely explained by a dying-back axonopathy, as suggested by several authors. neither a dorsal root ganglia neural loss could explain by itself our findings, because a diffuse csa reduction would have been expected. on the whole, these findings represent a peculiar feature in frda, but its pathophysiologic meaning remains unclear. inherited peripheral neuropathies are an important health concern for which there is currently no disease-modifying therapy. dogs are affected by a variety of peripheral neuropathies that are breed-specific, indicating a strong genetic component. the use of in-bred populations, such as pure-bred dogs, is advantageous for genetic dissection of disease. acquired peripheral neuropathy (apn) is an inherited late-onset generalized polyneuropathy with high prevalence in labrador retrievers. the most prominent features of apn, laryngeal paralysis and pelvic limb weakness, are associated with the longest peripheral motor nerves in the dog. the pathologic features of apn are similar to human peripheral neuropathy. our aim is to understand the genetic and pathologic features of apn for development of this condition as a naturally occurring large animal model for human disease. we performed a genome-wide association study (gwas) and short-read high-depth whole genome sequencing (wgs) to investigate the genetic underpinning of apn in the labrador. our gwas data indicates that apn is an autosomal dominant disease. the initial analysis from the wgs study resulted in a potential causal variant with an autosomal dominant pattern; this variant is associated with an axonal gene. the neuropathologic progression and histologic features of apn are poorly defined. using genetic markers from our gwas study, we are able to confidently identify labradors with pre-clinical apn, from which nerve biopsies can be obtained. preliminary analysis of biopsies from labradors suggest apn is an axonopathy. further histologic data from preclinical and symptomatic dogs is being obtained to further define the pathogenesis in this model. mutations in morc lead to an axonal form of neuropathy (cmt z). to date, nine families have been published with mutations in the morc gene, showing that this gene is frequently involved in cmt. while the recent genetic data clearly established the causative role of morc in cmt z, its phenotypic consequences in patients and role in neuronal biology remains to be clarified. therefore, we aim to look for altered genetic and biochemical pathways with a transcriptomic approach in order to investigate the role of morc in hereditary peripheral neuropathy. we have performed transcriptomic analysis using a human gene expression microarray (v x k, agilent technologies), in three hek- t cell lines: control, morc knock-down (kd) and the overexpression of the most common morc mutation, the p.r w (np_ ) (kr). differential gene expression assessment was carried out using limma moderated t-statistics. standard analysis techniques perform one test for each gene. thus, for each gene, a t-test statistic is reported together with its corresponding p-value. in this analysis we have used the conventional multiple testing p-value correction procedures proposed by benjamini hochberg to derive adjusted p-values. the preliminary results reveals that kd shows up-regulated genes involved in transmission of nerve impulse and cilium metabolism, suggesting that morc might act at this level in the peripheral nervous system. otherwise, kr shows a major alteration of main axonal metabolic pathways, including the overexpression of genes related to the generation of neuronal action potential, transport through the axon and its targeting in synapse formation. kr also shows a marked alteration of gene expression related to organization, assembly and cilium movement and with the axonemal dynein complex assembly. this study provides an important step towards understanding the pathomechanism underlying to morc p.r w and its role in cmt z. aifm encodes a mitochondria associated apoptosis inducing factor. mutations in aifm lead to a wide spectrum of neurodegenerative disorders: cowchock syndrome; a combined oxidative phosphorylation deficiency (coxpd ) with severe encephalomyopathy; x-linked deafness with peripheral sensory neuropathy; spondyloepimetaphyseal dysplasia (semd) with mental retardation; and an infantile motor neuron disease. previous studies showed severe defects in mitochondrial metabolism, related to redox function, mitochondrial fragmentation, and respiratory deficiencies. in addition, some mutations impair the protein expression of aifm and cause an increase in caspase-independent apoptosis. by targeted next-generation sequencing, we detected the aifm c. c>t (p.phe ser), in a year-boy. this mutation was confirmed in his year-old affected brother. electromyography and nerve conduction velocities studies revealed an axonal polyneuropathy with exclusive involvement of motor fibers, with an early childhood-onset. both children currently show normal cognitive and cranial nerves functions. the in silico structural modeling of human aifm showed that the mutation of a phenylalanine to serine at position disrupts the hydrophobic interaction between phe and pro , and consequently, it destabilizes an alpha-loop domain. cartoon of protein superposition between two different human aifm structures suggest that a lack of constraints in this region could affect the interaction between - helix and the - -hairpin regions, a very important stage for the functional activity of the aifm protein. patient-derived fibroblasts were used to investigate the pathological effect of the p.phe ser mutation: fibroblasts from patients show a similar mrna but different protein expression of aifm compared to healthy control fibroblasts. however, they have an aberrant morphology, from fibroblastic to polygonal shape, and they are larger than control fibroblast; mitochondria from mutant fibroblasts are markedly fragmented compared to controls; the viability of the patient's fibroblasts is lower, but it does not correlate with an increase in apoptosis. instead, it seems to be caused by an increase in the expression of genes activating the senescent program, like p and p . our study confirms that variable effect of different mutations on the protein function may contribute to the clinical variability observed in aifm patients. funds: isciii (pi / ); fundació per amor a l' art. sango k , takaku s , niimi n , yako h . diabetic neuropathy project, tokyo metropolitan institute of medical science, tokyo, japan. coculture models of neurons and schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained from the primary culture with embryonic or neonatal animals. because it is recognized that some biological properties of both neurons and schwann cells change with maturation and aging, culture systems of adult animal cells appear to mimic peripheral nerve degeneration and regeneration better than those of immature animal cells. we have established spontaneously immortalized schwann cell lines from long-term cultures of adult fischer rat peripheral nerves. one of these cell lines, designated ifrs , has been shown to retain distinct schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers, synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed pc cells. our current investigation focuses on the establishment of the coculture system of ifrs cells and nsc- motor neuron-like cells. nsc- cells were seeded at a low density ( x /ml) and maintained for a week in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (bdnf, ng/ml). after overnight exposure to mitomycin c (mmc, micro g/ml), nsc- cells were cocultured with ifrs cells ( x /ml) and maintained in serum-containing medium supplemented with bdnf ( ng/ml), ciliary neurotrophic factor (cntf, ng/ml) and coenzyme q ( micro m). under this culture condition, overgrowth of nsc- cells was prevented and gradual movement of ifrs cells toward the neurites emerging from nsc- cell bodies was observed. double-immunofluorescence staining carried out at day of the coculture showed myelin protein zero-immunoreactive ifrs cells surrounding the beta iii tubulin-immunoreactive neurites. this coculture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g. amyotrophic lateral sclerosis, charcot-marie-tooth diseases and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them. santos pp , torezani gs , maciero l , pagliarini lfd , romão tt , abunahman ms , ferreira is , bittar c , pupe c , nascimento ojm . universidade federal fluminense (uff), rio de janeiro, brazil. zika virus (zikv) is a flavivirus related to dengue, yellow fever and west nile viruses, and has been recently associated to the occurrence of neurological complications in children and adults. previous studies have linked zikv to the development of guillain-barré syndrome (gbs), myelitis, meningoencephalitis and ophthalmological manifestations in adults. guillain-barré syndrome (gbs) encompasses a spectrum of post-infectious neuropathies characterized by different distributions of weakness and sensory impairment. serum anti-ganglioside antibodies are often found and are related to different clinical patterns. recently we have encountered patients in rio de janeiro, brazil, with distal edema in lower limbs, acute weakness, pain and sensory disturbances during the acute stage of an acute febrile exanthematous illness. the symptoms persist for up to days, with complete resolution afterwards, without specific therapy. blood concentrations of muscle enzymes show normal values, and electromyography and nerve conduction studies (emg/ncs) are usually unremarkable. of note, all cases have had positive rt-pcr for zika virus, indicating an illness that occurred during the viremic phase of this arbovirus infection, and complete recovery within the expected timeframe for the resolution of the systemic viremia. there is a subset of patients who developed acute weakness very early after the initial viral symptoms, with clinical, laboratorial and electrophysiological findings that substantially differ from gbs. we describe three cases with similar features suggestive of an acute infective polyneuritis (aipn). one might hypothesize that zikv might lead to a direct neurotropic injury, significant enough to cause weakness and sensory complaints, but not severe enough to cause permanent damage, resolving in conjunction with decreasing blood viremia. we consider that these patients differ from classical gbs because their illness begins during the acute febrile stage of an infective illness and the clinical course is more rapid leading to complete resolution in a few days. savransky a , mozzoni j , massaro sanchez mp , reisin r , monges ms . department of neurology, hospital de pediatria j.p. garrahan, buenos aires, argentine; department of physical therapy, hospital de pediatria j. p garrahan, buenos aires, argentine; department of neurology, britain hospital, buenos aires, argentine. our objective is to describe a series of children with guillain barré syndrome (gbs) included in the igos protocol. as part of the igos multicenter protocol, pediatric patients meeting gbs diagnostic criteria, who consulted within the first weeks of symptom onset and had parental consent to participate in the study were included. patients were also offered to participate in the extended -year protocol. all patients were evaluated according to the igos protocol. patients were recruited between october and june . twenty-four patients, eight girls, participated. ages ranged from months to years (mean . years). all patients agreed to participate in the extended protocol, which was completed by of them. eight patients completed the -year follow-up and are still under evaluation. five patients were lost to follow-up.twenty-two had the classic variant of gbs and two miller fisher. radicular pain in the back or lower limbs was reported by %. twenty-three patients underwent lumbar puncture and albumin-cytological dissociation was found in . in all cases, csf was stored for proteomic studies. all patients underwent emg showing aidp in , and amsan and aman in each. one patient with aidp developed to cidp. in patients full-spine mri was performed and cauda equina enhancement was found in every case. three patients required ucip, two with invasive and one non-invasive ventilation. all patients were treated with gammaglobulin, with a second dose at weeks in cases with a poor response. all patients who followed the protocol were evaluated with the gbs disability score. median score was at baseline and between and at the -year assessment. we describe a series of children with gbs as a part of an international protocol including patients of different ages. pain was a frequent and early symptom and could be determined despite the young age of our patients. most patients fully recovered. we were invited to participate in igos kids to better assess this age group. saysavath k , somchit v , t. umapathi . mittaphab hospital, vientiane, lao pdr; setthathilath hospital vientiane; national neuroscience institute, singapore, singapore. laos people democratic republic is a country of . million people in south east asia. largely agricultural, its capital and metropolis is vientiane. adult neurological services are concentrated at mittaphab hospital, vientiane serviced by three neurologists. on the average about six cases of guillain-barré syndrome (gbs) are seen per year. it is believed that most patients do not seek medical attention. cases appear to cluster during the rainy season. most patients present late, often at the second week of illness when recovery is unapparent after seeking treatment from traditional medicine doctors and at district hospitals. a substantial number of patients seek treatment at hospitals across the border, in neighboring provinces of thailand. common antecedent symptoms are viral prodrome and diarrhea. miller fisher syndrome appears to be rare, possibly because of the mild deficits that do not prompt patients to seek medical attention. diagnosis is made largely from clinical features and from spinal fluid analysis. nerve conduction studies are not available. patients are often treated with steroids by internists. intravenous immunoglobulin and plasma exchange are not available. common complications include pneumonia, autonomic dysfunction (fluctuating blood pressure), pressure sores and depression. patients who develop respiratory failure are nursed at a twelve-bedded intensive care units. plans are afoot to set up a prospective gbs database, systematically study antecedent infections, including of flaviviruses, and develop low-volume plasma exchange as a feasible therapeutic modality. a gene therapy approach for treating cmt c neuropathy schiza n , markoullis k , richter j , tryfonos c , kagiava a , sargiannidou i , christodoulou c , kleopa ka , . neuroscience laboratory; department of molecular virology; neurology clinics, cyprus institute of neurology and genetics and cyprus school of molecular medicine, nicosia, cyprus. charcot-marie-tooth type c (cmt c) is the most frequent form among recessively inherited demyelinating neuropathies and results from mutations in the sh tc /kiaa gene. sh tc mutations cause loss of function of the sh tc protein suggesting that gene replacement therapy may be useful for treating cmt c. sh tc −/− mice develop all major aspects of the human pathology including early onset progressive peripheral neuropathy with hypo-and demyelination along with decreased motor and sensory nerve conduction velocities, offering a relevant model for testing treatments for cmt c. in order to develop a gene replacement strategy for cmt c, we generated a novel lentiviral vector, lv-mpz-sh tc .myc, to drive expression of the human sh tc cdna under the control of the myelin protein zero (mpz/ p ) promoter specifically in myelinating schwann cells. a c-terminus myc tag was included to facilitate expression analysis. a control vector (mock) was also produced in which the sh tc cdna was replaced by the egfp reporter gene. we first confirmed expression of hsh tc in hela cells transfected with the pcdna -cmv-sh tc .myc vector. immunofluorescence analysis confirmed a strong expression of sh tc specifically at the plasma membrane with additional localization in a dotted pattern intracellularly. for in vivo gene delivery we used both intraneural and intrathecal injections of the lv-mpz-sh tc .myc vector in -week to -month old sh tc −/− mice. expression of virally delivered hsh tc was assessed weeks after injection. immunofluorescence analysis showed hsh tc immunoreactivity in perinuclear schwann cell cytoplasm in sciatic nerve teased fibers of sh tc −/− mice following both intraneural and intrathecal delivery, while lumbar intrathecal gene delivery resulted additionally in expression of hsh tc in the lumbar roots. real time pcr analysis confirmed hsh tc mrna expression in both lumbar roots and sciatic nerves. thus, we have developed a novel lentiviral vector for schwann cell targeted gene delivery to treat cmt c and for testing possible therapeutic effects in the mouse model of the disease. hereditary neuropathies are a group of disorders which are characterised by the systemic impairment of peripheral nerves. more than neuropathies are associated with causative gene defects [ ]. charcot marie tooth (cmt a) neuropathy is the most frequent hereditary neuropathy, triggered by a mutation in the peripheral myelin protein gene (pmp ). cmt a leads to a primary loss of myelin sheath and afterwards to a degeneration of axons [ ] . symptoms appear with the degeneration of axons, whereas demyelination is thought to be largely asymptomatic. for that reason, we investigate in the mechanisms of axonal degeneration. for our analysis, we used purified axoplasma without detectable myelin proteins of the sciatic nerves of weeks old pmp -c mice. in this early stage of the axonal degeneration sarm was significantly increased. the nad + concentration in the axoplasma was dramatically reduced. this correlates to the previous finding that sarm promotes axonal degeneration by cleavage of nad + . additional studies showed an increase of the nad + -dependent axonal protective factor sirt in pmp -c sciatic nerves. nmnat- , known as the active component of the wallerian degeneration slow gene, was unaffected in axoplasma of pmp -c sciatic nerves. summarised, these results indicate that the pathway of sarm , nad + and sirt may play a critical role in axonal degeneration in neuropathy. we report two unrelated czech patients with cmt b , both sporadic cases in the family. patient is a year old man with congenital glaucoma after ophthalmological surgeries. his early motor development was normal. at the age of years parents noted gait problems, first neurological examination was at the age of years, when emg showed diffuse motor and sensory neuropathy with severely decreased ncv ( - m/s). he developed foot deformities (pes cavovarus) and underwent corrective orthopedic surgeries at the age of years. after several dna tests for demyelinating cmt the sfb gene was sanger sequenced and a novel missense mutation p.ile asn was found in homozygous state in the patient and in heterozygous state in both parents. the patient was later tested also by ngs of a panel of all genes to be causal for hereditary neuropathies and no other potentially causal variants were detected. patient is years old man, with normal early motor development. at the age of years parents noted gait problems with distal leg weakness which progressed into distal leg plegia at the age of years. hand weakness was noticed since the age of years. he has severe atrophies of distal muscles of all extremities, is self ambulant. at the age of , emg showed unrecordable responses from nerves of lower limbs and ncv was measurable only on ulnar nerve and was m/s. at the age of years increased intraocular pressure was diagnosed and he uses anti glaucoma eye drops. after many single gene tests for demyelinating cmt, we used ngs of a panel of genes known to be causal for hereditary neuropathies and two novel heterozygous, probably pathogenic variants affecting invariant splice sites were detected: c. - a>g and c. - _ del, both confirmed by sanger sequencing. the first variant is also in the father, but the second is probably de-novo (not detected in parents, despite correct parentity). sekiguchi y , kikuchi si , konno si , sekiguchi m . department of orthopaedic surgery, fukushima medical university school of medicine, fukushima, japan. carpal tunnel syndrome (cts) is the most common entrapment neuropathy. ultrasonography can be used to detect anatomic changes in cts. more recently, it has been shown that doppler ultrasonography can detect increased intraneural blood flow in cts. the purpose of this study was to determine the most suitable finding of pre-and postoperation in cts by ultrasonography. a total of wrists of patients with nerve conduction study (ncs) proven cts were evaluated with ultrasonography. we measured the median nerve's cross-sectional area (csa) and intraneural blood flow of median nerve by ultrasonography. the correlation between these ultrasonographic measurements, ncs severity and duration of clinical cts symptoms was analyzed. the csa (mean, mm ) was no significantly reduction after successful carpal tunnel release. morphologic median nerve changes may persist for a longer period regardless of successful surgery and clinical improvement. however, intraneural blood flow is increasing after successful carpal tunnel release. we conclude that doppler ultrasonography results strongly correlate with post operated cts improvement. hence doppler ultrasonography is a useful method for functional improvement of pre-and post cts operation. senger jl , chan km , olson jl , webber ca . university of alberta, edmonton, canada. the beneficial effects of a preinjury crush conditioning lesion (cl) on peripheral nerve regeneration is well-documented in animal models. no human studies have been attempted to date, given the ethical dilemma of deliberately injuring an intact nerve, and the difficulty in predicting the timing of a nerve injury. recent studies demonstrate that hour of electrical stimulation (es) produces effects similar to cl in neuronal cultures. this, coupled with a surgical environment favoring nerve transfers, in which an intact nerve is deliberately cut to reinnervated a denervated muscle, means that es may be clinically translatable to enhance regeneration. this study hypothesizes that es prior to nerve injury will enhance nerve regeneration. twelve sprague-dawley rats were divided into four groups based on conditioning-type to the mid-common peroneal (cp) nerve: es ( ), crush ( ), sham-es ( ), and naïve ( ). one week following conditioning, they underwent a cut/coaptation of the cp nerve at the sciatic trifurcation. post-cut day , nerves and dorsal root ganglia (drgs) were collected. axonal counts of nerves stained with nf- revealed similar regeneration between es and crush ( . vs. . mm, p= . ) that was superior to sham-stimulation ( . mm) or no-conditioning ( . mm, p< . ). a greater number of axons at the distal tip were present in animals that received either type of conditioning compared to the unconditioned cohorts. drgs were stained with neuronal injury marker growth associated factor- (gap- ), and satellite cell glial cells with glial fibrillary acidic protein (gfap). significant increase in gap- expression at three days was observed in es and crush cohorts compared to sham or naïve (p< . ) cohorts. the satellite glial cells of es and crush conditioning showed a significant increase in gfap expression ( . % and . % respectively) compared to sham ( . %) and naïve ( . %) drgs. by demonstrating similar improvements in axon regeneration, this proof of principle project suggests that es conditioning may produce regenerative outcomes comparable to the classical crush injury model. in turn, this suggests that es may be a promising method for delivering conditioning lesions in clinical trials for conditioning nerves prior to surgical intervention. we report a unique case of newly developed waldenstrom's macroglobulinemia (wm) in a patient with chronic inflammatory demyelinating polyneuropathy (cidp) with antibodies against myelin-associated glycoprotein (mag) and sulfatide who was undergoing treatment with intravenous immunoglobulines (iv-ig). subsequent rituximab infusions did not have a positive impact. patients with wm can develop demyelinating and axonal polyneuropathies and few patients have anti-mag and/or anti-sulfatide antibodies. anti-mag antibodies ( % of wm) are associated with sensorimotor axon loss and demyelination and anti-sulfatide ( % of wm) with sensory axonal loss. rarely, both antibodies can be present, with a more severe clinical phenotype. cidp associated with anti-mag and anti-sulfatide antibodies can represent independent entities, not associated with wm. there are no reports to date of patients with cidp associated with anti-mag and anti-sulfatide antibodies that developed wm during immunomodulatory therapy with iv-ig. in addition, subsequent rituximab infusions after the iv-ig were stopped have not been proven beneficial, as has been previously reported for anti-mag cidp patients. seventy-six year old right-handed gentleman presented with persistent numbness in his left foot, three months following artificial disc placement in his lumbar spine. gradually he developed sensory ataxia. no radicular signs were present on exam or impingement on serial spine mri's. ncs/emg studies were consistent with a cidp variant with severely prolonged distal motor latencies. serum anti-mag and anti-sulfatide antibodies were elevated. chronic therapy with iv-ig was able to partially stabilize the symptoms; however, six years later he newly developed wm. subsequent infusions with rituximab, after iv-ig was stopped, did not improve the clinical picture or the ncs/emg findings. wm can newly develop in an autoimmune setting, such as cidp associated with anti-mag and anti-sulfatide antibodies. in this particular case, there was an ongoing immunomodulatory therapy for our cidp patient, as he had monthly iv-ig infusions. this may reflect a possible induction of pathological b cell clone proliferation during the iv-ig treatment. subsequent rituximab infusions, after the iv-ig was stopped, did not improve the symptoms or the demyelination features on ncs/emg. he continues to be symptomatic despite efforts. charcot-marie-tooth disease (cmt) is an inherited neuropathy without known cure (prevalance: : ). duplication of the gene encoding the peripheral myelin protein of kda (pmp ) underlies the most common subtype cmt a. severely affected cmt patients suffer from sensory and motor symptoms with wheelchair-boundness. the clinical phenotype is highly variable and is determined by the amount of axonal loss, but the molecular mechanisms of the disturbed neuron-glia interaction are poorly understood. risk factors have not been investigated. therefore, cmt-net, a german network funded by the german ministry of education and research (bmbf, bonn, germany) includes interdisciplinary expertise from molecular biology, neurology, neuropathology and human genetics in order to identify genetic and non-genetic risk factors of disease severity of cmt by: (i) examining the mechanisms of the disturbed axon-glia-interaction and neuronal vulnerability, (ii) identification of genetic modifiers and (iii) novel therapeutic targets, (iv) validating outcome measures in children and adults, (v) establishing a biobank and (vi) exploring the disease burden via an internationally harmonised patient registry. cmt-net includes three service structures cmt-net will focus on cmt a, but also includes rarer subforms. we will provide the scientific basis for the development of translational approaches to therapy in patients. our approach bridges cutting edge molecular screening techniques, transgenic animal models of altered axon-glia interactions (fly, chick, mouse, rat), state-of-the-art genomic technologies and human patient trials in order to understand and treat the disease aggravation in cmt. sezer g , tekol y , sezer z , . erciyes university, betül ziya eren genome and stem cell centre, kayseri, turkey; erciyes university, school of medicine, pharmacology department, kayseri, turkey; erciyes university, good clinical practice and research centre, kayseri, turkey. analgesic effects of antidepressant drugs are well known for a long time, however, their systemic side effects limit their usage as an analgesics. venlafaxine is an antidepressant drug that has different structure. our purpose was to investigate whether systemic analgesic effect has been proved drug, venlafaxine, has local peripheral antinociceptive action. we applied vanlafaxine ( μl , , , μg) to male, sprague-dawley rats' paws by intraplantar injection and also by intraperitoneal route ( , , , mg/kg) in formalin test, a model for acute and tonic pain. we also pretreated another groups of rats with mg/kg naloxone (opioid receptor antagonist), mg/kg cpt (adenosine a receptor antagonist) or saline (ip.) before μg/paw venlafaxine injection. to check the effect is local or not, we determined the blood levels of venlafaxine in at different time points after both the local and systemic applications by gc-ms method. datas were expressed as number of flinches and total time for biting/licking of the injected paw over phase ( - min) or phase ( - min) and analyzed using the student's t-test. venlafaxine induced antinociception at , and μg concentrations by the local peripheral application and at , mg/kg doses by the systemic application in formalin test and the effects were comparable. pretreatment with naloxone diminished the effect of venlafaxine in the both phases, however, it was not statistically significant. pretreatment with cpt decreased venlafaxine induced antinociception only in phase . neither naloxone nor cpt changed formalin induced nociceptive behaviors alone. this is the first that determines the peripheral antinociceptive actions of venlafaxine in rat formalin test. with roles of opioid and adenosine a receptors in this action. our results suggest that venlafaxine has local peripheral antinociceptive effect and such an activity may led to trials for to use this drug as a cream-gel formulation for analgesia in clinics in the future. topical application might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects. shah a , hoffman em , klein cj , staff np . mayo clinic, rochester, usa. cipn is a common dose-limiting complication for patients with cancer. the long-term disease burden of cipn is compounded by increasing cancer survivorship, yet there are minimal data on long term outcomes following onset of cipn, especially in population-based studies. we utilized the rochester epidemiology project to examine incidence and disease burden of cipn among individuals of olmsted county, minnesota with neurotoxic chemotherapy exposure between and . clinical records were queried for the presence of neuropathic signs, symptoms and icd- diagnostic codes as well as for patient provided information on impairment with activities of daily living and use of pain medications. a total of individuals with incident exposure to neurotoxic chemotherapy agents between and were identified. based on aan criteria for identifying peripheral neuropathy, ( . %) of these individuals were determined to have cipn, while ( . %) controls did not. the median time from incident exposure to reported symptom onset was days (iqr . - ). patients with cipn received a neuropathy icd- diagnosis in merely cases ( . %). median survival following incident chemotherapy exposure among all cases and controls was . years with a significantly longer mean survival in cases with cipn as compared to that of controls ( . years vs. . years, p< . ). in addition to acute effects in cipn, individuals surviving greater than years following exposure to neurotoxic chemotherapy continue to self-report increased symptoms of numbness (or . , % ci . - . ) and pain (or . , % ci . . . ) of the extremities. through utilization of patient provided information, our study was able to collect data on long-term impairment associated with previous history of exposure to neurotoxic chemotherapy. our results are consistent with previous reports of the high incidence of cipn in the first two years following incident exposure. additionally, our results provide evidence of high incidence of cipn independent of individual chemotherapeutic agent used. additionally, our results indicate icd- -cm diagnostic code attribution may dramatically underestimate the magnitude of cipn. increased survival following exposure to neurotoxic chemotherapy and its long-term disease burden necessitates further study of among survivors. the utility of quantitative muscle ultrasound as a marker of disease severity in charcot-marie-tooth (cmt) disease subtypes was investigated. muscle ultrasound was prospectively performed on individual muscles from cmt patients ( cmt a, cmtx , cmt a) and compared to muscles from age and gender-matched controls. muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (fdi) and tibialis anterior (ta charcot-marie-tooth (cmt) disease is the most frequent inherited peripheral neuropathy, and there is currently no available cure. the most common subtype of cmt, cmt a, is completely associated with duplication of the pmp gene, which encodes peripheral myelin protein of schwann cells. previous studies of cmt a mainly relied on rodent models, and it is not yet clear how pmp overexpression leads to the phenotype in patients. based on induced pluripotent stem cell (ipsc) technology, we herein developed a brand new in vitro cell model of cmt a, called cmt a-hipscs, in the hopes of simulating the developmental progress of the disease and gaining new insights into its pathogenesis. here, we efficiently derived ncscs from cmt a-ipscs and assessed the potential of the isolated cmt a-neural crest stem cells (ncscs) to differentiate into peripheral neurons and schwann cells using defined media. we found that, unlike normal control ncscs, cmt a-ncscs rarely generated schwann cells. instead, cmt a-ncscs produced numerous endoneurial fibroblasts in the schwann cell differentiation system. we further established a pmp -overexpressing ipsc model, and obtained similar results when pmp -ncscs were subjected to schwann cell differentiation. these results suggest that the development of schwann cells in cmt a patients is interrupted by the duplication of pmp . with the exception of the demyelination-remyelination process, developmental disabilities of schwann cells should be considered as an underlying cause of cmt a. shimoi t , yamada t . international university of health and welfare, tochigi, japan, cmt japan, tokyo, japan. charcot-marie-tooth (cmt) disease is the most common hereditary motor and sensory neuropathy. our preliminary report suggests that a certain cmt patient has the recruitment disorder of motor units during muscle fatigue and this disorder may be a factor of "super fatigability" in motor neuropathy patients. if the "super fatigability" occurs, we would expect patients with this characteristic to become slower in recovery from muscle fatigue than patients without this characteristic. in order to verify this hypothesis, we measured characteristic of recovery from muscle fatigue in charcot-marie-tooth patients with electromyographic study. twenty three participants were asked to maintain their % of maximal voluntary isometric contraction (mvc) of elbow flexor until exhaustion as the fatigue exercise. in addition, the participants asked to perform s of their % of mvc at , , , , , , , , , s after the fatigue exercise as recovery tasks. the surface emg (semg) signals of biceps brachii muscle were determined during the exercise and tasks. muscle force, median power frequency (mdpf) and the root mean square of semg amplitude (rms) were used as objective parameters of muscle fatigue. borg scale was used as a subjective parameter of muscle fatigue. six of twenty three participants showed significant decrease of rms during the fatigue exercise. in consideration of this result, we compared alteration of mdpf in recovery task between six participants with decrease of rms (abnormal group) and seventeen participants with increase of rms (normal group). as the result, the abnormal group had at least s as the recovery time from muscle fatigue in contrast with s of normal group. the recovery time from muscle fatigue in subjective parameter was shorter than the time in objective parameters in each group. our data support the "super fatigability" hypothesis. and that hypothesis may induce "hidden muscle fatigue". comparison between complex regional pain syndrome type and based on electrophysiologic, imaging and clinical findings shin jy , moon jy , sung jj . seoul national university hospital, seoul, republic of korea. complex regional pain syndrome (crps) is a constant regional neuropathic pain characterized by various kinds of motor, sensory, and autonomic changes. conventionally, the crps is divided into type i and ii according to the absence and presence of nerve injury. but the pathogenesis of crps is not fully understood yet. and there is still no systematic comparative study between crps type i and ii. we compared between crps type i and ii using multimodal approaches including electrophysiologic, imaging, and clinical findings. the patients ( type i and type ii) diagnosed with crps using the international association for the study of pain (iasp) diagnostic criteria were included. type i and ii were divided by electromyography and nerve conduction study. we obtained clinical information such as continuing pain, allodynia, hyperalgesia, edema, temperature, skin color, sweating, trophic change from patients. all patients were evaluated by bone scan, thermography, quantitative sudomotor axon reflex test (qsart), quantitative somatosensory test (qst). the ratio of qsart and temperature threshold abnormality in type ii was higher compared to type i ( . % vs . %, . % vs . % respectively, p = . and . ), among clinical symptoms, sweating change significantly high in type ii compared to type i ( . % vs . %, p = . ). other electrophysiologic and imaging, clinical findings were not significantly different in both type. in this study, we identified that crps type i and ii are distinguished not only by the nerve injury but also by the sudomotor function, and qsart can serve as a good technique to differentiate between crps type i and ii. it is estimated that there are two distinct pathogenesis in crps. our results may be helpful to diagnose crps correctly and understand the pathogenesis of crps. sjogren syndrome (ss) is an autoimmune inflammatory disorder of exocrine glands resulting in xerophthalmia and xerostomia. ross syndrome is a rare entity characterized by tonic pupil, hyporeflexia, and segmental anhidrosis. we present a -year-old hispanic woman with debilitating sensory and autonomic neuropathies, and persistently elevated anti-ss-a and anti-ss-b antibodies, without the classic sicca complex. she initially developed diarrhea and an ear infection, then felt toe and later leg numbness, which eventually spread to her cheeks and tongue, over few months. four years later, her thumbs and index fingers started tingling. also, she developed orthostatic lightheadedness, tachycardia, segmental hypohidrosis of the right abdomen, and hyperhidrosis of the remaining trunk, intermittent erythema, chronic diarrhea, and a -pound weight loss. her exam demonstrated orthostatic hypotension, bilateral tonic pupils and light-near dissociation, sectoral palsy of the right iris sphincter, stocking-distribution diminished sensation to all modalities, pseudoathetosis, areflexia, dysmetria, intention tremor, romberg, and sensory gait ataxia. mri of neuraxis demonstrated t -weighted hyperintensity in the dorsal spinal cord from c to the lower thoracic level, with mild atrophy. electrodiagnostic testing was consistent with moderate-to-severe, length-dependent, asymmetric, sensory polyganglionopathies. csf showed oligoclonal bands. serology showed elevated antinuclear antibody ( : , reference < : ), ss-a ( , reference < u), ss-b ( , reference < u), and rheumatoid factor ( , reference < iu/ml) titers. the remaining workup was negative for infection (syphilis, hiv, htlv, hepatitis, lyme disease), paraneoplastic syndrome (anti-hu and ganglionic nicotinic acetylcholine receptor antibodies), pyridoxine intoxication, malignancy (chest/abdomen/pelvis ct, breast ultrasound, axillary lymph node flow cytometry, colon and esophagus biopsy), celiac disease, vitamin deficiency, autoimmune disease (anti-aquaporin and anti-gq b antibodies), and adrenoleukodystrophy. the patient received ivig and steroid with some gait improvement and currently takes mycophenolate. midodrine and fludrocortisone resolved her dizziness. this case highlights an important, treatable sensory ganglionopathy and systemic autonomic neuropathy due to sjogren syndrome, and illustrates the overlapping clinical triad of ross syndrome, which may guide future management. siles am , , assylbekova d , , diaz-manera j , , rojas-garcia r , , cortes e , , gallardo e , , illa i , , querol l , . neuromuscular diseases unit, neurology department, hospital de la santa creu i sant pau, univeristat autònoma de barcelona, barcelona, spain; centro para la investigación biomédica en red en enfermedades raras (ciberer), madrid, spain. inflammatory neuropathies are a heterogeneous group of peripheral nerve diseases that respond to immune-therapies. chronic inflammatory polyradiculoneuropathy (cidp) and multifocal motor neuropathy (mmn) are two chronic inflammatory neuropathies responding to intravenous immunoglobulins (ivig). b lymphocytes are involved in their pathogenesis. while widely used in clinical practice, ivig's mechanism(s) remain not completely understood. ivig are reported to lead to b-cell anergy and to increase regulatory t-cell function and frequency. regulatory b cells (bregs) are a rare subset of b lymphocytes that suppress immunopathology acting upon several target cells in the immune system. impaired breg yields have been described in a plethora of autoimmune conditions. the presence of regulatory b cells in inflammatory neuropathies and the effect of ivig therapy on their frequencies has not been studied. the aim of this study is to describe the frequencies of bregs in cidp and mmn and the effect of ivig on their frequencies. patients fulfilling diagnostic criteria for cidp or mmn and matching controls were included. pbmcs were obtained by gradient centrifugation before ivig infusion and one week after treatment. b-cells were isolated with negative selection magnetic beads, cultured and activated with the tlr agonist odn and anti-human igg+iga+igm. il- secretion capacity was assessed by flow-cytometry. twenty-eight patients were included of whom where cidp and mmn. of all patients included, received ivig and were suitable for pre and post ivig breg frequency comparisons. breg frequencies did not differ in patients (before ivig treatment) and controls (p= . , mann whitney test, two-tailed). however, the frequencies of bregs significantly increased one-week after treatment with ivig (p= . , wilcoxon matched pairs test, two-tailed). when stratifying by disease subtype, breg frequencies increased in cidp patients after ivig (p= . , wilcoxon matched pairs test, two-tailed) and in mmn (p= . , wilcoxon matched pairs test, two-tailed) although results did not reach statistical significance in mmn. this is the first study that studies the breg frequencies in cidp and the first study that addresses the effect of ivig on breg frequencies. our study provides the proof of principle that bregs could become a biomarker for response to ivig but this would need a larger and prospective study. siles am , , martínez-hernández e , diaz-manera j , , rojas-garcia r , , gallardo e , , illa i , , graus f , querol l , . paraneoplastic neuropathies (pn) are rare, immune-mediated disorders of the peripheral nerve with important prognostic implications. ectopical expression of neural antigens in the tumor leads to the development of onconeural antibodies. several autoantibodies associate to pn, including anti-hu, anti-caspr or anti-cv antibodies but a significant proportion of pn lack identifiable antigens. adhesion molecules that are autoantigens in other neuropathies, like contactin- , are present in several types of tumors. our study proposes a systematic screening of autoantibodies against neural cell-adhesion molecules and neural structures to detect novel antigenic reactivies in pn. thirty-five patients followed in our centre and at the neuroimmunology-multiple sclerosis unit at hospital clínic de barcelona, with pn fulfilling diagnostic criteria of possible (n= ; . %) and definite (n= ; . %) paraneoplastic disease were included. serum samples were obtained and tested by immunocytochemistry against contactin- (cntnt ), neurofascin (nf ) and the cntn /caspr complex. primary cultures of dorsal-root ganglia (drg) and rat schwann cells were incubated with patients' sera to detect antibodies targeting neural structures. ten individuals ( . %) presented with a tumor and a neuropathy involving both sensory and motor symptoms. the remaining patients ( . %) presented with a tumor and a classical sensory neuronopathy without anti-hu or any other onconeuronal antibody. among the latter, ( . %) patients were diagnosed with small-cell lung carcinoma. the rest of the individuals ( . %) associated diverse malignancies. we did not detect any sera reacting against cntn , nf or the cntn /caspr complex. in igg antibody screening experiments, patients ( . %) reacted against drg neurons, of them ( . %) reacting strongly, and patients ( . %) reacted mildly against rat schwann cells. in igm experiments, patients ( . %) reacted slightly against drg neurons and patients ( . %) against rat schwann cells, of them ( . %) featuring strong staining. experiments screening antibodies against motor neurons and immunoprecipitation assays are ongoing. overall, % of patients reacted strongly against either neurons or schwann cells. our study did not detect antibodies against the neural adhesion molecules cntn , nf and the cntn /caspr complex in patients with pn. however, a significant proportion of pn patients harbour antibodies targeting neural structures, which suggests that novel neoplasm-associated antigens remain to be discovered. simmons m , tao f , abreu l , zuchner s , li j . department of neurology, vanderbilt university school of medicine, nashville, tennessee, usa; hussman institute for human genomics, university of miami, miami, florida, usa. objective: despite of a shared genetic mutation of the trisomy of chromosome p (c p ), patients with charcot-marie-tooth type- a (cmt a) present with a high variability of their disease severities. the underlying cause for the variability is still unclear. in this study, we tested a hypothesis whether a second genetic mutation known to damage the nervous system is also present in cmt a patients with early onset and severe phenotypes. methods: from a cohort of patients with cmt a mutation (chromosome p duplication), we identified patients with an early onset (< or = years of age) of the disease. four of the eleven also had dna testing for a panel of known cmt-related genes and sequencing of mitochondrial dna in addition to the dna testing for c p duplication. results: besides the c p duplication, we identified three additional mutations in the four patients with early onset. the mutations were a missense mutation of arg his in mpz gene, an a g mutation in mitochondrial trna for glycin and a homozygous mutation of c p duplication. three of the four had symptomatic onset at birth. one showed symptoms at years of age. conduction velocities were severely reduced in all four patients (from to m/s). interpretation: traditional approaches to identify genetic modifiers, including snp association, assume that those modifiers are clustered in a small region of human genome and shared by the studied patients. however, our study suggests that genetic modifiers in cmt a may be highly diverse and scattered throughout the genome, which could make the conventional approach via the genetic variants association difficult. supported by grants from ninds (r ns ) and the national center for advancing translational sciences (ul tr sjogren's syndrome(ss) is a systemic auto-immune disease that apart from exocrine glands may affect any organ. involvement of peripheral nervous system results in wide spectrum of neuropathic manifestations. the aim of our study was to evaluate the clinico-electrophysiological patterns and pathological characteristics of neuropathy in sjogren's syndrome (ss) patients presented to neuromuscular clinic in a tertiary hospital from south india. this is a retrospective study from the departments of neurology, rheumatology and pathology from nizam's institute of medical sciences. twenty one patients with diagnosis of ss and peripheral neuropathy between to were analysed. clinical records, conventional nerve conduction studies, lip and nerve biopsy reports were collected.in patients with ss associated neuropathy,male to female ratio was : . in ( . %) neuropathy was the initial manifestation,while in ( %)exocrinopathy preceded neuropathy. the patterns of neuropathy included mononeuropathy multiplex(mnm) in patients ( %),ganglionopathy in ( %),length dependant, trigeminal, autonomic neuropathy and cidp in ( %)and cranial neuropathy in ( %).eighteen( %) were seropositive..schirmer's test was positive in ( . %).nerve biopsy showed vasculitis in patients, demyelinating and axonopathy in patients each.we conclude that neuropathy is frequently the initial presentation of ss.mnm is the common pattern followed by ganglionopathy. pattern of neuropathy helps in arriving at the diagnosis of ss. confirmation of ss is not by mere serology. schirmer's test and lip biopsy are equally essential for the diagnosis especially in seronegative patients when clinical index of suspicion is high. siskind ce , tesi rocha c . stanford health care, palo alto, ca, usa; stanford university, stanford, ca, usa. here, we report the first case of a human found to have a homozygous, presumed disease causing variant in the arl ip gene, causing charcot marie tooth disease (cmt) with central nervous system findings. the proband was born at term with apgars of and at and minutes. he was found to have iugr and was hospitalized for days for weight gain. he developed respiratory distress during the admission and was intubated. due to inability to extubate, he was transferred to stanford children's hospital, where he remained for three months. noted during admission was hypotonia, areflexia, minimal voluntary movment, sinus tachycardia, and dysautonomia. brain mri found polymicrogyria and cerebral underdevelopment with generally normal-appearing brainstem, with moderate ventriculomegaly. ncs found length dependent polyneuropathy with axonal degeneration. follow up muscle and nerve biopsy found immature muscle and amyelinating neuropathy the patient had normal plasma amino acid, acylcarnitine, lactate, pyruvate, urine organic acid testing, opththalmology exam, newborn screen, and normal array cgh. genetics ordered whole exome sequencing through baylor genetics laboratory (houston, tx, usa), which found a homozygous nonsense variant in arl ip : c. c>t, p.r x. a second child had been identified by baylor with two variants in this gene. that child had hypotonia, respiratory distress and seizures, and a muscle biopsy consistent with sma. the parents of that child chose to withdraw care at months of age. our patient's parents continued with aggressive therapies, including tracheostomy and g-tube for feedings. he had several subsequent hospitalizations for respiratory distress, possible seizure activity, and buldging anterior fontenelle, but now, at two years of age, has made developmental progress and is living at home with his family. he is able to smile, reaching for toys and swatting objects. he has little voluntary movement, and no longer responds to light touch stimuli. overall, this is the first picture of a child affected with a severe amyelinating form of cmt that causes weakness, hypotonia, and possible seizures, with the main concerning feature being the severe respiratory distress that may be life threatening, but can be managed with extreme care. charcot marie tooth (cmt) disease is the most common inherited peripheral neuropathy. patients frequently ask whether pregnancy will affect their cmt, whether cmt will affect their pregnancy, the optimal delivery and whether they or their child will have a higher risk of complications during pregnancy or delivery. so far few studies address these questions. currently no guidelines exist for the management of pregnancy, delivery and postnatal care in cmt patients. the aim of the study is assess the impact of pregnancy on cmt and assess how cmt affects pregnancy, delivery and care of the new born baby. we designed a retrospective questionnaire with expert help from an obstetrician with a special interest in pregnancy in patients with medical conditions. the questionnaire is divided into four parts (prior, during, after pregnancy and delivery) and includes questions on impairment, falls, pain, fatigue and respiratory complications during those periods; type of delivery, possible complications, details of anaesthesia and difficulties looking after the baby in the first months postpartum. so far women ( pregnancies) with cmt and related disorders have answered the questionnaire. % of patients had cmt a, the remaining had various subtypes of cmt and related disorders. patients reported deterioration of cmt symptoms during pregnancy in % of pregnancies with resolution of symptoms after pregnancy in % of pregnancies. of symptoms questioned walking ( %), balance ( %), and hand function ( %) deteriorated the most. there was an increased use of orthoses and walking aids during pregnancy. the majority of women ( %) had natural delivery, % were assisted and % had caesarian sections which was similar to the uk population ( %). no complications with anaesthesia were reported. the survey is currently ongoing. we plan to survey consecutive patients. data acquired from this survey will provide valuable information on current practice and will inform future guidelines and standard of care in charcot marie tooth disease. multifocal motor neuropathy (mmn) is a slowly progressive disorder in adults, characterized by asymmetrical limb weakness, mainly affecting the arms. despite beneficial effect of immunoglobulins, weakness gradually progresses. a major determinant of muscle weakness is the degeneration of affected motor axons. treatments aiming to reduce loss of motor axons require objective tools to quantify such an effect. therefore, we applied the compound muscle action potential (cmap) scan, which is an electrophysiological method that, with increasing transcutaneous stimulus-currents, successively activates all motor units (mus) in a muscle. it captures the contribution of enlarged mus due to reinnervation by the presence of relative large discontinuities in the scan. the aim of the present study was to identify pathophysiological changes of mu-loss and reinnervation in mmn patients by means of the cmap-scan. recordings were obtained from mmn patients. cmap-scan recordings were performed in the median nerve at the wrist where motor responses were recorded from the thenar muscle. we determined the number of largest cmap-scan discontinuities by means of a novel marker, d , where a low number is indicative of mu-loss and enlarged mus. furthermore, we applied the recently developed method of professor hugh bostock for obtaining a mu-number estimate from the cmap-scan. the median peak cmap amplitude was . mv (range . - . mv) and median d was (range - ). in three mmn patients with a normal maximum cmap amplitude (> mv) a reduced d (< ) was found indicative of mu-loss and enlarged mus. furthermore, d and the estimate of mu number were significantly related (r = . , p < . , n = ). the findings suggest that the cmap-scan is a sensitive tool in detecting the underlying pathological changes of reinnervation and mu-loss in mmn, more so than standard maximum cmap amplitude. it is quick and easy to perform and has the potential to be useful for follow-up studies. smith ag , thurgood b , revere c , hauer p , aperghis a , singleton jr . university of utah, salt lake city, utah, usa. corneal confocal microscopy (ccm) directly and quantitatively assesses corneal innervation including nerve fiber length (nfl) and density (nfd). ccm has shown promise as a diagnostic test. we have previously demonstrated that ccm has a diagnostic performance for diabetic neuropathy (dpn) similar to skin biopsy with assessment of intraepidermal nerve fiber density (ienfd) and nerve conduction studies (ncs). the responsiveness of these surrogate measures to dpn progression and their relation to clinically meaningful outcomes has not been well explored. diabetic patients undergoing annual retinopathy examination were recruited. each underwent ccm, ienfd, ncs including sural sensory and peroneal motor responses, the utah early neuropathy score (uens), the norfolk quality of life -diabetic neuropathy (nqol-dn, a validated neuropathy specific qol scale), and a minute walk test ( mwt). with dpn based on symptoms ( %) or signs underwent repeat testing at months and at months. at baseline, nqol-dn correlated with sural sensory amplitude (ssa) (− . , p< . ), peroneal motor conduction velocity (pcv) (− . , p< . ) and ienfd (− . , p< . ). no ccm metric was related to qol. mwt distance correlated with ssa ( . , p< . ), nfl ( . , p< . ) and nfd ( . , p< . ). over months, there was a significant worsening in dpn signs assessed by the uens (increase . +/− . , p< . ). ssa declined . uv (p< . ) and ienfd . fibers/mm (p< . ). there was no change in any ccm metric, pcv or nqol-dn. these findings suggest measures of distal axonal integrity are most sensitive to neuropathy progression, with ienfd having the greatest responsiveness. in contrast, ccm was not responsive to dpn progression. both ncs and ienfd (but not ccm) were significantly correlated with neuropathy-specific qol, whereas ncs and ccm measures correlated with physical functioning. the responsiveness of ienfd and ssa, and their relationship to qol support their selection as endpoints in dpn clinical trials. chronic inflammatory demyelination polyradiculoneuropathy (cidp) affects in , people, and is marked by chronic autoimmune infiltration of peripheral nerves and destruction of the myelin sheath. with current therapies, only % of cidp patients achieve complete remission. to produce more effective, mechanism-based therapies, we study mice with a partial loss of function g w substitution in the autoimmune regulator (aire) gene on the non-obese diabetic (nod) background (nod.aire gw/+ ) that develop spontaneous autoimmune peripheral polyneuropathy (sapp) resembling cidp. autoimmunity can result from defective immunosuppression. the potent, immunosuppressive cytokine interleukin (il- ) is increased in the peripheral blood mononuclear cells (pbmcs) of active phase cidp patients relative to remission phase patients. further, pbmcs from cidp patients produce il- in response to the myelin protein p . despite these findings, whether il- is important for cidp pathogenesis is not known. thus, we sought to determine the role of il- in sapp. il- was highly upregulated in sciatic nerves of nod.aire gw/+ mice with sapp, suggesting it may play an immunosuppressive role in pathogenesis. however, genetic ablation of il- in nod.aire gw/+ mice lead to a paradoxical delay in disease development. age-matched il- -deficient nod.aire gw/+ mice exhibited no sciatic nerve infiltrate and no reduction in nerve conduction during electrodiagnostic studies. interestingly, the delay in sapp was specific, since the incidences of five other autoimmune manifestations in il- -deficient nod.aire gw/+ mice were unchanged relative to il- -sufficient nod.aire gw/+ controls. importantly, il- -deficient nod.aire gw/+ mice did not have colitis, which is consistent with previous studies of il- deficiency on the nod background. il- is known to perform effector functions in autoimmunity by promoting b cell secretion of immunoglobulins. however, genetic ablation of b cells did not affect neuropathy development in nod.aire gw/+ mice, suggesting b cells are dispensable for pathogenesis and unlikely to mediate the protective effect of il- deficiency. il- -deficient nod.aire gw/+ cd + t cells, which are sufficient to transfer sapp, exhibited increased activation, increased interferon gamma secretion, and preserved nerve-specific t cell activation. these data suggest t cell activation and priming are unperturbed and not the mechanism of protection. in summary, our data showed that il- was paradoxically an effector cytokine in sapp. long exercise test (let) has been used especially in myotonic syndromes and muscle channelopathies. marked decrement in compound muscle action potential (cmap) amplitude after prolonged exercise was previously reported in patients with paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis. we describe a patient with secondary hypokalemic paralysis who showed abnormal let results. a -year-old man presented with ascending flaccid paralysis which evolved in a hyperacute fashion. the patient became quadriplegic after two hours. initial laboratory evaluation revealed severe hypokalemia, with normal thyroid function. we performed electrodiagnostic studies including long exercise test as proposed by mcmanis et al. nerve conduction study was normal, but marked decrement in cmap amplitude (up to % decrease after minutes) was noted after prolonged exercise. despite oral and intravenous potassium replacement, serum potassium level was not corrected as expected. the unusual clinical course prompted for evaluation of secondary etiologies. abdomen computed tomography scan revealed a . x . cm-sized mass in the left adrenal gland. aldosterone to renin ratio was elevated, suggestive of primary hyperaldosteronism. genetic study for cacna s mutation turned negative. after receiving laparoscopic adrenalectomy, the patient experienced no further attacks, and also was able to stop his antihypertensive medication. let may show abnormal results in condition with reduced membrane excitability, even without true channelopathy. according to international criteria, the diagnosis small fiber neuropathy (sfn) is based on clinical symptoms in combination with a reduced intraepidermal nerve fiber density (ienfd) in skin biopsy and/or abnormal temperature threshold testing (ttt). the sensitivity of skin biopsy is moderate to good, although ienfd is normal in about % of patients with sfn complaints. furthermore, ttt is a widely available diagnostic tool, but lacks specificity. corneal confocal microscopy (ccm) has been described and is used in clinical practice as an objective, non-invasive diagnostic tool to detect small nerve fiber damage in patients with diabetes mellitus. this study examines the applicability of ccm in patients with sfn, and the value of ccm as an additional diagnostic tool in sfn. we will include healthy participants to compare the results with the recently published ccm normative values, and patients referred to the sfn center maastricht with the clinical picture of sfn. corneal nerve fiber density (cnfd), branch density (cnbd), fiber length (cnfl), and the tortuosity coefficient (cnft) will be determined in all participants. the results will be compared with the ienfd and ttt. preliminary results will be presented. southanalinh k , university of health sciences, vientiane capital, lao p.d.r. located in south east asia, lao pdr is a landlocked country with a population of about . million inhabitants. the health indicators are among the lowest in south east asia. the total health caregivers in consisted of , persons corresponding to a ratio of . health workers per inhabitants. the main network for health care service provision remains the public system. its health care facilities consist of four central teaching and referral hospitals; five regional hospitals, including one teaching hospital; provincial hospitals; district hospitals, and about health centers. only one in seven sick people receives modern health care treatment. most people rely on self-medication and/or reliance on self-healing. neurological care is a very new field. knowledge of common neurological disorders among both the lao population and medical staff is only beginning to be spread. there are three neurologists in the country. six neurology residents are currently being trained in a three-year program supported by the association pour la promotion des neuro-sciences au laos (association for the promotion of neuro-sciences in laos) and the asean neurological association. indeed, resources are scarce. in the peripheral nerve diseases domain for example, we have only one electromyography machine that was only temporary used when emg experts from france and from singapore came to teach residents. a significant mismatch between the provision of specialized neurologic services and the needs for them exists, especially in rural areas. also, health insurance is not available for the majority. as a consequence, patients have to bear the costs themselves, which constitutes a limit to the access of available healthcare facilities. neurologic training centers, laboratory facilities and equipments are limited. optimizing available human resources, integrating primary, secondary, and tertiary healthcare tiers and making medical treatment more affordable are need to improve neurologic care in the developing world. in certain low-income countries with limited human and financial resources, it may be difficult for governments to apply some of these recommendations on their own. in these circumstances, it is suggested that countries work with international agencies, nongovernmental organizations or other partners to put their plans into practice. spina e , topa a , iodice r , tozza s , dubbioso r , ruggiero l , santoro l , manganelli f . department of neuroscience, odontostomatological and reproductive sciences, university of naples "federico ii", naples, italy. chronic inflammatory demyelinating polyradiculoneuropathy (cidp) is a disabling disease and about % of patients may become persistently disabled over time. our aim was identify clinical prognostic factors of long-term disability in a large series of cidp patients. we collected data from cidp patients with definite diagnosis according efns/pns criteria and positive response to first-line therapies (immunoglobulin or corticosteroids) including sex, age of onset, phenotype, disease duration, course of disease (monophasic/relapsing-remitting or chronic progressive) and disability at the time of diagnosis assessed using the modified rankin scale (baseline mrs). all patients had clinical assessment of disability through mrs within the last months (last mrs). ordinal logistic regression model was applied to evaluate the relationship among the clinical parameters and last mrs, considered as ordinal outcome ( - ). anova test for repeated measures was applied to test the overall effects of different course on disability accumulation while t-test was performed to evaluate inter-group differences for parametric variables. we found a significant relationship between last mrs and the course of disease [p< . , z= . , or: . ]. disability accumulation was greater in patients with chronic progressive course than those with monophasic/relapsing-remitting course of disease [p= . ]. moreover, patients with progressive course were older [p= . ]. our data suggest that chronic progressive course of disease may be a major negative prognostic factor for long-term disability in cidp patients. to note that a chronic progressive course of disease is also associated with an older age from the beginning and a more pronounced worsening over the course of disease. sprenger a , lichtenstein t , henning t , lehmann hc . department of neurology, university hospital of cologne, cologne, germany; institute of diagnostic and interventional radiology, university hospital of cologne, cologne, germany; department of neurology, university hospital of cologne, cologne, germany. an unresolved problem in the treatment of inflammatory neuropathies is the lack of valid and reliable diagnostic biomarkers to evaluate axonal damage. we investigated if "diffusion tensor imaging" (dti) and mri t w multi echo dixon imaging are eligible methods to determine proximal nerve injury in chronic inflammatory demyelinating polyneuropathy (cidp). in this prospective observational cohort study the sciatic nerve of cidp patients and age matched healthy controls was investigated. all subjects underwent multimodal mri imaging to determine fractional anisotropy (fa) and muscle fat fraction of the biceps femoris and quadriceps femoris muscle. patients were evaluated by mri, clinical examination and nerve conduction studies at baseline and after six months. the mean fractional anisotropy (fa) value was significantly lower in the sciatic nerve from cidp patients compared to controls. fat fraction of the biceps femoris and quadriceps femoris muscle were significantly higher in cidp patients compared to controls. mri outcome parameters remained unchanged after six months. our study demonstrates the utility of mri imaging to differentiate between "healthy" and functional constricted proximal nerve segments. we postulate that dti and dixon mri might be eligible methods to assess proximal nerve damage in cidp. the presence of peripheral myelin protein (pmp ) has been known for decades, but its functional role was uncovered only recently. recent characterization of pmp -deficient mice revealed a role of pmp in the lipid homeostasis of myelinating schwann cells. in this study, we analyzed the functional impact of pmp on myelination. to decipher the role of pmp , experimental demyelination was performed in myelinating dorsal root ganglia cultures, and in vivo re-myelination was assessed after experimental peripheral nerve damage. we used the myelinating dorsal root ganglia (drg) model in pmp -deficient schwann cell cultures, combined with an established de-and remyelinating protocol in order to analyze myelination in vitro. we also performed experimental nerve crush in pmp -deficient mice. morphometric parameters were defined for the in-vitro experiments and functional parameters such as nerve conduction velocity and the clinical score were additionally measured for the in vivo experiments. structural analyses of the drg cultures revealed fibers expressing myelin basic protein (mbp) and pmp , as well as fibers positive for mbp alone. in contrast to our previous in vivo data, we were also able to detect myelin segments that stained positive for pmp , but were negative for mbp. pmp -deficient drg-cultures demonstrated slightly greater nodal lengths than the control cultures. this trend was significantly augmented after in vitro de-and remyelination, which also resulted in decreased internodal lengths only now, while conserving an intact myelin structure. concomitantly, in vivo nerve crush gives rise to a more severe phenotype in pmp -deficient mice than in wild-type controls. consistent with this, nerve conduction studies showed a delay in remyelination, and analysis of semi-thin sections demonstrated an altered fiber structure in the peripheral nerve biopsies. together, these data suggest that in addition to its role in glial cell lipid homeostasis, pmp also plays a role in remyelination of the injured peripheral nervous system. anti-mag neuropathy remains a difficult diagnosis to treat given its limited therapeutic options. of all interventions, rituximab has emerged as the most effective, although its effect has been with mixed results, especially in patients with advanced axonal loss. lenalidomide is another promising immune modulating therapy, whose effect has been well demonstrated in neuropathy associated with poems (polyneuropathy, endocrinopathy, organomegaly, m-spike protein, and skin changes) syndrome, a condition that has several striking parallels to anti-mag neuropathy. the use of lenalidomide has not been previously described in anti-mag neuropathy. herein, we describe a case of lenalidomide-responsive anti-mag neuropathy in a patient with advanced axonal loss. suichi t , misawa s , sato y , beppu m , sekiguchi y , shibuya k , watanabe k , amino h , kuwabara s . department of neurology, graduate school of medicine, chiba university, chiba, japan; clinical research center, chiba university hospital, chiba, japan. polyneuropathy, organomegaly, endocrinopathy, m-protein, and skin changes (poems) syndrome is a rare cause of demyelinating neuropathy associated with plasma cell dyscrasia and vegf overproduction. several diagnostic criteria for the disorder have been published, but sensitivity/specificity analyses, and their validation have never been performed. the aim of this study is to establish valid diagnostic criteria for poems syndrome. consecutive poems patients, seen at chiba university hospital since , were screened. of these, we have set a gold standard group of poems syndrome, based on treatment response and exclusion criteria during -year follow-up, and patients was diagnosed as having definite poems syndrome. we also collected patients with cidp (demyelinating neuropathy control) and with multiple myeloma, primary amyloidosis, or mgus (m-protein control). criteria for poems syndrome was defined as having two of the three major criteria (polyneuropathy, m-protein, and elevated serum vegf level) and at least two of the four minor criteria (extravascular volume overload, skin changes, sclerotic bone lesions, and thrombocytosis) which were determined by logistic regression analyses. according to the criteria the sensitivity was %, and the specificity was %. our results indicate that the proposed criteria have an excellent diagnostic accuracy, and are useful in clinical practice, presumably leading to early diagnosis and treatment. intraepidermal electrical stimulation (ies) is a new technique to that assesses the function of a-delta fibers in the epidermis. using this technique, we previously reported that the epidermal pain threshold was two-hold in asymptomatic diabetic patients than in normal subjects (muscle nerve : - , ). subsequently, we reported that the elevated pain threshold negatively correlated with intraepidermal nerve fiver density (jpns : s , ). empirically, it is known that lowering the skin temperature makes it less likely to feel pain. therefore, it is necessary to investigate whether the results of ies are affected by skin temperature. the aim of this study was to investigate the influence of a low skin temperature on pain threshold. we recruited subjects with a mean age of . years. for nociceptive stimulation, we used an ies method with a concentric micro-needle electrode that was developed specifically for the selective stimulation of cutaneous a-delta fibers. we placed the ies electrode onto the extensor digitorum brevis and began stimulation with intensity strong enough for the subject to feel a pricking sensation, then reduced the current in steps of . ma until no sensation was felt. we defined pain threshold as the minimum electrical intensity at which a subject felt a pricking sensation. firstly, we measured pain threshold at skin temperature above degrees celsius. then, we put an ice pack on the extensor digitorum brevis for min to lower the skin temperature, and measured pain threshold at skin temperatures below degrees. mean pain threshold values above degrees and below degrees of skin temperature were . and . ma (p< . ), respectively. our data indicated an elevated pain threshold in epidermis with a low skin temperature. one of most common methods for nociceptive stimulation is painful co laser stimulation. some co laser stimulation studies reported pain threshold increased with a low skin temperature. our result is similar to that of co laser stimulation. pain threshold using ies is very easy and non-invasive technique. it may be useful for the evaluation of small fiber neuropathy. svačina mkr , röth p , bobylev i , sprenger a , zhang g , sheikh ka , lehmann hc . department of neurology, university hospital cologne, cologne, germany; department of neurology, university of texas, houston, usa. intravenous immunoglobulins (ivig) are an effective treatment in guillain-barré-syndrome (gbs). in most patients, the optimal ivig dose and regime is unknown. in serum and ivig preparations, immunoglobulin (ig) g form igg dimers, which are assumed to consist of idiotypic/anti-idiotypic antibody pairs. however, data about kinetics of igg dimer formation in gbs are lacking. to study igg dimer formation, c bl/ mice were injected with ivig and anti-gd b antibody or pbs. blood sera were collected h, h and week post injection. a third cohort received an anti-gd a/gt b antibody and blood was collected h post injection. igg was extracted and subtyped into polymeric, dimeric and monomeric fractions using the Äkta fplc system. dialysed dimeric and monomeric igg fractions were examined for the presence of anti-ganglioside antibodies by anti-ganglioside antibody elisa. further, blood samples from gbs patients were collected before (pre-ivig) and after treatment with ivig (post ivig). serum samples were examined for igg dimers and monomers using the Äkta fplc system. in the mouse model, a maximum peak of igg dimer formation was observed h post injection. in gbs patients' samples, igg serum levels and igg dimer content was significantly higher after treatment with ivig. we demonstrate here the feasibility to assess igg dimer formation in an animal model and in gbs patients' samples after treatment with ivig. h after ivig treatment appears to be the optimal time point to assess igg dimer formation. further studies are warranted to determine the utility of igg dimer formation as surrogate marker for treatment response in gbs. svaren j , moran jj , wu x , gutmann l , shy m . university of wisconsin-madison, madison, wisconsin, usa; university of iowa, iowa city, iowa, usa. development of outcome measures for clinical trials in cmt a is a major challenge given the slowly progressive nature of the disease. outcome measures can be used to measure a) target engagement for a given therapy, as well as b) disease process and c) disease burden. several candidate therapies have been shown to reduce pmp levels in cmt a rodent models and thereby ameliorate the symptoms of pmp overexpression. measuring pmp mrna reduction in human trials has so far been limited to analysis of skin biopsies by qrt-pcr, which did not demonstrate clear elevations of pmp mrna during, nor a reduction following, ascorbic acid trials. the analysis of skin biopsies is hampered by variable amounts of schwann cells (sc) in skin biopsies, as well as the variable amount of pmp in sc as previously established by immuno-em in cmt a skin biopsies (katona et al., ) . therefore, it is important to develop optimal normalization criteria to address the variability inherent in skin biopsy analysis. ideally this will employ normalization to sc-specific genes that are not altered by cmt a status. to optimize normalization, we have performed rna-seq analysis of skin biopsies from patient and control skin biopsy samples. analysis of these data after normalization to read depth indicated that pmp levels were . fold higher in cmt a patient samples compared to control skin biopsies. however, there was significant variability in pmp levels particularly in cmt a samples, which may be due to variable amounts of schwann cells in cmt a skin. using a combination of sc-specific genes for normalization, we were able to reduce the apparent variability and optimize the differential levels between cmt a and control skin biopsy samples. we also identified other sc-specific genes that were apparently induced in cmt a skin biopsies relative to control. these studies provide a new framework for gene expression analysis in skin biopsies, enabling more precise evaluation of pmp levels in clinical trials for cmt a as a measure of target engagement. in addition, the normalization framework may also be applicable to other types of cmt. chronic inflammatory demyelinating polyneuropathy (cidp) is the most common chronic autoimmune neuropathy, with an estimated prevalence of between and per , people. it can cause temporary disability in the affected individuals and may eventually lead to permanent disability or death. cidp is commonly treated with intravenous immunoglobulin (ivig) therapy or corticosteroids. octagam ® % is licensed for cidp in france, while octagam ® % is licensed for cidp in germany and belgium. this analysis presents data from three open, multicenter, non-interventional, single-arm, non-controlled studies of a post-authorisation safety surveillance (pass) program for the subset of patients receiving octagam ® % or % for neurological indications, focusing on patients with cidp. briefly, data from in-and out-patients in austria, france, germany, and uk treated with octagam ® for neurological disorders were collected by physicians and analyzed to assess safety and tolerability of the treatment. of patients included in the three studies, patients ( . %) received octagam ® for neurological indications, of which patients ( . %; mean age . years [range - ]) had cidp. the mean dose of octagam ® per course was . g/kg bw for patients with cidp; for the other neurologic indications, the dose ranged from . (for multiple sclerosis) to . g/kg bw (for guillain-barré syndrome). premedication was not needed in . % of these patients. the development of clinical appearance since last observation (mean: every . months) was assessed for of the cidp patients by their treating physicians. the majority of observations ( . %) assessed the patients as stable and . % showed even an improved clinical appearance. only . % of the observation periods resulted in deteriorations. adverse drug reactions were rare: of the infusions received by patients with neurological disorders, . % of infusions were associated with an adr ( . % of infusions in cidp patients). overall, treatment with octagam ® was effective and well-tolerated in patients with cidp. these results are consistent with data for the overall patient population (including patients with primary and secondary immunodeficiencies, dermatological and other diseases). szepanowski f , szepanowski lp , kleinschnitz c , kieseier bc , stettner m . department of neurology, medical faculty, university duisburg-essen, essen, germany; department of neurology, medical faculty, heinrich-heine-university, düsseldorf, germany. lysophosphatidic acid (lpa) is a pleiotropic signaling lipid that acts as ligand for at least six specific g protein coupled receptors. schwann cells (sc) are known to mainly express the lpa receptor subtype. an emerging body of in vivo evidence has linked lpa with injury induced peripheral nerve demyelination as well as neuropathic pain. however, the molecular mechanism underlying its demyelinating effect has remained largely unclear. myelinated dorsal root ganglia (drg) cultures were treated either with lpa, lpa + am (lpa antagonist) or vehicle. we assessed myelin basic protein, tumor necrosis factor alpha (tnf-alpha) as well as the sc differentiation marker sox by immunocytochemistry. additionally, myelin was investigated by sudan black staining. to better understand the relevance of lpa signaling for demyelination in vivo, we performed sciatic nerve crush in c bl/ mice treated with am at mg/kg in order to study schwann cell expression of tnf-alpha, sox and sox , a marker for sc dedifferentiation, by immunohistochemistry. in drg cultures, lpa caused a significant reduction of myelin as demonstrated by both sudan black staining and immunocytochemical analysis of myelin basic protein. demyelination was paralleled by an upregulation of tnf-alpha as well as downregulation of sox . lpa mediated effects were found to be blocked by addition of the lpa receptor antagonist am . in c bl/ mice, am treatment prior to crush injury increased sox expression in scs in the distal nerve stump while reducing the number of cells expressing sox . these data indicate that lpa may be a critical factor to shift scs towards an injury-associated phenotype and contribute to the onset of wallerian degeneration. szepanowski lp , szepanowski f , kleinschnitz c , stettner m . department of neurology, university hospital essen, essen, germany. glyphosate-based formulations comprise the world's most commonly used herbicides. in non-resistant plants, glyphosate exerts toxic effects most likely via inhibition of aromatic amino acid synthesis by interfering with the shikimate pathway. while glyphosate is the active ingredient, herbicidal formulations contain several adjuvants, including polyethoxlated alkylamines (poeas). although glyphosate has long been considered safe for use in humans and animals, several studies have implicated glyphosate and/or the commonly used adjuvants in cytotoxicity, carcinogenicity and endocrine disruption. furthermore, glyphosate-based herbicide has been reported to mediate neurotoxicity in immature rat hippocampus involving glutamate excitotoxicity. however, it remains unclear whether glyphosate alone or in combination with its adjuvants may have detrimental effects on myelin integrity in the peripheral nervous system. myelinated dorsal root ganglia (drg) cultures were treated over the course of ten days with either pure glyphosate or a glyphosate-based herbicide at concentrations of . %, . % and . %. the concentration of the glyphosate-based herbicide was matched with regard to glyphosate content ( %). controls were treated with equal amounts of vehicle adjusted for the ph. subsequently, cultures were stained with sudan black and myelin content was assessed by determining the number of internodes per neurons. while glyphosate, regardless of its concentration, did not show any effect on myelin content, the glyphosate-based herbicide caused significant demyelination in a concentration-dependent manner. notably, at . %, drg cultures were completely devoid of myelin and appeared severely necrotic. these data raise the possibility that not glyphosate itself, but rather the adjuvants in glyphosate-based herbicide formulations may cause demyelination. the open question whether demyelination is a direct effect of the adjuvants or a consequence of increased cellular glyphosate uptake due to permeabilization warrants further investigation. tan cy , tan mp , yeoh ky , goh kj , shahrizaila n . department of medicine, university of malaya, kuala lumpur, malaysia. in guillain-barré syndrome (gbs), autonomic dysfunction is common and accounts for significant morbidity and mortality. there have been many studies investigating the electrodiagnosis of gbs but few have studied autonomic dysfunction in gbs. the current study comprehensively investigates quantitative autonomic function in patients with gbs and its variant. ten gbs patients were prospectively recruited and the results were compared to age-and gender-matched healthy controls. a series of autonomic function tests including computational (power spectrum analysis of heart rate variability (hrv) and baroreflex sensitivity (brs) at rest) and challenge tests (deep breathing, eyeball compression, active standing, valsalva manoeuvre, isometric exercise and ice-water hand immersion) were performed. parasympathetic function was represented by high frequency (hf) hrv, heart rate responses to deep breathing, eyeball compression, valsalva manoeuvre and active standing. sympathetic function was represented by low frequency (lf) hrv, blood pressure responses to active standing, sustained handgrip and ice-water hand immersion. in the frequency domain analysis of hrv, low frequency (lf: . ± . vs . ± . ; p= . ), high frequency (hf: . ± . vs . ± . ; p= . ) and total power spectral densities (psd: . ± . vs . ± . ; p= . ) were significantly reduced in patients compared to controls. the mean up slope ( . ± . vs . ± . ; p= . ), down slope ( . ± . vs . ± . ; p= . ) and total brs slope ( . ± . vs . ± . ; p= . ) were significantly lower in the gbs group. the diastolic rise in blood pressure upon ice-water hand immersion was significantly lower in gbs group compared to controls ( . ± . vs . ± . ; p= . ). our findings suggest that computation dependent tests (hrv and brs) were sensitive at detecting autonomic dysfunction and baroreceptor reflex insensitivity in gbs patients. in contrast, ice-water hand immersion was the only reliable challenge test making it useful as a bedside measure of autonomic function in gbs patients. sjögren's syndrome (ss) is an autoimmune disease that affects both east and west. nevertheless, we still have limited knowledge of how autoantibodies in ss affects the peripheral nervous system. in this study, we investigated the peripheral neuropathy in ss and sicca complex using the nerve excitability test, to elucidate how peripheral nerves are affected. we have enrolled a total of patients with ss or sicca complex. of these, two patients were excluded due to co-morbid carpal tunnel syndrome. each patient received clinical evaluation, examination for ssa/ssb antibodies titer, the nerve excitability test, conventional thermal quantitative sensory test, and conventional nerve conduction study. compared to normal control subjects, motor nerve excitability test of ss patients with positive ssa or ssb antibodies (n = ) were found to have increased rheobase (p< . ), increased relative refractory period (rrp) (p< . ), increased refractoriness at . ms (p< . ), increased accommodation toward depolarizing current in threshold electrotonus (te) (p< . ), and decreased superexcitability (p< . ). the sensory axonal study in seropositive ss also revealed increased rrp (p< . ), increased refractoriness at . ms (p< . ), and increased accommodation toward hyperpolarizing current in threshold electrotonus (te) (p< . ). meanwhile, in seronegative ss and sicca complex (n = ), we found no significant axonal properties changes. the present study revealed that peripheral nerves are affected differently in seropositive ss and in seronegative ss/sicca complex. in seropositive ss, motor axons tended to be depolarized, and both sensory and motor axons have increased refractoriness. the findings suggested that ssa and ssb antibodies might play a role in the inactivation of transient sodium channels. the effects of the antibodies on transient sodium channels might be the basis of peripheral neuropathies and even cardiac arrhythmias and heart block in ss. charcot-marie-tooth disease type a (cmt a), caused by the pmp duplication on chromosome p . , is the most common subtype of inherited peripheral neuropathies and affects in , individuals worldwide. while sharing the same genetic cause, cmt a patients often present great variability in their phenotypic presentation and disease severity. the cause of the phenotypic variability is largely unclear. in this study, we performed genome-wide association study (gwas) to identify novel genetic modifiers of various phenotypes in cmt a. dna samples from cmt a patients were genotyped on illumina omniexpress platform. after standard quality control, the dataset includes k markers in individuals ( individuals from european ancestry, and individuals from asian ancestry). we focused our analyses on the european population. logistic regression in plink was used to analyze the association between the clinical outcomes and patients' genotypes in an additive model. for cmt neuropathy score (cmtns), the analysis was performed using linear regression in plink, adjusting for patients' age. the analyses yielded several suggestive association signals. an association peak on chromosome was identified in difficulty with eating utensils (lead snp rs , chr : , p= . e- , odds ratio= . ). the peak is located within a non-coding gene linc . hearing loss showed an association peak on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ), located in an intergenic region near the megf gene. in foot plantar flexion, an association signal was identified in the dscam gene on chromosome (lead snp rs , chr : , p= . e- , odds ratio= . ). cmtns showed an association signal on chromosome (lead snp rs , chr : , p= . e- , beta= . ), located within an intergenic region close to dffb, c orf , and linc . while these suggestive signals require further validation, our study provides novel insights into the genetic architecture of cmt a. novel genetic modifiers may serve as potential targets for therapeutic interventions in the future. teng a , ohnmar , kalpana p , chai yh , umapathi t . yong loo lin school of medicine, national university of singapore, singapore; department of neurology, national neuroscience institute, singapore. we present an intriguing diagnostic puzzle, that was eventually cracked serendipitously. a -year-old man was seen for bilateral ptosis. evaluation for myasthenia gravis was negative. nevertheless, a diagnosis of ocular myasthenia gravis was made and he was put on pyridostigmine. he did not respond. the ptosis progressively worsened. he sought a second opinion. at this evaluation, he was noted to have complex ophthalmoplegia without diplopia, bilateral facial weakness and mild bulbar weakness. he had no sensory complaints. the limb examination was remarkable for slightly reduced reflexes, normal strength, and other than increased vibration threshold at the toes, intact sensory examination. repeat serological and electrodiagnostic work-up for myasthenia gravis was negative. a myopathic disorder such as chronic progressive external ophthalmoplegia was considered. serum creatine kinase and lactate were normal. he underwent biceps muscle biopsy which showed increased cox-negative and sdh positive fibers, supporting the then clinical impression of a mitochondrial cytopathy. at this point, he underwent blepharoplasty to improve his vision. routine histological examination of the levator palpebrae muscle showed amyloid deposits. this prompted a review of the earlier biceps biopsy, which revealed amyloid deposits that were not appreciated before. at this point, the significance of the patient and his son's history of lattice corneal dystrophy became apparent. he also reported that his late mother had similar facial appearance as his. the patient's nerve conduction study showed length-dependent sensory axonal polyneuropathy, right carpal tunnel syndrome and bilateral facial neuropathy. he had no definite symptoms of autonomic neuropathy. cardiac evaluation was unremarkable. the final diagnosis of familial gelsolin amyloid polyneuropathy was made. genetic confirmation for the patient and his family is being planned. we highlight the key clinical features of gelsolin neuropathy. the symmetric cranial neuropathy can resemble a muscle or neuromuscular junction disorder and the relative sparing of the cardiac muscle, somatic and autonomic nerves contrasts with transthyretin-related amyloid polyneuropathy. neuropathy is one of the most common long-term complications of diabetes. furthermore, % to % of diabetic neuropathy patients will develop neuropathic pain. the pathophysiology of neuropathic pain in diabetic peripheral neuropathy is complex and not fully understood. a potential mechanism is a change in voltage gated sodium channels, such as nav . . loss of function mutations in this channel cause insensitivity to pain, whereas gain of function mutations have been linked with different pain syndromes including small fiber neuropathy. in a cohort of patients with diabetic peripheral neuropathy we investigated whether mutations in nav . were associated with diabetic neuropathic pain. twelve nav . variants were identified in nine participants all within a cohort of participants with painful diabetic peripheral neuropathy. five of these variants were previously associated with pain disorders: v l, m l; w r, r h, l v. among the other variants two of them met the criteria of potential pathogenicity based on predictive algorithms and were further studied. functional analysis by whole cell patch clamp showed that one of these variants (m t) drastically impairs the inactivation of the channel by shifting the steady-state fast-inactivation towards more depolarizing potentials. there were no phenotypic difference between those participants with pathogenic variants and those participants without pathogenic variants. no rare nav . variants were found in participants with painless diabetic peripheral neuropathy. these observations suggest that mutations in nav . may contribute to painful diabetic peripheral neuropathy. tholance y , rosier c , f bouhour , psimaras d , kuntzer t , taieb g , créange a , delmont e , camdessanché jp , antoine jc . university hospital, saint-etienne, france; university hospital, lyon, france; university hospital, paris, france; university hospital, lausanne, switzerland; university hospital, montpellier, france; university hospital, creteil, france; university hospital, marseille, france. dysimmune sensory neuronopathies (snn) encompass paraneoplastic snn and snn associated with systemic autoimmune diseases such as sjögren syndrome, lupus or inflammatory bowel or rheumatic diseases but also a number of apparently idiopathic cases. biomarker antibodies are well-known in paraneoplastic snn but are lacking in non paraneoplastic cases. from a mono-center retrospective study we identified in anti-fgfr antibody as a potential biomarker of dysimmunity in patients with idiopathic or systemic autoimmune disease associated sensory neuropathy. the identified patients were more frequently women and had a non lenthg dependent neuropathy suggestive of snn. anti-fgfr antibody was the only immunological marker in / of cases at initial work-up although / of patients eventually developed with time systemic autoimmune disease. to confirm the incidence and the clinical pattern of patients with anti-fgfr antibodies we launched a prospective multicenter french study including patients with a sensory neuropathy suspected to be a snn of no paraneoplastic, genetic or metabolic origin. we present here the results on the first included patients compared to healthy blood donors. anti-fgcr antibodies were searched by elisa using the trk intracellular domain of the protein (invitrogen©). we found patients positive for anti-fgfr antibody ( . %). these patients were women and men aged . years as a mean ( - ). the neuropathy was acute and subacute in one patient respectively and progressive in the others. six patients fulfilled the diagnosis criteria of snn and the last one had a sensory neuropathy in the lower limb with abnormal sensory action potentials in the four limbs suggesting snn without reaching the requested criteria. one patient developed uveitis which is a new symptom with anti-fgfr ab. an unclassified dysimmune context was present at the initial work up in patients and one patient developed sjögren syndrome with follow-up. as a whole the clinical pattern of these patients is consistent with that of the initially published series. the lower prevalence of positive sera may be due to more stringent criteria used for elisa but needs to be confirmed on the complete prospective series. peripheral neuropathy research registry (pnrr) neurological assessment was scored using the total neuropathy score clinical version (tnsc), comprising pinprick and vibration sensibility, deep tendon reflexes, strength and patient symptom report. compound sensory action potential (csap) amplitudes were recorded antidromically at the lateral malleolus, stimulating the sural nerve at the mid-calf. of the total sample, % reported lower limb neuropathy, with % of patients reporting 'quite a bit' or 'very much' severity of tingling and numbness in their feet. the average sural csap amplitude was . ± . v and % of patients had sural amplitudes below the lower limit of normal for age. the total tnsc score correlated with the pro fact-gog ntx score (r = −. , p<. ) and sural amplitude (r = −. , p <. ). vibration sensibility correlated with the overall fact-gog ntx score (r =−. , p <. ), and sural amplitude (r = −. , p <. ). sural amplitude correlated with patient reported severity of numbness and tingling in the lower limbs (r =−. , p <. ) but not with the overall fact-gog ntx score. patient reports of neuropathic symptoms in the lower limb correlate with both objective neurophysiological and clinical measures of neuropathy severity. identifying links between objective neurophysiological markers and patient reported outcomes are critical to assess the impact of clinical interventions. tomaselli pj , gouvea sp , nyshyama kfs , nicolau n jr , lourenço cm , marques w jr , . division of neuromuscular diseases, department of neurosciences and behaviour sciences, clinical hospital of ribeirão preto, university of são paulo, ribeirão preto, brazil; neurogenetics, department of neurosciences and behaviour sciences, university of são paulo, ribeirão preto, brazil. mutations in the gab junction beta -protein gene (gjb ) are the second most frequent cause of charcot-marie-tooth disease (cmt), accounting for approximately % of cmt cases worldwide. the gjb codes for connexin protein (cx ). in the peripheral nervous system, the cx is expressed in the schwann cells and allows intercellular traffic of ions and small molecules between opposed cells. we analysed retrospectively detailed clinical and neurophysiological data of five families carrying novel gjb mutation submitted for testing at our neurogenetics laboratory. mutations were identified by bidirectional sanger sequence analysis of gjb coding region. we identified a total of subjects from five different kindreds with novel mutations (p.a v, p.l w, p.l q, p.f s, p.r l). these five novel mutations segregate with phenotype, are located in highly conserved amino acids among gjb and other gab junction protein sequences and among different species, are not present in any public database (exac, dbsnp and genome database), and were not found in normal brazilian controls. in silico analysis, predict these variants to be pathogenic, there was no male-to-male transmission; males were more severely affected than females. four out seven female have subclinical neuropathy and were only identified after clinical and electrophysiological evaluation. the conduction velocities were in the intermediated range in the males patients and higher in the females included in this study. we describe five new pathogenic mutations causing cmtx in a brazilian population and expand the number of causative mutations in the gjb gene. funded pure neural leprosy (pnl) is a slowly progressive, predominantly sensory patchy neuropathy presenting with positive and/or negative sensory symptoms, which are usually followed over time by distal asymmetrical weakness. despite rare, monomelic involvement in leprosy has already been reported. we sought to describe the clinical and electrophysiological patterns of an unusual leprosy neuropathy presentation. clinical data were retrospectively collected from nine patients who had monomelic involvement and were referred for further investigation to the emg lab. seven out nine patients were male. four patients had a brachial plexus like presentation and five have a lumbosacral plexus like presentation. the initial complaint was hypoesthesia in four patients, tingling in two patients and hypoesthesia with tingling in two patients. severe pain was observed in just one patient. all individuals from the group of patients with lumbosacral plexus-like presentation and three with brachial plexus-like presentation had no sensory nerve action potentials (snaps) for all nerves tested in the affected limb with or without motor involvement. one patient with brachial plexus-like presentation had focal slowing of conduction velocity with temporal dispersion of both median and ulnar nerves in the affected limb. one patient with plexus-like presentation had snaps with low amplitude of all nerves in the affect limb. the diagnosis of leprosy was confirmed by nerve biopsy findings, anti-pgl antibody, and positive response to specific treatment. nerve biopsy was performed in four patients, and the bacillus was found in two. the anti-pgl antibody was positive in four patients. plexus mri was performed in two patients and was normal. we found the distribution of motor and sensory symptoms were restricted to on limb in this group of patients. as a typically patchy disorder pnl may affect any nerve, although the reason why damage are restrict to only one limb has to be elucidated. the description of these cases increases the clinical spectrum of leprosy neuropathy. this possibility should be considered in the differential diagnosis of patients with plexopathy from endemic areas after excluding other causes. funded ataxia with oculomotor apraxia type (aoa ) is a very complex disorder characterized by an early-onset progressive cerebellar ataxia with cerebellar atrophy and peripheral neuropathy and it is caused by recessive mutations in the aprataxin gene (aptx ). when the neuropathy is present, it has been described in % of cases as primarily axonal. we describe a case of aoa due compound heterozygous mutations in aptx associated with demyelinating neuropathy. the patient was born from healthy and non-consanguineous parents and presented in the first decade with progressive cerebellar ataxia, multidirectional ophtalmoparesia, oculomotor apraxia, choreiform movements of limbs and peripheral neuropathy. he had normal cognition and stopped walking at age of . blood tests were unremarkable with normal levels of leucocytes, serum proteins, immunoglobulin, cholesterol, vitamin e, and alfa-feto protein. brain mri showed severe cerebellar atrophy. the motor conduction velocity in the upper limbs was slow with preserved amplitudes. the distal latencies and the minimal f wave latencies were prolonged. there was no evidence for superimposed acquired demyelinating neuropathy. direct sequencing of the aptx gene revealed two variants, c. - a>g, p? and c. g>a; p.w x. the first variant is novel and affects a highly conserved acceptor splice site of exon . the other variant is the most common portuguese variant, the nonsense mutation w x is located in exon . parental dna was tested and confirmed the variants were in different alleles. the presence of a demyelinating neuropathy in aoa suggests that phenotypic variability in this condition may be larger than previously considered. at the same time, it increases the differential diagnosis of inherited conditions with cerebellar ataxia and demyelinating neuropathy. finally, this finding opens the functional effects of the aptx gene. funded by: cnpq, fapesp, faepa, pronas (ministry of health). topa a , spina e , iodice r , tozza s , ruggiero l , dubbioso r , esposito m , santoro l , manganelli f . university of naples "federico ii", naples, italy. we report our -year experience of subcutaneous immunoglobulin (scig) in a cohort of patients with chronic inflammatory demyelinating polyneuropathy (cidp) from a tertiary care neuromuscular center. we analyzed data from cidp patients ( males and females, mean age: ± . years; mean age at onset: . ± . years; disease duration: . ± . years) treated with scig and with a follow-up period of months. all patients were previously responders to intravenous immunoglobulin (ivig). eight patients had a typical cidp and five patients had an atypical variant of cidp. five patients switched to weekly maintenance scig therapy (continuous regimen) because of short-lasting response to ivig therapy. eight patients with a longer lasting response to ivig received scig with a pulsed regimen similar to that used for ivig (from to cycles per year); seven of them because of difficulty in hospitalization and one for allergic reaction to ivig. changes in clinical status were assessed over the period of follow-up by using clinical evaluation of muscle strength, modified rankin scale, overall neuropathy scale and inflammatory neuropathy cause and treatmentsum score. in patients we evaluated also six minute walking test, hole-peg-test and meter walking test. all the five patients treated with a continuous regimen of scig remained clinically stable throughout the follow-up period. among the patients receiving pulsed scig treatment, out of ( %) responded to scig similarly to ivig, while three patients ( . %) worsened and needed to be treated again with ivig and the other one ( . %) stopped any therapy. subcutaneously administered immunoglobulin were well tolerated and no patients complained of adverse events. in conclusion, our findings confirm that continuous scig therapy is efficacious in maintaining clinical stability in patients with short-lasting response to ivig. moreover, our data suggest that pulsed therapy with scig may represent an alternative therapeutic option for the treatment of a subset of cidp patients. touvier t , ferri c , mastrangelo r , glimcher l , , wrabetz l , , , d'antonio m . myelin biology unit, division of genetics and cell biology, san raffaele scientific institute, dibit, milan, italy; department of cancer immunology and virology, dana-farber cancer institute, boston, usa; department of medicine, harvard medical school, boston, usa; hunter james kelly research institute, university at buffalo, buffalo, usa; department of biochemistry, university at buffalo, buffalo, usa; neurology, jacobs school of medicine and biomedical sciences, university at buffalo, buffalo, usa. mpz glycoprotein is an abundant product of terminal differentiation in myelinating schwann cells. the mutant mpzs del causes charcot-marie-tooth (cmt) b disease in humans and a similar demyelinating neuropathy in transgenic mice. mpzs del protein is retained in the endoplasmic reticulum (er) of schwann cells and induces an unfolded protein response (upr) characterized by activation of perk, atf and ire /xbp pathways. we have previously reported that activation of chop and gadd , two downstream mediators of perk, is pathogenetic in mpzs del mice (pennuto, ; d' antonio, ) but the role of the other upr branches remains to be investigated. in this study, we investigated the role of the er stress sensor enzyme ire and of xbp -a transcription factor specifically activated by ire -in mpzs del pathogenesis. we generated a new mouse model with schwann cells-specific ablation of xbp and in parallel we exploited mpzs del dorsal root ganglia (drg) explant cultures in which xbp signaling is modulated by gain/loss of function approaches. we observed that absence of xbp dramatically worsens hypomyelination and electrophysiological/locomotor parameters in young and adult mpzs del neuropathic animals. interestingly we observed that perk, atf and ire -mediated ridd signalings are upregulated in neuropathic animals lacking xbp . this suggests that activation of xbp targets have an essential role in limiting mpzs del toxicity, which cannot be compensated by other stress responses. moreover, we demonstrated in mpzs del drg cultures that inhibition of xbp splicing by u c (cross, ) decreases myelination whereas activation of xbp splicing by quercetin (wiseman, ) slightly ameliorates myelination. altogether, these data demonstrate that xbp pathway has a critical adaptive role in mpzs del neuropathy and suggest that activation of this pathway may be beneficial for cmt b and perhaps for a broad range of neuropathies characterized by upr activation. tozza s , bruzzese d , iodice r , esposito m , dubbioso r , ruggiero l , topa a , spina e , santoro l , manganelli f . department of neuroscience, reproductive sciences and odontostomatology, university of naples "federico ii", naples, italy; department of public health, university federico ii of naples, naples, italy. in cmt a patients, the clinical impairment progressively increases over time and correlates with the axonal loss. evidence has suggested that the decline of physical performance in cmt a patients may reflect a process of normal ageing. the aim of our study was to describe, by a case-control cross-sectional design, the progression of physical impairment with ageing in cmt a patients. we enrolled cmt a patients ( m; range - years) and sex-and age-matched healthy controls. to assess physical performance, all patients and controls underwent -meter walk test ( mwt), -minute walk test ( mwt) and -hole peg test ( hpt) of their dominant (d) and non-dominant (nd) sides. moreover, to assess clinical disability, impairment and quality of life in the cmt a group we used the charcot-marie-tooth neuropathy score (cmtns), the mrc sum score and the short form- (sf- ) questionnaire. the linear regression model was used to evaluate the changes over time of clinical measures in patients and controls. the chow test was used to determine whether the ageing had a different impact on clinical measures for the two groups. physical performance worsened with ageing in both patients and controls, but with a greater slope for cmt a patients [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. the rate of deterioration of physical performance was not different between patients and controls until the th year of age. after the th year of age the rate of deterioration became greater in cmt a group [difference in slopes: mwt, . (c.i. . to . ), p< . ; mwt, − . (c.i. - . to − . ), p< . ; hpt-d, . (c.i. . to . ), p< . ; hpt-nd, . (c.i. . to . ), p< . ]. moreover, in cmt a patients also cmtns, mrc sum score and sf- worsened with ageing and with a greater rate of deterioration after the th year of age. our study demonstrates that clinical decline in cmt a patients goes parallel to the normal ageing process until the th year of age, whereupon the clinical deterioration accelerates. tsouni p , devic p , moura b , planque e , bédat-millet al , devaux j , steck aj , delmont e , hottinger af , kuntzer t . dcn, chuv, lausanne, switzerland; centre de référence maladies neuromusculaires, hospices civils de lyon, lyon, france; cabinet médical, epinal, france; département de neurologie, chu de rouen, rouen, france; cnrs, crn m-umr , université aix-marseille, marseille, france; centre de référence maladies neuromusculaires et sla, hôpital la timone, marseille, france. new therapeutic options in immuno-oncology have allowed significant progress in the management of melanomas. treatment usually consists of a combination of two monoclonal antibodies targeting cytotoxic lymphocyte-associated protein and programmed cell death- . as a result, the immunologic barrier protecting tumor cells is overcome allowing an antitumor response. we report cancer patients with immune checkpoint inhibitor-induced neuropathies as a complication of this immunomodulating oncologic treatment. case reports: our index patient developed severe myalgia days after introduction of ipilimumab-nivolumab followed by painful paresthesias of the face and extremities day after the nd cycle of treatment. generalized areflexic quadriparesis (mrc ) with gowers'sign and distal loss of vibratory sensation were found. a -day course of ivig plus corticosteroids (cs) had no effect. three monthly ivig courses were necessary to improve the deficits at months. a survey of sfnp members revealed other patients with similar acute courses but with phenotypes varying from sensorimotor deficits with areflexia and myalgia to purely sensory ataxic forms following immunomodulating treatment. work-up including anti-nodal antibodies were negative in patients. from the other , had abnormal csf and had necrotizing myopathy. detailed repeat ncs demonstrated signs of nerve hyperexcitability and of demyelination or conduction blocks. evolution was slowly favorable following ivig and cs. discussion: our report underscores that atypical acute generalized demyelinating neuropathies are induced by these novel treatments. they may be associated with severe myalgia or other systemic toxic effects. discontinuation of the oncologic treatment depends on severity of symptoms. outcome was slowly favorable following ivig or cs, albeit slower than in the case of primary inflammatory neuropathies, probably given the long half-life of the monoclonal antibodies. gap junctions (gjs) are membrane channels found in most tissues connecting adjacent cells or different cell compartments as in schwann cells. they are involved in electrical connectivity and metabolic homeostasis allowing the passage of small molecules such as ions, second messengers, nucleotides and peptides. an important functional role of peripheral nerve connexins is suggested by their involvement in x-linked inherited neuropathy as well as in acquired neuropathy caused by oxaliplatin. although gjs play a role in electrical connectivity their specific role in the formation of the sciatic nerve compound action potential (cap) remains unclear. the aim of this study was to investigate the role of peripheral nerve connexins in the electrical responses of the mouse sciatic nerve under normal and stress conditions. for this purpose we used sciatic nerves of three different mouse models, the cx knockout (ko), cx ko and the cx /cx double knockout (dko) mice. using our ex vivo model for extracellular recordings we exposed sciatic nerves from different genotypes to three different gj blockers: octanol, -beta-glycyrrhetinic acid (gra) and octanoic acid (oa) and recorded the cap. amplitude and duration of the cap were used as an indication for the effects of the different blockers on the cap formation. all gj blockers caused a gradual decrease of the cap without any changes in the duration of the cap in all genotypes, suggesting progressive disturbance of axonal membrane excitability in the absence of one or two gj proteins. comparison of the three genotypes showed that cx may play a dominant role in the maintenance of the cap formation since nerves from cx ko mice proved to be more sensitive to the gj blockers compared to the cx ko nerves showing a faster decline of the cap amplitude. moreover the effect of gj blockers was similar in cx ko and dko nerves. finally, the effect of gj blockers on the dko nerves implies the presence of another gj protein. in conclusion, our results confirm the direct functional involvement of cx gj channels and cx hemichannels in the cap formation and indicate the existence of at least one more connexin in peripheral nerve. ca( +)-dependent anti-ganglioside antibody in seronegative guillain-barrÉ syndrome uchibori a , gyohda a , chiba a . kyorin university, tokyo, japan. we have reported ca + -dependent igg anti-ganglioside gq b antibodies in gq b-seronegative patients with fisher syndrome and its related disorders (fs-rd). in patients with fs-rd who were gq b-seronegative in conventional assays using phosphate-buffered saline without ca + , % turned seropositive for gq b-related antigens in assays using ca + -added tris-buffered saline. objective: we investigated whether ca + -dependent anti-ganglioside antibodies was present also in ganglioside-seronegative patients with other clinical disease types of guillain-barré syndrome (gbs) other than fs-rd. methods: the subjects were the following: patients with final clinical diagnosis as gbs (acute motor axonal neuropathy [aman], n = , and acute inflammatory demyelinating polyradiculoneuropathy [aidp], n = ), and patients with final clinical diagnosis as sensory ataxic neuropathy (san), n = . all subjects were ganglioside-seronegative in the conventional assays. we assayed serum igg antibodies against various gangliosides (including asialo-gm ) in elisa using tris-buffered saline as a basal buffer in both ca + -added and -non-added conditions. increase of the optical density (od) more than . in ca + -added condition compared with ca + -non-added one was taken significant, i.e. positive for ca + -dependent antibody. results: ca + -dependent antibody was negative in all aman and san patients. in aidp, the antibody titers (ods) against gainac-gd a were significantly increased in patients, and those against asialo-gm were increased in other patients in ca + -added condition. however, the titiers of those ca + -dependent antibodies were all at low level. conclusions: in clinical disease types of gbs other than fs-rd, ca + -dependent igg antibodies against ganglioside were detected in a few patients with aidp, but the positive rate and the antibody titers were low compared with case of gq b-seronegative fs-rd. ca + -dependent antibodies against ganglioside are considered to be more specific for gq b. the aim of the study was to investigate the relationship between median sensory conduction of median nerve and ulnar nerve in patients diagnosed with carpal tunnel syndrome. two hundred and eighty-six hands with carpal tunnel syndrome and hands in control group were investigated. patients were staged clinically and electrophysiologically. diagnosis of carpal tunnel syndrome was made according to the presence of paresthesia, pain in the innervation area of the median nerve, weakness and atrophy in the median nerve innervated muscles, positive phalen and tinel tests. median motor and sensorial nerve conduction study, including first, second, third finger and palm, and ulnar motor and sensorial nerve conduction of fifth finger studies were performed to all patients and control group. the ratio of distal latency and velocity of nerve conduction of first, second, third and palmar branches to fifth finger was calculated. distal latency of first, second, third finger and palm of patients with cts are longer and velocity is more slowly than controls. in addition to these findings, the velocity of fifth finger is also slower and distal latency of this one is longer than healthy subjects. the most sensitive method of classifying the carpal tunnel syndrome as normal, mild and moderate is the ratio of distal latency and velocity of second finger (p< . ). carpal tunnel syndrome is the most common encountered neuropathy. in nerve conduction studies can be used the ratio of distal latency and velocity of second finger as determine the degree of carpal tunnel syndrome. the most surprising finding of this report is the nerve conduction studies of fifth finger. the subclinical susceptibility of fifth finger can be explained by overusing of wrist. ursino g , gemelli c , grandis m , reni l , bellone e , geroldi a , gotta f , mandich p , ferrara m , schenone a dinogmi university of genoa, genoa, italy; irccs-aou san martino hospital genoa, genoa, italy. charcot-marie-tooth (cmt) neuropathy represents a clinically and genetically heterogeneous group of hereditary peripheral neuropathies characterized by chronic motor and sensory impairment. to date mutations in up to genes may cause cmt. the aim of this study is to describe our large population of cmt patients, and, within this, highlight specific phenotypes. the cmt clinic in genova, started in . during the years, patients underwent complete neurological, rehabilitative, neurophysiological examinations and genetic testing. the patients were routinely tested for common genes (as pmp , gjb , mpz, mfn ), while in specific cases we followed the candidate gene approach testing single genes based on the genotype-phenotype correlation. however, the ngs techniques were used when routine genetic testing was negative and a clear genotype-phenotype correlation could not be identified. cases are present to date in our database of cmt patients. in ( . %) patients, in spite of a clinical diagnosis of cmt, a genetic diagnosis is still lacking; ( . %) patients had alternative diagnosis (i.e hereditary spastic paraparesis etc.). instead, in patients ( . %) a defined genetic diagnosis was reached, of them being females ( . %) and males ( . %). among these, except for the more common cmt a, hnpp and cmt x phenotypes, we frequently observed patients affected by cmt b and cmt f. according to most literature data, we observed ( . %) patients with cmt b and patients ( . %) affected by cmt f. at the first visit, the cmt b phenotype was clearly length-dependent: . % patients showed impairment of the lower limbs and saving of the upper limbs; in terms of severity of the neuropathy, the mean cmtns was . and the mean age was . years. similarly, % of patients affected by cmt f, present with the same phenotype; the mean cmtns at the first visit was . and the mean age was . years. in conclusion, based on the experience of the genova cmt clinic, we describe a large population of cmt patients and a specific phenotype in cmt b and f patients, characterized by involvement of the lower limbs and selective sparing of the upper ones, which may help in addressing the diagnostic algorithm. non-freezing cold injury (nfci) develops following sustained exposure to cold temperatures, resulting in tissue cooling but not freezing. this can result in persistent sensory disturbance of the hands and feet including numbness, paraesthesia and chronic pain. both vascular and neurological aetiologies of this pain have been suggested but remain unproven. we prospectively approached patients referred for clinical assessment of chronic pain following non-freezing cold injury between february and november . of patients approached consented to undergo detailed neurological evaluations including: questionnaires to detail pain location and characteristics, structured neurological examination, quantitative sensory testing, nerve conduction studies and skin biopsy for intra-epidermal fibre assessment. of the study participants all had experienced nfci whilst serving in the united kingdom armed services and the majority were of african descent ( . %) and male ( . %). many patients reported multiple exposures to cold. the median time between initial injury and referral was . years. pain was principally localised to the hands and the feet, neuropathic in nature and in all study participants associated with cold hypersensitivity. clinical examination and quantitative sensory testing were consistent with a sensory neuropathy. in all cases large fibre nerve conduction studies were normal. the intra-epidermal nerve fibre density, however, was markedly reduced; ⋅ % of subjects having a count at or below the ⋅ centile of published normative controls. using the neuropathic pain special interest group (neupsig) of the international association for the study of pain (iasp) grading for neuropathic pain % had probable and ⋅ % definite neuropathic pain. chronic non-freezing cold injury is a disabling neuropathic pain disorder due to a sensory neuropathy. why some individuals develop an acute painful sensory neuropathy on sustained cold exposure is not yet known but individuals of african descent appear vulnerable. screening tools, such as the dn questionnaire, and treatment algorithms for neuropathic pain should now be used in the management of these patients. funded by the wellcome trust and the uk ministry of defence guillain-barré syndrome (gbs) is highly heterogeneous regarding clinical presentation, course, electrophysiological subtype and outcome. in part this variety is associated with differences between geographical regions, although this had not been investigated in a single comparative study. one aim of igos is to define the influence of the geographical origin on the heterogeneity of gbs. in igos all gbs patients within weeks of onset can participate, independent of age, variant, severity and treatment. in february , patients were included from countries. the first inclusions in igos were used in this analysis. seventy-five patients ( %) were excluded because of alternative diagnoses (n= , including a-cidp), protocol violations (n= ) or missing data (n= ). of the remaining patients, % were males and % female with a median age of years (iqr - ). at entry % presented with tetraparesis and % with paraparesis. during follow-up % needed mechanical ventilation and % died. of all gbs patients % received treatment ( % ivig, % pe). the remaining % received supportive care only (mild gbs or low social-economic status). antecedent events were reported in % of patients, including upper respiratory tract infection ( %) and gastro-enteritis ( %) as the most frequent events. the pure motor form was the predominant subtype in patients from bangladesh ( %). in europe/americas and asia (without bangladesh) the predominant subtype was the sensorimotor form ( % in europe/americas and % in asia). in asia (without bangladesh) there was a relatively larger proportion of patients with mfs/mfs-overlap syndrome ( %) than in other regions ( - %, p< . ). the proportion of patients able to walk unaided at months after follow-up was % in europe/americas, % in bangladesh and % in asia (without bangladesh). kaplan-meier analysis comparing electrophysiological subtypes of gbs (as reported by neurologist) showed that patients with inexcitable nerves or axonal neuropathy needed more time to regain the ability to walk unaided than the patients with demyelinating or equivocal result (log-rank test, p < . ). these findings demonstrate the extensive geographical differences in gbs. future igos studies will investigate the role of genetic and environmental factors that additionally could explain these differences. van den bergh pyk , attarian s , grapperon am , nicolas g , cassereau j , rajabally ya , delmont e , woodard jl , piéret f and the university of louvain gbs electrodiagnosis study group * . corticosteroids are considered as one of the first line treatments for chronic inflammatory demyelinating polyneuropathy (cidp). different types of corticosteroids are used and there are no comparative studies assessing the improvement rates, remission rates, tolerability and the side effect profiles of these treatment regimens. in addition, there are currently no reliable predictors of favorable treatment response to steroids, which would greatly ease the choice of first line treatment. in this retrospective study we will compare efficacy, tolerability and safety of three different corticosteroid regimens used as first line treatment in three large cidp centers in netherlands, serbia and italy. treatment naïve cidp patients who received either pulsed dexamethasone, pulsed methylprednisolone or daily prednisone will be included in the study. data will be extracted from patient charts. the primary outcome is the percentage of treatment responders at months after start of first treatment, in which treatment response is defined as subjects who improved after treatment and are either without treatment after six months or are still being treated with the first chosen therapy. secondary endpoints include the remission rates and in case of a relapse, the mean duration of remission to relapse; the discontinuation rate within months of treatment due to inefficacy, adverse events or intolerance; and the frequency of adverse events and serious adverse events (sae) during treatment or within month after stopping treatment. furthermore, we will explore the value of previously reported potential predictors of treatment response. results will be presented at the peripheral nerve society meeting . van lieverloo g , , musters a , adrichem m , esveldt r , doorenspleet m , klarenbeek p , van schaik i , de vries n , eftimov f . academic medical center, department of neurology, amsterdam, the netherlands; academic medical center/university of amsterdam, department of clinical rheumatology and immunology, amsterdam, the netherlands. following reports that pathogenic antibodies are present in a minority of patients with chronic demyelinating inflammatory neuropathy (cidp), we studied whether oligoclonal expansions of b-cell clones are present in patients with cidp. recently, we developed a new method for b-cell receptor (bcr) repertoire landscaping based on high throughput sequencing (hts) of rna extracted from blood. bcr repertoire was analyzed in patients with cidp: patients with active disease and starting treatment (group ), patients with stable disease using intravenous immunoglobulin (ivig) treatment in which treatment withdrawal was attempted (group ), and patients in remission (i.e. no treatment in the last months, group ). clinical parameters and sampling was performed at baseline (group , and ), at months after start of treatment (group ), at months or earlier in case of relapse in group and at baseline only in group . most cidp patients had highly expanded bcr clones, regardless of disease activity and response to treatment. however, in group , the most expanded b-cell clones at baseline showed no overlap with the expanded bcr clones after improvement. based on these preliminary data expanded bcr clones are observed in the peripheral blood of most cidp patients, regardless of disease activity (active, stable disease or remission/cure). functional characterization of these expanded clones remains to be performed. van rijs w , fokkink wjr , tio-gillen ap , brem md , jacobs bc , huizinga r . erasmus mc, university medical centre, rotterdam, the netherlands. myeloid cells, including monocytes, macrophages and dendritic cells, are critically involved in the induction of adaptive immune responses, clearance of pathogens and in the initiation of tissue repair. in the guillain-barré syndrome (gbs), macrophages are present in the peripheral nerve, where they phagocytose (damaged) myelin and axons. dendritic cells (dc) are increased in the cerebrospinal fluid of patients with gbs. however, the composition and phenotype of monocytes and dc subsets in the peripheral blood is unclear and it is unknown if these cells can be used as biomarker to monitor disease activity or response to treatment. here we investigated the frequency and phenotype of six myeloid subsets in the peripheral blood mononuclear cells (pbmc) using advanced -color flow cytometry. pbmc were isolated from patients with gbs, before and after immunomodulatory treatment, and age and gender-matched, healthy controls. the frequency of total monocytes, determined as percentage of cd + cells, was increased in gbs patients compared to controls (p< . ). the monocyte population was skewed towards more intermediate (cd +cd +; p< . ) and less non-classical (cd -cd +; p< . ) monocytes. classical (cd +cd -) and intermediate monocytes as well as cd c+ dc expressed significantly higher levels of cd compared to healthy controls. in contrast, the expression of cd and siglec- was significantly higher in the non-classical monocytes of gbs patients compared to controls. no differences were observed in the expression of cd , cd , cd and siglec- . immunomodulatory treatment strongly reduced the frequency of non-classical monocytes and all dc populations in cd + pbmc. the expression of cd , cd c and hla-dr was reduced in classical monocytes after treatment. in addition, siglec- expression was reduced in several monocyte and dc populations after treatment. in summary, our data identify significant changes in the monocyte compartment in gbs. the decrease in non-classical monocytes may suggest that these cells have migrated to peripheral tissues, promoting the differentiation of classical monocytes into intermediate monocytes. further analysis should reveal whether these changes are related to preceding infections, disease severity and response to treatment. in mme. the two mutations were absent from control databases (e.g. exac), affected highly conserved aminoacids and were predicted to have deleterious effects by in silico analysis. unfortunately, both parents were deceased and we were therefore unable to prove that the two mutations reside on separate alleles. mme encodes the metalloprotease neprilysin whose role in peripheral nervous system is still unclear. higuchi and colleagues have described japanese cmt families with late-onset sensory-motor axonal neuropathy and recessive loss-of-function mutations in mme. we report two novel missense mutations in mme in a case of late-onset cmt . we hypothesize an autosomal-recessive mode of inheritance as most likely given the clinical phenotype and the absence of a family history. anti-contactin- (cntn ) antibodies were recently identified in a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (cidp) showing acute/subacute onset of severe sensory-motor neuropathy and poor response to intravenous immunoglobulin (ivig) and corticosteroids. these antibodies belong to the igg isotype and interact with cntn -neurofascin (nf ) complex at paranodes leading to loss of nodal integrity. a -year-old man presented with acute onset of distal weakness in the lower limbs, four limbs paraesthesias and sensory ataxia. at clinical examination ankle swelling was also observed. nerve conduction study showed a demyelinating polyneuropathy and cerebrospinal-fluid examination revealed cyto-albuminologic dissociation. sural nerve biopsy disclosed diffuse loss of myelinated fibres. at routine blood test serum albumin was reduced and proteinuria was gr/ hours, thus leading to the diagnosis of nephrotic syndrome. kidney biopsy showed changes consistent with membranous glomerulonephritis, together with sub-epithelial deposits of immune complexes and complement deposition. treatment with ivig and corticosteroids did not improve neurological status, while membranous glomerulonephritis showed moderate response to ivig. a six-month course of cyclophosphamide was started leading to normalization of renal function and muscle strength and partial improvement of sensory ataxia. the patient did not require any further treatment and after years his condition remains stable. cntn antibodies were tested on a recently collected patient's serum and resulted positive on both elisa and cell-based assay. the patient here reported showed the typical clinical features of anti cntn -associated cidp including older age, acute onset, severe motor impairment and sensory ataxia. the contemporary occurrence of membranous glomerulonephritis was reported in only one other case. contactin- is expressed at low levels in the kidney and a direct damage of anti-cntn antibodies could be hypothesized. alternatively, renal damage might have been secondary to unspecific immune complexes deposition. a good response to anti-cd rituximab was recently reported in patients with cidp associated with anti-cntn and anti-nf antibodies. notwithstanding this single-case observation, our report suggests that also cyclophosphamide may be considered an effective therapy in anti-cntn antibodies-associated cidp and membranous glomerulonephritis, leading to persistent clinical remission. velasco r , , besora s , santos c , sala r , izquierdo c , simó m , gil-gil m , , jiménez l , pardo b , calvo m , palmero r , clapés v , bruna j duloxetine is the only agent demonstrated effective in treating pain related with chemotherapy-induced peripheral neuropathy (cipn). patients with symptomatic cipn treated with duloxetine were retrospectively collected in a single-institution. aim of the study was to evaluate the drug's efficacy and rate of compliance. only patients with cipn with distressing positive symptoms (pain, numbness and/or paresthesia), and non-progressive disease were included. cipn was graded employing the total neuropathy score (tns © ) and national cancer institute-common toxicity criteria. response to duloxetine was assessed with patient global impression of change (pigc) scale ( : no benefit; : excellent response). consecutive first one-hundred cipn patients treated with duloxetine were analyzed. median age was ( - ). , , and received platinum, taxane, bortezomib and vincristine-based regimen, respectively. median tnsc © was ( - ). severity of neuropathy was grade ( %), grade ( %), and grade ( %). sixteen patients were on treatment with other analgesic agents. median time from finishing chemotherapy to duloxetine initiation was months [ - ]. median pigc score was [ - ]. among responders, . % and . % scored low ( - ) and high ( - ) benefit, respectively. fifty-seven ( %) patients discontinued early duloxetine due to intolerable side effects (n= ) the goal of this study was to determine whether predicted fork stalling and template switching (fostes) during mitosis deletes exon in peripheral myelin protein kd (pmp ) and causes a gain-of-function mutation associated with peripheral neuropathy in a family with charcot marie tooth disease type e. two siblings previously reported to have genomic re-arrangements predicted to involve exon of pmp were evaluated clinically and by electrophysiology. skin biopsies from the proband were studied by rt-pcr to determine the effects of the exon re-arrangements on exon mrna expression in myelinating schwann cells. transient transfection studies with wild type and mutant pmp were performed in cos and rt cells to determine the fate of the resultant mutant protein. both affected siblings had a length-dependent demyelinating neuropathy with severely slow nerve conduction velocities (< m/sec). rt-pcr studies of schwann cell rna from one of the siblings demonstrated a complete in frame deletion of pmp exon (pmp delta ). transfection studies demonstrated that pmp delta protein is retained within the endoplasmic reticulum and not transported to the plasma membrane. our results confirm that that fostes mediated genomic rearrangement produced a deletion of exon of pmp , resulting in expression of both pmp mrna and protein lacking this sequence. in addition, we provide direct experimental evidence for endoplasmic reticulum retention of the mutant protein suggesting a gain-of-function mutational mechanism consistent with the observed cmt e in this family. pmp delta is another example of a mutated myelin protein that is misfolded and thus likely to contribute to the pathogenesis of the neuropathy. in poems and cidp, distal limb nerve conduction studies are limited in identifying demyelination and detecting treatment effects in severely affected patients. blink reflex r latency may help to not only identify demyelination but also provide a meaningful treatment outcome measure especially in severely affected patients. poems and cidp patients having undergone routine nerve conductions and blink reflex testing were identified. correlation between r latency, limb nerve conduction studies and neuropathy impairment scores (nis) was calculated with treatment. blink reflexes were performed in patients ( poems, cidp; nis range: - points). overall, r latency prolongation occurred in . % of patients ( . % poems, . % cidp). patients with r prolongation (> ms) had more severely affected nerve conductions in both poems (ulnar cmap . mv vs . mv, p= . ) and cidp (ulnar cmap . mv vs . mv, p< . ). r latency correlated with nis severity in poems better than cidp (r = . vs . , p=< . vs. . ). follow-up nis and r latency evaluations after treatment were available in patients ( poems, cidp). the r latency changes were concordant with the nis changes in % of poems and % of cidp patients. in severely affected patients [ulnar cmap amplitude ≤ . mv ( . %: / )] except for one, all had prolonged r (> ms), allowing for treatment follow up and initial diagnosis. blink reflex r latencies are valuable in defining demyelination in severely affected poems and cidp patients, but also provide a sensitive, early treatment outcome measure among those same severely affected patients. watson dj , martinez c , wallenhorst c , hubsch a , shebl a , simon tl . csl behring llc, king of prussia, usa; institute for epidemiology, statistics and informatics gmbh, frankfurt, germany; csl behring ag, bern, switzerland; csl behring gmbh, marburg, germany. chronic inflammatory demyelinating polyneuropathy (cidp) rarely occurs in children. clinical trials in pediatric patients have not been performed and there are little data on therapy with intravenous immunoglobulin (ivig). we performed an observational trial to investigate the risk of adverse events of special interest (aesi) with ivig, i.e. hemolytic anemia, aseptic meningitis, acute renal failure, thromboembolic events and anaphylactic reactions. the study cohort was derived from the us premier perspective database and consisted of patients < years with a diagnosis of cidp (icd- cm diagnosis code . ) treated with the ivig privigen ® (csl behring, bern switzerland) between jan and dec . we identified pediatric cidp patients: preschool children (age - years at first treatment for cidp), children ( - years) and adolescents ( - years); females and males; white, one black and two allocated to "other race". six patients had a history of other ivig use for guillain-barré syndrome, one patient for myasthenia gravis and one for immunodeficiency before the diagnosis of cidp. the mean privigen ® dose calculated from the cumulative quantity of privigen ® per treatment episode and the corresponding age-and gender specific median of the us population body weight estimate was . g/kg body weight. the number of recorded treatment episodes per patient ranged from to . using an at-risk period of days for hemolytic anemia, and days for other aesi after each privigen ® administration, no aesi were observed in the patients with cidp with a total of person-days at-risk for ha and person-days at-risk for other aesi. this observational study shows that ivig (privigen ® ) is used for treatment of cidp in pediatric patients with or without concomitant conditions and revealed no particular safety issues in this patient group. hsn- is a peripheral neuropathy most frequently caused by missense mutations in the sptlc or sptlc genes, which code for two subunits of the enzyme serine palmitoyltransferase (spt). spt catalyzes the first and rate limiting step of de novo sphingolipid synthesis. it has been shown that mutations in spt cause a change in enzyme substrate specificity which results in the production of two atypical sphinganines, deoxysphinganine (dsp) and deoxymethylsphinganine (dmsp), rather than the normal enzyme product, sphinganine (sp). levels of deoxysphingolipids are elevated in the blood of hsn- patients and this has been shown to cause the peripheral nerve damage characteristic of the disease, which affects both sensory as well as motor axons. however, the underlying pathomechanism of how deoxysphingolipids damage neurons remains elusive. here, dsp and dmsp-mediated neurotoxicity was examined in primary mouse motor and sensory neurons, by assessing cell survival and neurite outgrowth following exposure to different concentrations of sp, dsp or dmsp. the abnormal enzyme products were found to have a rapid and dose-dependent neurotoxic effect in primary neurons. we also explored the potential mechanisms that underlie deoxysphingolipid neurotoxicity, by characterizing mitochondrial function and changes in calcium handling. we found that mitochondrial dysfunction and calcium handling deficits may be key mediators of abnormal sphingolipid neurotoxicity, in both motor and sensory cell models. specifically, we revealed mitochondrial abnormalities, signs of endoplasmic reticulum stress and dysfunction of store-operated calcium channels. we propose that early deficits in mitochondria and calcium handling may underlie deoxysphingolipid neurotoxicity and thus present potential therapeutic targets for hsn- . months. one patient was excluded because the decrease in hba c was not contemporaneous with weight loss. the mean and median decrease in bmi per month was . and . respectively. the mean and median interval between surgery and decrease in hba c was . and . days respectively. records of these patients were scrutinized and classified as: 'probable tind': acute painful neuropathy and acute dysautonomia with temporal relationship to the decrease in hba c; 'possible tind': acute painful neuropathy or acute dysautonomia or temporal relationship to decrease in hba c is uncertain; 'unlikely tind': when an alternative explanation exists for symptoms. only one patient was classified as 'possible tind': a -year-old woman who developed neuropathic pain in both lower limbs month after a rapid hba c decline of . % and about months after bariatric surgery. she had no documented autonomic symptoms. our study is limited by small cohort size, retrospective design and reliance on hospital records. nonetheless, it demonstrates that besides nutritional neuropathies, tind should also be considered in dm patients who develop peripheral neuropathy after bariatric surgery. in another study, we found tind is uncommon in a general cohort of diabetic patients. the occurrence of 'possible tind' in only dm patients corroborates earlier data that weight loss may act in tandem to increase the risk of tind. woolums bm , tabuchi m , sung h , sullivan jm , mamah c , yang m , blum id , wu mn , sumner cj , lloyd te . johns hopkins university, baltimore, usa. dominant missense mutations in the gene encoding the cation channel transient receptor vanilloid, family member (trpv ) cause inherited neuropathies including charcot-marie-tooth disease c (cmt c). in vitro, mutations in trpv that cause cmt c cause a gain of trpv channel function and increased intracellular calcium which subsequently leads to cellular toxicity. however, the mechanisms by which cmt c mutations in trpv lead to neuronal dysfunction in vivo remain poorly understood. we generated transgenic drosophila that express either wild-type or a cmt c causing trpv mutant (trpv r c ) to assess the effect of trpv r c on neuron function in vivo. selective expression of trpv r c in drosophila ccap neurons (n ccap ) results in a failure of drosophila wing expansion that is blocked by genetically inactivating the channel pore, demonstrating the requirement of channel function in mediating this phenotype. perforated patch clamp analysis of n ccap reveals that trpv r c causes a calcium-dependent increase in n ccap neuronal excitability. this hyperexcitability is restored to control levels by application of a trpv selective antagonist. high level expression of trpv r c causes synaptic and dendritic degeneration, both of which are rescued genetically by inactivating the channel pore or pharmacologically by feeding larvae a trpv selective antagonist. we conducted a genetic screen in n ccap and found that camkii knockdown potently suppresses the trpv r c mediated wing expansion phenotype and selectively rescues degeneration of synapses but not dendrites. we also find that trpv r c , but not controls, disrupts mitochondrial transport in axons by increasing the number of stationary mitochondria. our data demonstrate that trpv r c elevates neuronal intracellular calcium which disrupts mitochondrial transport and mediates neurodegeneration through compartment-specific calcium-mediated signaling pathways, and supports the further investigation of trpv antagonists as potential therapeutics for the treatment of cmt c. mood disorders, including anxiety and depression, are commonly observed among chronic pain patients with prevalence estimates ranging from to %. comorbidity between mood and chronic pain disorders has been linked to altered limbic regulation of the hypothalamic-pituitary-adrenal (hpa) axis. stress activates the hpa axis and can initiate and/or exacerbate symptoms related to both chronic pain and mood disorders. previous studies from our laboratory have investigated the influence of early life stress on mechanical pain hypersensitivity, visceral hypersensitivity and behavioral evidence of mood disorder later in life. here, we are testing the hypothesis that chronic stress exposure in adulthood can increase somatic and visceral sensitivity and anhedonic behaviors in a mouse strain with a genetic predisposition to anxiety. adult, female a/j mice were exposed to repeated foot shock stress for continuous days and tested for alterations in mechanical sensitivity, sucrose preference, visceromotor response (vmr) during urinary bladder distension, and serum corticosterone levels. mice that underwent shock stress had a significantly decreased mechanical withdrawal threshold in the hind paw compared to their baseline and sham group measurements. sucrose preference was measured prior to shock exposure and throughout the shock paradigm as an indicator of anhedonic behavior. mice that underwent shock stress displayed a trend toward decreased sucrose preference, indicating anhedonia, in comparison to mice in the sham group that did not display anhedonia. mice that underwent shock stress displayed significant increases in vmr during bladder distension compared to sham mice. finally, serum corticosterone levels were significantly higher in the mice that underwent shock stress compared to sham mice, indicating a stress-induced increase in hpa axis output. together these data suggest that chronic stress exposure can induce mechanical allodynia, visceral hypersensitivity, and depression-like behaviors in an anxiety-prone mouse strain. future studies will incorporate gene expression in the hypothalamus, amygdala, and hippocampus, as well as investigation of possible downstream peripheral neuroimmune modulation and neuronal morphology changes. guillain-barré syndrome (gbs) is an acute monophasic immune-mediated neuropathy. as prognostic markers of gbs, modified erasmus gbs outcome score (megos), erasmus gbs respiratory insufficiency score (egris), and Δigg have been reported. however, the proportions of subtypes of gbs are known to be different between the western countries and japan, it remains to be elucidated whether those markers can also be applied to gbs in japan or not. we here investigated retrospectively gbs patients and determined the megos and the egris of those cases. among them, Δigg could be obtained in cases. we evaluated the prognosis using gbs outcome score (functional grade: fg) at months; we called good prognosis when fg at months was less than and poor prognosis when that was or more. as a result, in cases with higher score than at megos on admission cases ( %) had poor prognosis and in cases with higher score than at megos at day of admission cases had poor prognosis. in cases with higher score than on egris cases ( %) needed the mechanical ventilator. patients with good prognosis had higher Δigg(average: mg/dl) than patients with poor prognosis(average: mg/dl). we calculated the cut-off value of Δigg in patients, which was mg/dl. patients with higher Δigg than mg/dl could significantly walk independently at six months (p< . ). patients ( %) had poor prognosis in patients with lower Δigg than mg/dl. ( %) of patients were treated with the single cycle of intravenous immunoglobulin (ivig). other patients ( %) were treated with the combined therapies, such as intra venous methylprednisolone and/or plasmapheresis or the second cycle of ivig in addition to ivig. although there was no difference in prognosis between patients with the single cycle of intravenous immunoglobulin (ivig) and patients with the combined therapies, in the patients who had fg> and megos > on admission, the combined therapies made better prognosis than the single course of ivig (p< . ). we found that megos, egris and Δigg were also available in japan. the efficacy of the combined therapies in severe gbs patients should be investigated in the future large scale prospective studies. yiu em , , , burns j , , , menezes mp , , ryan mm , , and for the paediatric cmt best practice guidelines consortium. royal children's hospital melbourne, melbourne, victoria, australia; murdoch childrens research institute, melbourne, victoria, australia; university of melbourne, melbourne, victoria, australia; university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia. charcot-marie-tooth disease (cmt) often presents during childhood. common symptoms include weakness, limb pain and cramps, foot deformity, and balance impairment. guidelines for the optimal management of common problems experienced by children with cmt do not currently exist. development of these guidelines will provide an evidence base for the management and monitoring of children with cmt. a series of systematic literature reviews utilising the grade (grades of recommendation, assessment, development, and evaluation) approach were conducted to answer pre-specified key clinical questions related to the management of paediatric cmt. these included treatment recommendations for symptoms such as weakness, pain, balance impairment, joint deformity, and impaired upper limb function, and anticipatory monitoring for associated complications such as hip dysplasia. this yielded minimal to no evidence for the pre-specified clinical questions, and evidence-based management recommendations could not be made. consensus-based statements will therefore be formulated via a three-round delphi process, to be conducted in . the delphi panel will consist of local and international medical and allied health professionals who have experience in the management of children with cmt. efficacy of pxt in the treatment of adult patients cmt a (n= ) was shown in a multicenter, randomized, double-blind, placebo-controlled phase ii study (attarian et al. ) . pxt taken x/day, orally, for consecutive months was well tolerated and safe. significant improvement of disability was observed for the highest tested dose, thought indicative for an early, meaningful change in disease course (meta-analysis by mandel et al., ) . this formed the rationale to initiate a multicenter, randomized, double-blind, placebo-controlled pivotal phase iii study (clin-icaltrials.gov: nct ) of pxt in mildly to moderately affected cmt a patients in december . the primary objective is to assess the efficacy of doses of pxt compared to placebo on disability as measured by the mean change from baseline overall neurology limitations scale (onls) score at month and . furthermore, efficacy on the proportion of responders (i.e. improvement of onls), impairment (cmtns-v ), functional tests ( -mwt, qmt, -hpt), electrophysiological parameters (cmap, snap and ncv) and quality of life (eq- d, vas) are secondary endpoints. pursuant this study, patients will be eligible for a -month extension study, in which pxt assigned patients will continue with the previously assigned dose, whereas placebo patients will be randomized to one of the two pxt doses. the study is conducted in investigational sites in countries (eu, canada and us). in december patient randomization was completed (n= ). the screen failure rate was %, as expected ( patients were screened). the independent dsmb recommended to continue the study as planned following a safety analysis on the first patients treated for > months. preliminary baseline characteristics are based on patients (data not cleaned). the study population had a mean age of . ± . years (range - ; male . %) of which . % had a confirmed genetic diagnosis of cmt a. the mean cmtns-v was . ± . and the mean motor nerve conduction of the ulnar nerve was . ± . m/s. ten patients withdrew from the study, due to adverse events unrelated to study treatment. the last patient completing the study is expected in march . zhang g , ghosh p , lin j , ghauri a , sheikh ka . department of neurology, university of texas health science center at houston, houston, tx, usa. assessment of epidermal nerve fiber density and its structure integrity is critical for the diagnosis and evaluating the effectiveness of potential therapies in small fiber neuropathies. currently, skin biopsies, at multiple sites, are most commonly used to assess these diseases. these studies are expensive and time consuming due to cumbersome processing and quantification techniques and serial biopsies over time are often not feasible due to costs and patient acceptance. moreover, vast majority of normative data for skin biopsies in humans are available only for few distal sites and a significant proportion of patients with small fiber neuropathies have focal or regional symptoms not involving the commonly biopsied sites in the leg. live imaging could overcome these limitations and provide a noninvasive real time assessment of epidermal nerve fibers all over the body. we previously found that anti-ganglioside antibody (aga) is an effective neuronal delivery vector for transport of various cargos, such as fluorescent dyes, to peripheral nerves. in the current proof of concept study, we examined whether non-invasive multiphoton microscopy can be used to probe/image the epidermal nerve fibers in living animals after systemic and/or local delivery of fluorescently conjugated aga. we found that the individual nerve endings in skin and cornea are distinctly labeled, and visualized under two-photon microscope. the epidermal nerve fiber labelling by fluorescent-tagged aga was further validated using transgenic mice selectively expressing yellow fluorescent protein in their nervous system. in-vivo multiphoton imaging provide a tool with potential for dynamic longitudinal evaluation of small fiber neuropathies, including nerve degeneration and regeneration, without tissue removal. thus, the use of multiphoton microscopy in conjunction with fluorescently labeled aga as neuronal vector can have many research and clinical applications, such as labeling and live visualization of epidermal nerve fibers to assess small sensory nerve fibers in health and disease. zhou y , tavori h , lee s , al salihi m , fazio s , notterpek l . mcknight brain institute, university of florida, gainesville, florida, usa; knight cardiovascular institute, oregon health and science university, portland, oregon, usa. the majority of hereditary neuropathies are due to abnormalities in peripheral myelin protein (pmp ), whose genetic variants include increased expression (gene duplication), haploinsufficiency (gene deletion), or point mutations. phenotypic heterogeneity in clinical presentation is common among hereditary neuropathy patients even within the same family, the cause of which has not been determined. to investigate the role of pmp in the pathogenesis of the neuropathies we have generated and characterized pmp null (pmp −/− ) mice (amici et al., ) whose peripheral nerves show alterations in lipid metabolism (lee et al., ) . to examine the molecular changes underlying these abnormalities we determined the expression of cholesterol synthesis (srebp pathway), and cholesterol uptake, transport and efflux genes (lxr pathway). in affected nerves, we found the cholesterol synthesis pathway inhibited, while the lxr pathway, and particularly apoe and abca , upregulated at the mrna and protein levels. since pmp is expressed at low levels in the liver, the central organ for the regulation of cholesterol in the body, we studied liver tissue form pmp −/− mice. liver from pmp −/− mice showed significant hepatomegaly, clear features of microvesicular steatosis, as well as marked increase in lxr pathway genes and proteins (abca and apoe), as compared to wt. ultrastructural studies identified lipid droplets and significantly enlarged mitochondria in the liver of male pmp −/− mice, which is not due to mitochondria fusion, as the levels of mfn and remained similar to wt. as disturbed hepatic cholesterol homeostasis induces the activation of kupffer and stellate cells, we determined the extent of inflammation in nerve and liver tissues from pmp −/− mice with leukocyte (cd b) and macrophage markers (cd ). in nerve sections, we detected an increase in the number of cd b+ cells, which was confirmed by western blots. in the liver of pmp −/− mice we found a significant increase in cd -reactive kupffer cells and elevated levels of tnf-alpha. the severe dysregulation of cholesterol metabolism in nerve and liver, including neuroand hepatic inflammation in the absence of pmp − suggest that dysregulated cholesterol metabolism and inflammation may act as a disease modifier in pmp -dependent neuropathies. our aim was to ascertain frequency of and risk factors for tee, arterial (ate) and venous thromboembolic events (vte) in neuromuscular patients receiving regular ivig. we performed a retrospective case-note review of inflammatory neuropathy patients receiving regular ivig treatment. we collected the following data over a month study period this analysis suggests tee incidence is higher in ivig treated patients than comparable population-based rates. examination of tee occurrence in age and vascular risk factor matched ivig-treated and ivig-naïve individuals is required to appreciate the excess risk associated with ivig treatment. référence des maladies neuromusculaires et la sla, hôpital de la timone ha) and whether temporal dispersion (td) parameters are helpful. fifty-eight patients diagnosed according to asbury and cornblath ( ) were prospectively included between january and september . edx and were performed - (mean= ) and - days (mean= ) after disease onset, respectively. there were no differences in classification consistency between ho and ha at edx (p= . ) and edx (p= . ), but more patients were classified as aman when comparing ra with ho and ha at edx (p< . ). at edx , ra classified more patients as equivocal with ho (p< . ) and as aman with ha (p< . ) ) or edx (ho, fe p= ; ra, fe p= . ; ra with td, fe p= . ). gm , gd a, gd b and gq b igg antibodies were tested (willison at edx , only ho showed maybe more antibodies with aman compared to aidp (fe p= . , phi=− . ) and with aman compared to equivocal cases since correlation with factors associated with axonal gbs, in casu rcf and antibodies, is far from exclusive, the usefulness of edx subtype classification using specific criteria sets, remains doubtful. the frequency of rcf indicates that nodal/paranodal alterations may represent the main pathophysiology in more gbs patients than currently thought ruiz m , lessi f , cacciavillani m , riva m , salvalaggio a , campagnolo m , briani c . neurology, department of neuroscience, university of padua, padua, italy; hematology and clinical immunology unit, department of medicine, university of padua, padua, italy; cemes, data medica group, padua, italy.arsenic trioxide (ato) is highly effective in treatment of acute promyelocytic leukemia (apl). it is licensed in italy for treatment of relapsed-refractory apl and for first line chemotherapy in low risk patients. ato most frequent side effects are well described, but less is known on ato induced. we describe apl patients who were treated with all-trans retinoic acid (atra)/ato as first line therapy. the characteristics of ato induced neuropathy was prospectically analyzed by neurological evaluation using both the total neuropathy score, clinical version (tnsc) (a validated scale for chemotherapy induced peripheral neuropathy) and neurophysiological assessment. patients have been evaluated at baseline, at the end of the induction phase, at the end of ato/atra treatment and year after discontinuation of treatment. baseline neurophysiology was performed at the end of induction phase. both patients were men, respectively and -yr-old. none of the patients had previous history of neuropathy. baseline tnsc was (no clinical signs of neuropathy) in both patients. neurophysiological evaluation performed after the end of induction cycle did not reveal signs of peripheral neuropathy in both patients. patient received mg of ato during induction, total , mg. patient received mg of ato during induction, total , . both patients developed leg numbness during consolidation cycles and patient also hand numbness. tnsc at the end of therapy was in patient and in patient . neurophysiology at the end of therapy detected signs of sensitive axonal neuropathy in both patients. they received full doses of ato consolidation ( . mg/kg/day for days/week, on alternate months for total months and tretinoin weeks on weeks off). during the first year of follow-up both tnsc and neurophysiology year after the end of consolidation cycle were consistent with full recovery. our patients developed sensory axonal neuropathy during ato therapy, that clinically manifested during consolidation cycles and improved up to complete recovery during follow-up. published case reports show that outcomes may not be as good as in our patients. a multicenter prospective study evaluating the characteristics of ato-induced neuropathy in apl is ongoing. ruiz m , campagnolo m , salvalaggio a , cacciavillani m , taioli f , fabrizi gm , briani c . department of neuroscience, neurology unit, university of padua, padua, italy; data medica group, emg unit, cemes, padua, italy;mutations in the mitochondrial copper-binding protein sco , cytochrome c oxidase assembly protein, have been reported in several cases of fatal infantile cardioencephalomyopathy with cox deficiency. we identified compound heterozygous variants in sco in two unrelated patients with isolated length dependent axonal sensorimotor polyneuropathy of variable clinical severity (axonal autosomal recessive charcot-marie-tooth disease type , cmt ) by whole exome sequencing. although peripheral neuropathy has been described as a secondary feature in a few cases of fatal infantile cardioencephalomyopathy, the disease onset, clinical phenotype and survival in our patients differ significantly from the previously described cases. our patients developed predominantly motor neuropathy; moreover, they are still alive and they have not developed cardiomyopathy, which is the main phenotype and cause of death at early infancy in reported patients. both of our patients harbor mutations adjacent or near the conserved copper-binding motif (cxxxc), including the common reported pathogenic variant e k and the novel change d g. in addition, each patient carries a second mutation located in the same loop region of the protein, p t and r q. western blots from fibroblasts from these cmt patients showed reduced levels of cox , a subunit of complex iv, indicating cox deficiency. our findings demonstrate that cmt can be the predominant phenotype associated with sco mutations, pointing to a broader phenotypic heterogeneity. the mechanism linking mitochondrial respiratory chain dysfunction to isolated axonal loss of variable severity remains to be elucidated. the muscarinic acetylcholine (ach) type receptor (m r) is a metabotropic g protein-coupled receptor expressed by adult sensory neurons. cholinergic signaling through muscarinic receptors can modulate axonal plasticity in invertebrates and lower vertebrates. we have recently shown that selective (pirenzepine) and specific (muscarinic toxin : mt ) m r antagonists elevate neurite outgrowth and protect from small and large fiber neuropathy in adult sensory neurons in various animal models (calcutt, et al., ) . furthermore, we demonstrated that excessive cholinergic signaling due to m r overexpression caused a significant reduction in neurite outgrowth (calcutt, et al., ) . the mechanism of m r-antagonist driven neurite outgrowth remains poorly understood, however, we have proposed that ach constrains axonal outgrowth via m r activation. cholinergic signaling is mediated via recruitment of trimeric g proteins, of which g alpha- and g alpha- regulate cytoskeleton dynamics by control of tubulin polymerization. activated gtp-bound g-alpha proteins destabilize microtubules by increasing the intrinsic gtpase activity of tubulin. we have therefore tested the hypothesis that cholinergic signaling regulates neurite outgrowth via modulation of g protein mobilization and the dynamics of the tubulin cytoskeleton. we found that over-expression of m r in adult sensory neurons induced dissolution of the tubulin cytoskeleton in distal neurites. g alpha- expression in adult sensory neurons was significantly higher (p< . , -fold) than g alpha- . subsequent knockdown the peripheral neuropathy research registry (pnrr) is a multicenter collaborative research project sponsored by the foundation for peripheral neuropathy to advance the science in distal symmetrical polyneuropathies (dsp). the registry was designed to prospectively characterize clinical phenotype and natural history of patients with dsp and obtain biofluids to identify new causes and genetic modifiers of dsp with careful genotype/phenotype correlations, and to develop biomarkers. the enrollment in the registry is still ongoing but an interim analysis was carried at the end of december . eligible study participants were years or older with a diagnosis of idiopathic, diabetic, chemotherapy-or hiv-induced peripheral neuropathy. they were examined by a physician at one of the six consortium members (johns hopkins university school of medicine, icahn school of medicine at mount sinai medical center, beth israel deaconess medical center, northwestern university medical center, university of utah medical center and kansas university medical center). the collected data set included ( ) a detailed questionnaire, discussing their symptoms, medical history and family history, ( ) a standardized neurological examination form, ( ) electrodiagnostic evaluation and ( ) diagnostic laboratory testing. blood samples (whole blood, plasma and serum) were collected for future biomarker and genotyping evaluations. at the end of , complete data sets and blood samples were collected from patients. % had diabetic pn, % had hiv-associated pn, % had chemotherapy induced pn and % were diagnosed with idiopathic pn. detailed analysis of clinical presentation, examination findings and diagnostic investigations will be discussed at the presentation. standardized phenotyping with linked bio-specimen banking will help establish the minimum data set required for neuropathy diagnosis and support genotype-phenotype correlations with next generation sequencing technologies and development of novel biomarkers. pnrr data will improve our understanding of disease mechanisms paving the way for new therapeutic discoveries in painful and non-painful neuropathies. chemotherapy-induced peripheral neuropathy (cipn) is a major side effect of treatment, typically presenting as a sensory neuropathy. symptoms include pain, paraesthesia, and numbness in the extremities, resulting in functional impairment. increasingly, patient reported outcomes (pros) are utilized to accurately examine the impact of cipn symptoms on patient function. however, the links between objective neurological assessment and pro measures remain ill defined. this study aimed to identify links between neurophysiological measures and pros in patients treated with neurotoxic chemotherapy. assessments were conducted in patients (f= , mean age . ± . years) who had completed neurotoxic chemotherapy on average . ± months previously (platinum-based n= , taxane-based n= or taxane/platinum combination therapy n= ). patients reported the presence and severity of neuropathic symptoms via the fact-gog ntx , a validated patient questionnaire. clinical the most important neurological side effect of a large number of anti-cancer drugs is a painful peripheral neuropathy. mainly chemotherapeutics that interfere with microtubules, including the plant derived vinca-alkaloids such as vincristine, are well known to cause chemotherapy-induced peripheral neuropathies (cipn). to date, few treatments are available and focus on symptom alleviation and pain reduction rather than on preventing the neuropathy all together. for the first time, we highlight the potential of specific histone deacetylase (hdac ) inhibitors as a preventive therapy for cipn, using novel rodent models for vincristine-induced peripheral neuropathies (vipn), characterized by a sensory axonopathy. one reason so few therapies are available, is because the exact pathophysiological mechanisms are poorly understood. mounting evidence proposes axonal transport, a pathway frequently disturbed in neurological disorders, as a major player in the pathophysiology of vipn. proper axonal transport requires dynamic microtubules which are highly modulated by post-translational modifications. since vincristine interferes with the polymerization of microtubules, we reason disturbances in microtubule dynamics, and by extension axonal transport, could contribute to vipn. we illustrate that increasing acetylation of -tubulin after hdac -inhibition, can restore vincristine-induced defects in axonal transport in cultured dorsal root ganglion neurons. also in vivo, -tubulin acetylation was restored in the saphenous nerve and dorsal root ganglia, two sensory tissues that are affected by vincristine. ultimately, this correlates to a reduced severity of the neurological symptoms, both on the electrophysiological and on the behavioral level. moreover, we discovered that hdac -inhibition was not only protective against neurotoxicity, but also reduced tumor progression in a mouse model for acute lymphoblastic leukemia. taken together, our results show that hdac -inhibition is an ideal strategy to prevent vipn with beneficial effects both on the neurotoxicity as well as on tumor growth.approximately two-thirds of cidp subjects need long-term corticosteroids or intravenous immunoglobulins (ivig), with ivig being slightly preferred based on safety profiles. subcutaneous ig (scig) is an alternative option for ig delivery but has not previously been investigated in a large-scale clinical trial in cidp.we performed a randomized, double-blind trial in cidp investigating . and . g/kg weekly doses of scig igpro (hizentra ® , csl behring) versus placebo in subjects for maintenance treatment. ivig-dependent adults with definite or probable cidp according to efns/pns criteria were eligible. the primary outcome was the percentage of subjects with a cidp relapse ( -point deterioration on adjusted incat disability score) or who were withdrawn for any other reason during the -week scig-treatment period. multiple secondary endpoints were assessed. superiority of at least one igpro dose over placebo was tested one-sided using the cochran-armitage trend test for the primary outcome and the jonckheere-terpstra tests for secondary outcomes.the primary outcome occurred in % of high-dose scig, % of low-dose scig, and % of placebo subjects (p < . ); cidp relapse occurred in % of high-dose scig, % of low-dose scig and % of placebo subjects (p < . ), respectively. both scig doses were superior to placebo (low-dose vs placebo p = . ; high dose vs placebo p < . ). median incat score, mrc sum score, and grip strength remained stable in scig subjects. high-dose scig prevented the r-ods decline seen with low-dose scig and placebo (p < . ). all placebo subjects deteriorated on measures of strength and disability.causally related adverse events occurred in ( %) subjects ( % placebo, % low dose, and % high dose).scig igpro was efficacious and safe as maintenance treatment. mutations in metalloendopeptidase (mme) gene have been associated with autosomal-recessive late-onset charcot-marie-tooth type- (cmt ). to date, all patients have had at least one truncating mutation, either in homozygosity or in trans with a missense mutation. more recently, loss-of-function and missense heterozygous mutations were also identified in autosomal-dominant cmt. we report the case of a previously healthy caucasian woman, born to healthy unrelated parents, who presented at the age of thirty-nine with numbness and cold sensation in the lower limbs. subsequently she developed progressive gait disturbance and impaired hand dexterity. her homozygous twin presented at the same age with similar symptoms. the family history was otherwise uneventful, in particular neither neuropathy nor dementia were described. neurological examination at the age of fifty-three revealed a steppage gait, distal upper and lower limb atrophy and weakness, distal sensory loss and bilateral pes cavus. deep tendon reflexes were normal in the upper limbs and absent in the lower limbs. nerve conduction studies revealed an axonal sensory and motor neuropathy. a sural nerve biopsy revealed a reduction in myelinated nerve fibers and active axonal degeneration. targeted sanger sequencing of mpz , gjb , gdap , nefl, fkrp, bscl , hspb and mfn were negative. sureselect focused exome sequencing was therefore performed and identified two missense heterozygous mutations [c. g>a,p.c y;c. t>c,p.y h] or lack of efficacy (n= ). most frequently reported adverse events were cognitive ( %), gastrointestinal ( %) and genitourinary ( %). discontinuation due to perception of lack of efficacy was more frequently reported by men ( % vs % p= . ). women presented higher punctuations on pigc scale compared with men ( . ± . vs . ± . , p= . ). pigc scores were significantly higher in patients receiving taxane ( . ± . ) than platinum ( . ± . ) agents (p= . ). no significant differences according severity of neuropathy neither type of chemotherapy were observed in drop-out and retention rates. patients with long-lasting cipn (> months) reported lower pigc scores ( . ± . vs . ± . , p= . ) and higher frequency of suspension due to adverse events ( % vs %, p= . ) and less rate of continuation of duloxetine ( % vs %, p= . ). more than one-third of patients with disturbing cipn discontinued duloxetine prematurely due to intolerable side-effects. low tolerability, male gender and long-lasting cipn may limit duloxetine usefulness in the treatment of symptomatic cipn. verboon c , jacobs bc , and the igos consortium. department of neurology, erasmus medical centre, rotterdam, the netherlands; department of immunology, erasmus medical centre, rotterdam, the netherlands.the efficacy of intravenous immunoglobulin (ivig) in guillain-barré syndrome (gbs) has only been demonstrated in severely affected patients who are unable to walk independently. although there is no proof that ivig is effective in milder forms of gbs, some neurologists are treating these patients with ivig considering that even milder forms of gbs may result in poor recovery, residual deficits, fatigue or pain.we determined the effectiveness of a single course of ivig ( g/kg in - days) in relatively mild forms of gbs in the ongoing observational international gbs outcome study (igos). the gbs disability score, mrc sum score and patient reported outcome measures (prom) were compared at and weeks. ordinal logistic regression analysis was used to determine the effect of ivig on the gbs disability score, adjusted for previously identified prognostic factors.data were analyzed from the first patients enrolled in igos by december , including patients with mild gbs at entry, of which patients ( %) were treated with supportive care, while patients ( %) received ivig (start ivig after onset of symptoms in days, median , iqr - ). at baseline, patients in the ivig treated group compared to the untreated group less frequently had pure motor gbs ( % versus %, p< . ) and axonal damage or unresponsive nerves ( % versus %, p= . ), but a worse gbs disability scores at nadir (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ). at weeks, the median mrc sum scores and prom were not significantly different between treated and untreated patients. however, more patients in the ivig group showed complete recovery of muscle strength at weeks than patients in the control group ( % versus %) (p= . ) and more frequently showed full neurological recovery on the gbs disability scale ( % versus %, p= . ). additional results will be presented at the conference.based on the results of this interim analysis in observational data, we conclude that patients with a relatively mild form of gbs may benefit from a single course of ivig. despite treatment with intravenous immunoglobulin (ivig), many patients with guillain-barré syndrome (gbs) recover insufficiently. we primarily aimed to determine whether a second ivig course ( g/kg in - days) in patients with a poor prognosis improves outcome on the gbs disability scale after weeks. we included patients from the prospective, observational international gbs outcome study (igos) treated with ivig and who had a poor prognosis on the modified erasmus gbs outcome score (megos). of patients enrolled in igos, patients were eligible; patients ( %) were treated with one ivig course (control group); patients ( %) received an 'early' second ivig course ( - weeks after start first course) and patients ( %) a 'late' second ivig course (within - weeks). one week after study entry, patients receiving an 'early' or 'late' second ivig course had significantly worse gbs disability scores and mrc sum scores than controls, implying the need for adjustment of baseline characteristics. the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' group, and . ( % ci . - . ) for the 'late' group, suggesting worse outcomes with a second course of ivig compared to controls. at months, patients ( %) in the 'early' second ivig group, patients ( %) in the control group and only ( %) in the 'late' second ivig group were able to walk unaided (p= . ). the adjusted common odds ratio for a better gbs disability score at weeks was . ( % ci . - . ) for the 'early' second ivig group and only . ( % ci . - . ) for the 'late' second ivig group. in gbs patients with a poor prognosis, we did not find a beneficial effect of a second course of ivig after weeks follow-up. our results suggest that an 'early' administered second ivig course might improve outcome at weeks. given the limitations of this observational study, a randomized controlled trial with a larger number of gbs patients with a poor prognosis being treated early in the disease course is needed to confirm or refute these results. vidal c , bhatt n , agudelo c , mahapatra a , saporta ma . department of neurology, university of miami miller school of medicine, miami, usa.multifocal motor neuropathy (mmn) is an inflammatory demyelinating chronic neuropathy characterized by progressive asymmetric weakness in the distribution of two or more peripheral nerves, without objective sensory loss or upper motor neuron signs. the cardinal neurophysiological finding is conduction block outside the usual sites of nerve compression. supportive clinical criteria include high titers of anti-gm antibodies, good response to ivig, increased cerebrospinal fluid (csf) protein (< g/dl) and magnetic resonance imaging (mri) with diffuse swelling of the brachial plexus. we report a case of a year old woman with a two month history of progressive muscular weakness which began in the right upper extremity, described as difficulty in gripping objects, writing and typing on the computer, followed by progressive lower extremity weakness with difficulty rising from a chair and left foot drop. she also complained of pain in her right trapezius and scapular area. she denied any tingling or numbness. on neurological examination there was right scapular winging and asymmetric weakness predominantly involving the right upper and left lower extremities. reflexes were absent throughout. lumbar puncture revealed albuminocytological dissociation (wbc and protein . g/dl). ncs revealed slow conduction velocities for both ulnar and median nerves and a median nerve conduction block at the forearm segment. sensory nerve action potentials were normal for all tested nerves. needle emg revealed acute denervation of right periscapular muscles. brachial plexus mri showed symmetric bilateral thickening from trunks to peripheral nerves. anti-gm igg/igm were negative. two days after lumbar puncture and the beginning of ivig, patient presented binocular diplopia on extreme left lateral gaze. brain mri with and without contrast showed no intracranial pathologies. after five days of ivig, patient was discharged with significant clinical improvement and recovery of the right scapular winging. diplopia improved a week after discharge. we report an atypical multifocal motor neuropathy case. although this patient fulfills all clinical criteria for mmn, we report some features usually not found in mmn such as scapular winging and a mild and transient left vi nerve palsy. mmn should be included in the differential diagnosis of scapular winging. vlckova e , , raputova j , , srotova i , , sommer c , Üçeyler n , birklein f , rebhorn c , rittner hl , kovalova e , , nekvapilova e , , belobradkova j , olsovsky j , weber p , dusek l , jarkovsky j , bednarik j , . despite many studies addressing biomarkers for pain in diabetic polyneuropathy (dpn), little is known about why it affects only a certain proportion of dpn patients. the somatosensory system plays a key role in the pathophysiology of neuropathic pain (neup) and subgroups with different sensory profiles might respond differently to pain treatment. we aimed to characterize sensory phenotypes of patients with painful and painless diabetic neuropathy and to assess demographic, clinical, metabolic, electrophysiological and psychological parameters related to the presence of neup in a large cohort of well-defined dpn subjects.this observational cross-sectional multi-centre cohort study (performed as part of the ncrnapain eu consortium) of subjects with dpn (non-painful, ndpn, n= ; painful, pdpn n= ) associated with diabetes mellitus of type and (median age years, range - years; women) comprised detailed history taking, neurological examination, laboratory tests, quantitative sensory testing, nerve conduction studies, neuropathy severity scores, and neuropsychological questionnaires. all parameters were analysed with regard to the presence and severity of neup. the presence and severity of neup were positively correlated with severity of neuropathy and thermal hyposensitivity (p< . ). a minority of pdpn patients ( . %) had a sensory profile indicating thermal hypersensitivity; this was associated with less severe neuropathy and better response to pain therapy. the presence of neup was also associated with female gender (p< . ) and with higher cognitive appraisal of pain as assessed by the pain catastrophizing scale (p< . ), while parameters related to diabetes (duration, hba c, microangiopathy) showed no influence on neup presence and severity. this study confirms the necessity of comprehensive dpn phenotyping and underlines the importance of the severity of neuropathy that should be taken into account in the stratification of patients with pdpn for analgesic treatment and drug trials.people with charcot marie tooth disease (cmt) experience slowly progressive muscle weakness, sensory loss and musculoskeletal changes over time. this leads to disability and risk of comorbidities due to inactivity. exercise is important to maintain general health but may also help to improve symptoms of cmt. we conducted a randomised controlled crossover trial of aerobic exercise to ascertain the effect of training on fitness levels, muscle strength, function and general well-being. in addition, we monitored the safety of training and feasibility of participation in this type of exercise in local community gyms. motivation, confidence and barriers to exercise were explored using qualitative interviews. the recruitment target was people. in total people with cmt a were approached to participate and were unable to commit to the trial or did not meet the study criteria on initial screening. thirty-one people underwent more detailed screening but three failed to meet the study criteria, five people withdrew before starting and three withdrew part way through the study. the data for the people who started the study were analysed using a random effects model. there was a % participation level in the training and it was well tolerated with no increases in pain or blood serum creatine kinase. an increase in vo peak (ml/min/kg) was observed in the cmt group with (pre training: n= , . ± . , % ci . to . ; post training: n= , . ± . , % ci . to . ; pre control: n= , . ± . % ci . to . ; post control: n= , . ± . , % ci . to . ) . there was wide between subject variation leading to a small overall effect size with cohen d of . ( % ci:- . to . ). a tentative regression model showed no effect of group or time point. there were no major changes in other measures of impairment, function or patient reported outcome measures. this pilot study showed that a community based model of training had a small effect on cardiopulmonary fitness, and was well tolerated with good participation. ankle-foot orthoses (afo) are commonly prescribed for children with charcot-marie-tooth disease (cmt) to manage foot drop, however the type and severity of functional impairment results in gait deviations that might require alternate orthotic designs. the aim of this study was to identify d gait patterns of children with cmt based on severity of functional weakness (based on heel walk, toe walk and foot drop items during gait using the cmt pediatric scale) to inform a design pipeline for d printed orthoses. d gait data were captured with an -camera vicon nexus motion capture system using the lower body plug-in-gait model in children with cmt ( male; ± . yrs, ± . cm, ± . kg), of various cmt types: cmt a, cmt e, cmt f, cmt a, cmt c, cmtx , cmtx and compared to typically developing children ( male; . ± . yrs, ± . cm, ± . kg). data were subdivided into three groups denoting increasing severity of dorsiflexion and plantarflexion weakness: no difficulty heel or toe walking (cmt nd ), difficulty heel walking (cmt dh ), difficulty toe and heel walking (cmt dth ). the cmt nd group showed a near-normal gait pattern. the only significant differences noted at the ankle were reduced peak dorsiflexion in stance (p< . ), indicating that an orthotic intervention may not be required. in addition to reduced peak dorsiflexion, the cmt dh group demonstrated significantly reduced dorsiflexion in swing (foot-drop) and a reduced dorsiflexor moment in loading response (p< . ). this suggest, the cmt dh group would require a flexible afo to allow activation of the plantarflexors during push-off, prevent foot-drop and restore a heel rocker in loading response. in contrast, the cmt dth group presented with significantly delayed and increased peak dorsiflexion in stance and reduced plantarflexion and power at push-off (p< . ). they also had significantly increased mean knee extensor moment (p< . ) revealing early signs of 'crouch gait'. therefore, the cmt dth group would require a rigid afo to limit the amount of dorsiflexion and assist movement of the ground reaction force anterior to the knee during stance. three distinct gait patterns at the ankle were identified in children with cmt, indicating patient-specific orthotic design pathways to target specific functional impairment. wojciechowski e , , chang a , cheng t , , little d , , menezes mp , , hogan s , burns j , . university of sydney, new south wales, australia; sydney children's hospitals network (randwick and westmead), new south wales, australia.children with cmt are often prescribed ankle-foot orthoses (afo) to manage lower limb impairment such as foot drop and foot deformities. they are handmade by plaster cast followed by thermoplastic moulding. this traditional approach provides limited design options, can be costly, with long outpatient wait times. d printing, also known as additive manufacturing, has the potential to transform the way orthoses are prescribed, designed and manufactured. the aim of this review was to evaluate the evidence of d printing afos compared to traditional manufacturing methods, for children with cmt. there are currently no studies evaluating the application of d printing afos for children with cmt. however, a small, but emerging evidence base exists for d printed afo's in adults from studies including healthy participants and populations with rheumatoid arthritis, post-polio syndrome, foot drop and ankle weakness secondary to injury. samples sizes ranged from - participants for studies related to in-shoe orthoses and from - for studies related to afos. the methods of d printing included sterolithography, selective laser sintering and fused deposition modelling using materials such as nylon , nylon , polylactide, polycarbonate and abs. d printed afos were comparable to traditional manufactured orthoses in terms of patient-perceived comfort, temporal-spatial parameters, plantar pressure measurements and d gait analysis. however, the effects of long-term usage and durability of d printed afos has not been investigated. d printing orthoses have potential advantages including increased design possibilities, improved productivity, higher compliance, and reduced labour needs. disadvantages include redesigning clinical pathways, limited evidence base for clinical effectiveness, limited biocompatible materials, occupational safety considerations and a high level of expertise required for software operation and fabrication of devices. further research is required to determine the feasibility of d printed afos for children with cmt, and the most appropriate and effective printing pathway, materials to improve health outcomes of affected patients. wong shj , koh sj , lee bjh , chng ysk , pawa c , subramaniam t , cheng ksa , umapathi t . lee kong chian school of medicine, nanyang technological university, singapore; national neuroscience institute, singapore; yong loo lin school of medicine, national university singapore, singapore; khoo teck puat hospital, singapore.treatment-induced neuropathy of diabetes mellitus (dm) (tind) is a complication of rapid glycaemic control. individuals present with neuropathic pain and autonomic dysfunction within weeks of improvement in glucose control. the use of both insulin and oral hypoglycaemic agents has been associated with tind. its severity is determined by the rate and quantum of hba c decline. other predisposing factors include anorexia and weight loss. we studied the incidence of tind in dm patients who have undergone massive weight loss and hba c decline after bariatric surgery. we screened electronic records of patients ( dm, non-dm) who underwent bariatric surgery between and . dm patients fulfilled the tind hba c criteria of a decrease of ≥ % over months or ≥ % over key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections.